WO2021057726A1 - SCREENING OF FC SPECIFICALLY BINDING TO FCγR BY USING MAMMALIAN DISPLAY - Google Patents

Abstract

Disclosed are a method for combining mammalian display and technologies such as flow sorting and next-generation sequencing to screen a Fc variant having enhanced binding ability to specific FcγR, and the Fc variant obtained by the screening.

Description

Screening of FcγR specific binding to Fc using mammalian display Technical field

The present invention relates to Fc region variants, polypeptides containing the Fc region variants, and pharmaceutical compositions containing the polypeptides, as well as Fc screening methods using mammalian display technology and next-generation sequencing. The introduction of amino acid mutations into the Fc region can enhance the binding ability to FcγR receptors, such as FcγRIIB or FcγRIIIA, when compared with the Fc region of wild-type human IgG.

Background technique

Antibody drugs are now playing an increasingly important role in disease treatment. The Fc receptor in the human body is a type of receptor that can bind to the Fc segment of an antibody, which is essential for the efficacy of some antibody drugs. Nowadays, most antibody drugs are of the IgG type. The receptor that binds to IgG in the human body is called FcγR, which is mainly divided into FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), FcγRIIIB (CD16B), FcRn. There is also a genetic polymorphism of amino acid 158 (F158 or V158) in FcγRIIIA, and a genetic polymorphism of amino acid 131 (H131 or R131) in FcγRIIA. Among them, FcγRI (CD64), FcγRIIA (CD32A), FcγRIIIA (CD16A) transmit activation signals to the cell through ITAM in their intracellular domain, and FcγRIIB (CD32B) transmits inhibitory signals to the cell through the ITIM motif contained in its intracellular domain. .

The antibody targets the antigen through its Fab segment, and the antibody Fc can bind to the FcγRIIIA expressed on the surface of natural killer cells, thereby causing natural killer cell-mediated ADCC to kill target cells. FcγRIIA can mediate the ADCP effect of macrophages. FcγRIIB plays an important role in the agonistic activity of agonistic antibodies of the anti-TNF receptor family.

Changing the binding ability of antibody Fc and FcγR by means of point mutations has been shown to be used to improve the efficacy of antibody drugs. For example, the XmAb platform owned by Xencor Company changes the effector function of the antibody by substituting amino acids in the Fc region of the antibody. Based on the reported structure of the Fc/FcR complex, this platform combines the strategies of "directed diversity" and "quality diversity", and uses computer simulations to simulate amino acid mutations that are important for binding. For example, through the Fc/FcR contact interface, optimize the corresponding region of Fc. After the amino acid, the Fc variant candidates were constructed into complete alemtuzumab and expressed and purified, and the semi-automatic AlphaScreen assay developed by Xencor was used to detect the binding ability to FcR. The anti-CD19 antibody XmAb5574 developed by Xencor, through the mutation of S239D/I332E on Fc, can enhance the ADCC effect by 100 to 1000 times in vitro. In the B cell depletion experiment in rhesus monkeys, Rituximab containing the S239D/I332E mutation is better than wild-type Shows better killing effect (ADCC).

MacroGenics has an Fc optimization platform (MacroGenics’ Fc Optimization platform), which has been successfully applied to the development of margetuximab and enoblituzumab. The Fc optimization platform involves: constructing an IgG1 constant region (including CH1, hinge region, CH2, and CH3 region) mutation library into a yeast expression vector by error-prone PCR, using YeastDisplay to display the mutation library, and finally FACS sorting and Yeast clones with strong FcγRIIIA binding ability were subcloned into eukaryotic cells for expression, and identified by ELISA. MGAH22 (margetuximab) developed by MacroGenics is a monoclonal antibody against cancer cell antigen her2. Its Fc region contains five mutations of L235V/F243L/R292P/Y300L/P396L, which increases its affinity with CD16A (V158) from 415nM to 89nM, the affinity with CD16A (F158) increased from 1059nM to 161nM, and showed better efficacy than wild-type in CD16A humanized mouse breast cancer model.

Chugai Pharmaceutical Company screened more than 500 mutants (obtained through comprehensive mutagenesis) and determined that P238D or L328E can increase the binding of FcγRIIB while weakening the binding to other receptors. Using the P238D mutant as a template for further mutations, through screening about 400 mutants, it was finally confirmed that E233D, G237D, H268D, P271G, Y296D, and A330R could increase the binding to FcγRIIB. These mutations were further combined to obtain a V12 variant (E233D/G237D/P238D/H268D/P271G/A330R), which has a 217-fold enhanced binding to FcγRIIB, which is equivalent to FcγRIIA (R131) compared to wild type, and binds to other receptors The force is reduced.

Li Fubin and others found that after increasing the binding ability of Fc and FcγRIIB (CD32B), anti-CD40 agonistic antibodies can exert a better curative effect in mouse colon cancer models.

Based on increasing the binding capacity of Fc to specific Fc receptors, antibody drugs can exert better efficacy. There are currently some screening methods for Fc.

William et al. constructed 4 Fc mutation libraries and built them into the phagemid phage expression vector. Fc can be expressed on the surface of the phage. Using a well plate coated with FcRn on the surface, the phage For binding, the phage is eluted at pH 7, and after several rounds of enrichment, the phage that binds to FcRn under acidic conditions (pH=6) can be selected. This method takes advantage of the large capacity of the phage library, which can reach 10 ⁷ -10 ⁸ , but as a protein expressed in mammalian cells, the formation of disulfide bonds and glycosylation of Fc on the surface of phage are not very effective. Good completion (Dall'Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, Brewah YA, Wu H, Kiener PA, Langermann S (2002) Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences .J Immunol 169:5171-5180).

Jeffrey et al. amplified the antibody CH1-CH3 region by error-prone PCR, introduced random mutations into it, and constructed it into the pYD1 expression vector and transformed EBY100 yeast to construct a 1.8 x 10 ⁷ library . In the induction medium, the antibody CH1-CH3 segment was expressed on the surface of yeast, and then the biotinylated FcγR and mouse anti-hFc F(ab)2-FITC antibody were used for staining and flow-sorted 1.2% positive cells Group from which Fc variants are identified. Although the use of eukaryotic yeast single-cell organisms in this method can be closer to the expression environment of human antibodies, there are still certain differences from fully human-expressed antibodies, especially the glycosylation modification of yeast and human cells is very different. The glycosylation modification has a great influence on the affinity of Fc and FcγR. For example, the removal of fucose from Fc glycosylation can significantly enhance the affinity of Fc and FcγR. (Fc Optimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells In vitro and Controls Tumor Expansion In vivo via Low-Affinity Activating FcγReceptors).

In summary, improving the binding ability of Fc with corresponding receptors through point mutations is essential for improving the efficacy of antibody drugs. Therefore, it is necessary to develop a new Fc screening method, which has an improved screening success rate and efficiency, and can more effectively select Fc variants that meet the mammalian application environment of Fc drugs.

Brief description of the invention

The present invention is based at least in part on the inventor’s discovery: Combining mammalian display with flow sorting, next-generation sequencing and other technologies can efficiently screen out Fc variants with altered binding ability to the desired FcγR .

Therefore, the present invention provides a new Fc screening platform, which includes: (1) constructing a mammalian cell display Fc mutation library; (2) sorting (especially sorting by flow cytometry) displaying library members (3) Perform deep sequencing and cluster analysis on the library members obtained by sorting; and optionally (4) Test/verify the desired binding properties of the Fc variants obtained by screening.

The present invention also provides Fc variants with altered FcγR binding properties and/or altered effector functions (such as ADCC activity or cell activation effect) obtained by the screening method of the present invention.

The screening platform of the present invention has the following advantages:

On the one hand, although phage display screening and yeast display screening systems have been reported in the prior art, these systems express Fc polypeptides in a non-natural environment. In contrast, in the screening platform of the present invention, the Fc region is expressed in its natural environment, that is, mammalian cells. Therefore, in the method of the present invention, it can be ensured that all cellular components involved in antibody/Fc synthesis and processing (folding, disulfide bond formation, glycosylation, etc.) under normal conditions can be used in physiological forms and concentrations. For the expressed Fc region polypeptide. Thus, through the screening method of the present invention, Fc variants that are more suitable for production and application in mammalian cells will be obtained.

On the other hand, although there are reports in the literature that full-length antibodies can be displayed on mammalian cells (see, for example, CN 104011080A), all relevant reports are used to screen antibody variable regions that bind to specific antigens, and there is no report on the use of displayed full-length antibodies. Long antibodies or Fc constant regions are used to screen specific applications of Fc variants. Compared with the prior art, the present inventors surprisingly found that independent of the antibody variable region, the Fc region expressed alone can not only be displayed on the surface of mammalian cells in a correctly folded functional form, but also that the Fc region variants and Fc region can be displayed on the surface of mammalian cells. The changes in the binding properties of receptors can be effectively screened/identified by sorting and displaying Fc receptor binding signals on the cell surface, followed by deep sequencing and cluster analysis. The screening platform of the present invention has the following advantages:

-Due to the purpose of efficient packaging, the length of the viral expression vector will be limited. Compared with the display of full-length antibodies, the method of the present invention, which displays much smaller Fc region polypeptides, is more conducive to the operation of the vector and promotes the effective packaging of the vector in the viral particles, and accordingly also promotes the screening efficiency;

-By combining the mammalian display system with flow cytometry and deep sequencing, the throughput and effectiveness of Fc variant selection are significantly improved, and it is suitable for identifying mutations in the Fc region that are more effective for the improvement of desired properties Regional and dominant amino acid positions;

-As demonstrated in the examples of the present application, the Fc variants obtained by the display and screening method of the present invention, before and after assembly into a full-length antibody, exhibit a fairly consistent expected Fc/FcγR binding activity.

On the other hand, most of the screening methods in the prior art require that after the library is screened, the library obtained by screening is subjected to single clones, and then expression, property detection and sequencing are performed on the selected single clones one by one. In these techniques, the monoclonal step significantly limits the screening throughput of the library, limiting the analysis of library members to only a few selected monoclonal library members, such as dozens or at most hundreds of members.

Different from the prior art library screening method, in the method of the present invention, instead of the monoclonal step of screening members of the library, the present inventors used deep sequencing and cluster analysis to analyze the cell population after sorting. Expanding from dozens or hundreds of conventional library members to thousands or tens of thousands of library members greatly improves the throughput of the entire screening method, significantly reduces the time and cost of screening, and increases the available screening Types of mutant variants. In addition, by combining library screening with the results of deep sequencing, the combination of positive and negative screening and the comparison of the results of deep sequencing of the screened cells can be used to find another with enhanced binding capacity of one or more FcγRs and unchanged or weakened. One or more Fc mutants with FcγR binding ability. At the same time, by analyzing the degree of enrichment of sorting, the method of the present invention can reduce the number of screening cycles. For example, as shown in the examples, cell sorting can be reduced to 1-2 cycles, and Fc with desired properties can still be effectively obtained. Variants.

Brief description of the drawings

Fig. 1 is a schematic diagram of the expression cassette of the membrane display type Fc expression vector. In the figure, IL-2 signal sequence is the IL2 protein signal peptide; PDGFRTM is the transmembrane region of the PDGFR protein; Linker is the linker sequence; hinge is the antibody hinge region; Fc is the C segment region of the antibody heavy chain, including CH2 and CH3 regions , And optionally the CH4 zone.

Figure 2 shows a diagram of 293FT cells displaying different Fc molecules on their surface, stained with biotinylated FcγRIIB (antigen), streptavidin-PE and anti-FLAG-FITC antibodies and subjected to flow cytometric analysis.

Figure 3A shows a flow chart of the first round of sorting of mutant libraries 1-4 driven by FcγRIIIA (F158). Figure 3B shows the cell staining and flow cytometric analysis after the proliferation of the cells sorted in the first round. Figure 3C shows a diagram of proliferating cells after two rounds of FcγRIIIA (F158) sorting for mutation library 1, mutation library 2, and mutation library 3, respectively stained with FcγRIIIA (F158) and FcγRIIIA (V158) and flow cytometric analysis; And, the proliferating cells of the mutant library 4 after a round of FcγRIIIA (F158) sorting were stained with FcγRIIIA (F158) and FcγRIIIA (V158) and flow cytometric analysis respectively.

Figure 4 shows the second-generation sequencing results of mutant library 2, mutant library 3, and mutant library 4 after FcγRIIIA (F158) staining and flow sorting compared with the original library without sorting. The horizontal axis represents the category count of the sequences contained in the library, and the vertical axis represents the cumulative status of the corresponding sequence in the total number of sequences.

Figure 5 shows the corresponding mutations detected by second-generation sequencing in mutant library 2, mutant library 3, and mutant library 4 after FcγRIIIA (F158) staining and flow sorting compared with the original library without sorting The degree of amino acid enrichment of the region.

Figure 6 shows the changes in the binding strength of some mutants obtained from the screening of FcγRIIIA (F158), FcγRIIIA (V158) and FcγRIIB relative to wild-type Fc. The mutants are some high-frequency Fc mutants that appear in mutation library 2, mutation library 3, and mutation library 4 after being enriched by FcγRIIIA (F158). The bonding strength is measured by surface plasmon resonance.

Figure 7 shows that using surface plasmon resonance measurements (Figure 7A) and using cell-based assay measurements (Figure 7B), compared with wild-type Fc (WT), Fc variants are in FcγRIIIA (F158), FcγRIIIA (V158), and Changes in the binding strength of FcγRIIB. The Fc variant is a combination of mutations of mutation library 2 and mutation library 4 selected in FIG. 6.

Figures 8A-C show the ADCC effect of antibodies containing mutations in the Fc region measured on human peripheral blood mononuclear cells, wherein the detected antibody contains the combined mutations in Figure 7 in the Fc region.

Fig. 9A shows a diagram of mutation library 1, mutation library 2, mutation library 3, and mutation library 4 after each round of FcγRIIB sorting and proliferation, staining and flow cytometric analysis with FcγRIIB, respectively. Fig. 9B shows a flow cytometric analysis diagram of mutant library 2 and mutant library 4 after two rounds of FcγRIIB enrichment, respectively stained with FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA (H131), and FcγRIIA (R131).

Figure 10 shows the flow cytometry of mutant library 2 stained and negatively screened with FcγRIIIA (F158) and FcγRIIIA (V158) after two rounds of FcγRIIB sorting.

Figure 11 shows the flow cytometry of mutant library 4 stained and negatively screened with FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA (H131) and FcγRIIA (R131) after two rounds of FcγRIIB sorting.

Figure 12 shows that mutant library 1 and mutant library 3 after four rounds of FcγRIIB staining and flow sorting, and mutant library 2 and mutant library 4 after two rounds of FcγRIIB staining and flow sorting, and the original without sorting. Library, comparison on the results of second-generation sequencing. The horizontal axis represents the category count of the sequences contained in the library, and the vertical axis represents the cumulative status of the corresponding sequence in the total number of sequences.

Figure 13 shows that after mutation library 1, mutation library 2, mutation library 3, and mutation library 4 are FcγRIIB stained and flow sorted, they are in the corresponding mutation regions through next-generation sequencing and the original library that has not been sorted. Comparison of the degree of amino acid enrichment.

Figure 14 shows the comparison of the second-generation sequencing results of mutant libraries 2 and 4 after two rounds of FcγRIIB staining and flow sorting, and then negative screening, with the original library that has not been sorted. The horizontal axis represents the category count of the sequences contained in the library, and the vertical axis represents the cumulative status of the corresponding sequence in the total number of sequences.

Figure 15 shows that mutant library 2 has undergone two rounds of FcγRIIB staining and flow sorting, followed by FcγRIIIA (F158) or FcγRIIIA (V158) negative screening, and then passed through second-generation sequencing. , Comparison of the degree of amino acid enrichment in the corresponding mutation region.

Figure 16 shows that mutant library 4 has been subjected to two rounds of FcγRIIB staining and flow sorting, followed by negative screening of FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA (H131), and FcγRIIA (R131), followed by second-generation sequencing. Compared with the original library without sorting, the degree of amino acid enrichment in the corresponding mutation region is compared.

Figure 17 shows that, in Example 11, some high-frequency Fc mutants appearing in the mutant library 1-4 after passing the FcγRIIB positive sieve and/or the subsequent negative sieve, compared with wild-type Fc (WTFc), have surface plasmon resonance During the measurement, changes in the binding strength of FcyRIIIA (F158), FcyRIIIA (V158), FcyRIIA (H131), FcyRIIA (R131), and FcyRIIB were shown.

Figure 18 shows that the Fc variants containing the mutation combination of mutation library 2 and mutation library 4 in Figure 17 and wild-type Fc (WT) measured by surface plasmon resonance in FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA ( H131), FcγRIIA (R131) and changes in the binding strength of FcγRIIB.

Figures 19A and B show the degree of activation of the Jurkat-CD40 cell line or Jurkat-4-1BB cell line by CD40 agonistic antibodies or 4-1BB agonistic antibodies containing different Fc variants.

Figure 20 shows the plasmid map of the lentiviral expression vector pCDH used in the Examples.

Figure 21A-B shows the sequence of the natural human IgG1 constant region and the amino acid numbering according to the EU numbering system.

Figure 22A-B shows the sequence of the primers used to prepare the Fc mutation library.

Figure 23 shows the plasmid map of the vector pFUSE used for Fc polypeptide expression in the Examples.

Figure 24 shows the amino acid sequence (SEQ ID No: 1) and coding nucleotide sequence (SEQ ID No: 2) of the parent IgG1 Fc region used in the construction of the Fc variant in the Examples.

Figures 25A-C show the cell activation effect mediated by CD40 agonist antibodies containing wild-type Fc regions or containing different Fc region variants.

Figure 26 shows the effect of using fucose-depleted cell CHO as a display platform on the binding fluorescence signal of Fc polypeptide receptor FcγRIIIA displayed on the cell surface.

Brief description of sequence listing

SEQ ID NO:1: the amino acid sequence of the parent IgG1 Fc region used to construct the Fc variant of the embodiment;

SEQ ID NO: 2: the nucleotide sequence encoding the parental IgG1 Fc region sequence (SEQ ID no: 1);

SEQ ID NO: 3: Amino acid sequence of IL2 protein signal peptide;

SEQ ID NO: 4: the amino acid sequence of the transmembrane region of the PDGFR protein;

SEQ ID NO: 5: the amino acid sequence of the FLAG tag.

SEQ IID NO: 6: Sequence of Herceptin's VH-CH1

SEQ ID NO: 7: Light chain sequence of Herceptin:

SEQ ID NO: 8: The sequence of Rituximab's VH-CH1

SEQ ID NO: 9: Light chain sequence of Rituximab

SEQ ID NO: 10: Amino acid sequence of CD40 agonistic antibody heavy chain (with wild-type hIgG1 Fc region)

SEQ ID NO: 11: Amino acid sequence of CD40 agonistic antibody light chain

SEQ ID NO: 12: Utomilumab agonistic antibody heavy chain amino acid sequence (with wild-type hIgG1 Fc region)

SEQ ID NO: 13: the amino acid sequence of the light chain of the Utomilumab agonistic antibody.

Detailed description of the invention

Unless explicitly stated to the contrary, the implementation of the present invention will adopt conventional chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology methods in the art. For descriptions of these methods, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated in July 2008); Short Protocols in Molecular Biology: A Compendium of Methods, from Current Protocols in Molecular Biology. Associates and Wiley-Interscience; Glover, DNACloning: A Practical Approach, vol. I&II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation( B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols in Immunology QE Coligan, AMKruisbeek, DH Margulies, EMShevach and W. Strober, eds., 1991); Annual Review of Immunology; and periodicals such as Advances in Immunology.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.

The term "about" when used in conjunction with a numerical value means to cover a numerical value within a range having a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value.

When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "including" means to include the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also covers the case consisting of the stated elements, integers or steps. For example, when referring to a polypeptide "comprising" a specific sequence, it is also intended to encompass the polypeptide consisting of the specific sequence.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which does not include the heavy chain constant region CH1. The Fc region of an immunoglobulin usually contains two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. Generally, the dimerization of two identical heavy chains of immunoglobulins is mediated by the dimerization of the CH3 domain, and by linking the CH1 constant domain to the hinge region of the Fc constant domain (e.g., CH2 and CH3) The disulfide bonds to stabilize. Therefore, in the present invention, the Fc region can be the last two immunoglobulin constant regions of IgA, IgD, and IgG, or the last three immunoglobulin constant regions of IgE and IgM, and optionally the N-terminal direction of these constant regions On the hinge area.

In the present invention, in one embodiment, the Fc region contains CH2 and CH3 domains. In a preferred embodiment, the Fc region further comprises amino acid residues of the hinge region. In one embodiment, the Fc region is a human IgG heavy chain Fc region, extending from Glu216 of the heavy chain to the carboxy terminus, wherein the C-terminal lysine (Lys447) located in the Fc region may or may not be present. In yet another embodiment, the Fc region preferably includes a complete IgG hinge region (EU numbering positions 216-230), and dimers are formed through disulfide bonds in the hinge region. In one embodiment, the two chains forming the Fc dimer comprise part or all of the hinge region and CH2 and CH3 domains, respectively.

As used herein, the term Fc region includes native sequence Fc-regions and variant Fc regions. In one embodiment, the Fc region may be any IgG Fc region, preferably a mammalian or human IgG Fc region, such as IgG1, IgG2, IgG3 or IgG4 Fc region. In one embodiment, the amino acid sequence of the Fc region of human IgG1 starts from the hinge region and ends at the carboxy terminus of the CH3 region, as shown in FIG. 21. The natural or wild-type human IgG1 Fc region is intended to encompass these natural allelic forms herein.

In this context, the Fc region may be this region in isolation, or this region in an antibody, antibody fragment, or Fc fusion protein.

In this article, unless otherwise indicated, the numbering of amino acid residues in the Fc-region or constant region is performed according to the EU numbering system, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. The portion of this document describing this numbering system is incorporated herein by reference. Regarding the EU number of the Fc region or constant region, it can also be easily obtained from the EU number query website: http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html. Herein, according to this numbering system, specific amino acid residues in the constant region of an antibody IgG are mentioned. For example, "I332" refers to the isoleucine at position 332 of EU. The amino acid mutation at a specific position in the constant region is represented by (original amino acid, amino acid position, mutated amino acid). For example, "I332E" means that the isoleucine (I) at EU position 332 is replaced by glutamic acid (E). When referring to a combination of mutations, the combined mutations are connected by a plus sign (+). "K326I+I332E" means that the Fc region contains both mutations K326I and I332E. When there are multiple mutation possibilities in a specific position, it is represented by the symbol "/" in this document. For example, the mutation "K326I/S" means that residue K at position 326 can be replaced with an I or S residue.

"Fc protein" or "Fc polypeptide" are used interchangeably herein to refer to a protein or polypeptide comprising an Fc region. In addition, according to the context used in the expression, it can be understood that the expression can also refer to a polypeptide consisting essentially of an Fc region, or a polypeptide consisting of an Fc region.

In one embodiment, the Fc protein is an Fc polypeptide displayed on the outer surface of the cell membrane. Preferably, in the present invention, the Fc protein is displayed on the outer surface of the cell membrane by including a vector encoding the Fc protein of the present invention displayed on the surface of the cell membrane. In one embodiment, the vector is a viral vector, preferably a lentiviral vector. In one embodiment, a vector containing a polynucleotide encoding an Fc protein is introduced into a mammalian cell, and the mammalian cell is cultured under conditions suitable for expressing an Fc protein-encoding nucleic acid, thereby producing a vector that displays the Fc protein on the cell surface. mammal.

In yet another embodiment, the Fc protein is a soluble protein that does not bind to the cell membrane and includes a hinge region and CH2 and CH3 regions.

As used herein, "Fc region variant" and "variant Fc region" can be used interchangeably, and refer to the introduction of one or more elements at any position of the Fc region relative to the Fc region before modification (ie, the parent Fc region). An Fc region with multiple amino acid modifications (ie, amino acid substitutions, deletions, and/or insertions). In some embodiments, the parental Fc region is a natural immunoglobulin Fc region, that is, a wild-type Fc region. In another embodiment, the parent Fc region is an Fc region into which a mutation has been introduced in the wild-type Fc region. In one embodiment, the parental Fc region comprises the amino acid sequence of SEQ ID NO:1. In yet another embodiment, the parental Fc region includes SEQ ID NO; 1 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % Or above percent sequence identity. In some embodiments, the parent Fc region is an amino acid sequence in which amino acid mutations that change Fc properties are introduced in the wild-type Fc region such as SEQ ID NO:1, wherein the Fc properties can be selected from, including but not limited to, and Specific Fc receptor binding affinity, Fc heterodimerization, Fc glycosylation pattern. The amino acid mutation may be a mutation known in the art or obtained by the screening method of the present invention. The use of such parental Fc regions in the methods of the present invention may be desirable in some situations, for example, when it is desired to further improve certain Fc properties, or to superimpose multiple different Fc properties.

In this context, "Fc variant protein", "Fc variant polypeptide", and "Fc variant" are used interchangeably and refer to a polypeptide comprising a variant Fc region. The cell surface display method of the present invention can be used to display and screen Fc variant proteins. The "mammalian cell display Fc mutation library" of the present invention is a cell collection containing a plurality of mammalian cells displaying different Fc variant proteins on the cell surface.

"Fc partner" refers to any molecule that can bind to the Fc region of an antibody and form an Fc/Fc partner complex, such as a protein or polypeptide from an organism. Fc partners include but are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, protein A, protein G and viral FcγR. Fc partners also include Fc receptor homologs (FcRH). FcRH is an Fc receptor homologous to FcγR (Davis et al., 2002, Immunological Reviews 190:123-136, which is incorporated herein as a reference). Preferably, the Fc partners are FcRn and FcγR. In this application, in most embodiments of the Fc variant screening method of the present invention, FcγR is used as an example for description, but as will be clear to those skilled in the art, in these embodiments, other Fc partners can be used instead of examples. Sexual FcγR is performed.

As used herein, "Fc receptor" may be any Fc receptor that binds to the Fc of an antibody, including but not limited to Fcγ receptor and FcRn receptor.

It is known that there are three types of human Fcγ receptors (FcγR), FcγRI, FcγRII and FcγRIII for IgG antibodies. These three types of receptors can be divided into subtypes, including FcyRIA, FcyRIB, FcyRIIA, FcyRIIB, FcyRIIC, FcyRIIIA and FcyRIIIB. In addition, several allelic variants with different IgG subtype binding capabilities have been found, such as FcyRIIIA-F158 and FcyRIIIA-V158; and FcyRIIA-H131 and FcyRIIA-R131.

All FcγRs bind to the same region on IgG Fc—the junction between the hinge region and CH2, but have different affinity. For example, FcγRI is a high-affinity binding receptor, while FcγRII and III are low-affinity binding receptors. The binding site of antibody and FcRn is located at the junction of CH2 and CH3.

In mammals, humoral immunity is mostly mediated through the interaction between the Fc region of antibodies and C1q and the complement cascade. The cellular immune response is mostly mediated by the interaction between the Fc region of the antibody and the Fcγ receptor. FcyRI, FcyRIIA, FcyRIIIA and FcyRIIIB are activating FcyR. And FcγRIIB is an inhibitory FcγR. The intracellular signal transduction of the activating receptor is mediated by phosphorylation of the receptor's intracellular ITAM motif, which leads to effector functions such as ADCC, ADCP, and inflammatory response by inducing the release of cytokines. The cell signaling of the inhibitory receptor FcγRIIB is mediated by phosphorylation of the ITIM motif in the receptor cell, and plays a role in balancing the active signaling pathway. The interaction of antibodies with FcγR and C1q mainly depends on the hinge and CH2 amino acid sequence and the glycosylation of the CH2 region.

In one aspect, therefore, the present invention relates to the use of the mammalian cell display Fc mutation library of the present invention to screen for Fc variants, wherein the Fc variants have fine-regulated Fc/Fc receptor binding properties, thereby having improved Antibody properties, such as improved effector function.

In this regard, one embodiment of the present invention relates to altering FcγR-based effector functions. For example, in one embodiment, the present invention relates to an Fc region variant and a screening method thereof, wherein the Fc region variant has an enhanced binding ability to FcγRIIIA F158 and/or V158 relative to a wild-type Fc region, and preferably Improved ADCC activity, such as enhanced ADCC activity and/or greater ADCC responsive population (ie, a larger proportion of individuals in the population respond to antibodies containing the Fc variant). In still another embodiment, the present invention relates to an Fc region variant and a screening method thereof, wherein the Fc region variant has an enhanced binding ability to FcγRIIB relative to a wild-type Fc region. In yet another embodiment, the present invention relates to an Fc region variant and a screening method thereof, wherein the Fc region variant has an altered binding ratio of FcγRIIB to FcγRIIIA relative to the wild-type Fc region. In a preferred embodiment, relative to the wild-type Fc region, the Fc region variant has enhanced FcγRIIB binding and at the same time has substantially equivalent or reduced binding capacity to FcγRIIIA. In a preferred embodiment, the antibody comprising the Fc region variant exhibits an enhanced cell activation effect, and preferably also exhibits a reduced ADCC activity.

Another aspect of the present invention involves changing the binding ability of the Fc region to the neonatal Fc receptor (FcRn). FcRn plays an important role in IgG cell trafficking and serum half-life. In one embodiment, therefore, the present invention relates to an Fc region variant and a screening method thereof, wherein the Fc region variant has an altered binding to FcRn relative to a wild-type Fc region, and thus has an altered circulating half-life.

In yet another aspect, the present invention relates to further engineering the affinity of Fc and FcγR while changing FcRn binding and the corresponding antibody half-life. In one embodiment, therefore, the present invention relates to an Fc region variant having an altered FcRn binding ability and an altered FcγR binding ability and a screening method thereof, wherein the Fc region variant preferably has an enhanced half-life and at the same time an improved Effector function.

As used herein, "antibody" refers to a protein comprising a heavy chain and/or light chain variable region, such as a full-length antibody, or an antibody fragment such as a single chain scFv antibody, Fab, F(ab)2', Fab'. Preferably, the antibody of the present invention further comprises an Fc region, which may be a natural Fc region or an Fc region variant comprising one or more amino acid substitutions, deletions and/or insertions, preferably an Fc region variant. In one embodiment, the antibody is a full-length antibody comprising a heavy chain and a light chain, wherein the Fc region is connected to the heavy chain variable region VH and CH1 through a hinge region. In another embodiment, the antibody is formed by linking antibody fragments to the Fc region. In one embodiment, the antibody fragment is a scFv, wherein the scFv is connected to the Fc region through a hinge region.

"Fc fusion protein" refers herein to a protein comprising an Fc region fused to other polypeptides. Other polypeptides can be polypeptides that can specifically bind to the target molecule, such as immunoglobulin polypeptides, such as the heavy chain and/or light chain variable regions of an antibody that can bind to the target molecule, or the solubility of a receptor that can bind to the target molecule. section. Therefore, antibodies such as scFv-Fc forms of antibodies and immune fusions belong to the category of Fc fusion proteins.

The term "effector function" refers to those biological activities attributable to the Fc-region of an antibody, which vary with antibody class. There are five main antibody classes known: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 . The IgGFc region can mediate several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and the half-life/clearance rate of antibodies and antigen-antibody complexes. In some cases, depending on the purpose of treatment, these effector functions are ideal for therapeutic antibodies, but in other cases they may be unnecessary or even harmful. Therefore, in one embodiment, the present invention provides a variant Fc region having amino acid residue changes in the Fc region, thereby altering the effector function of an antibody, and a screening method thereof. For example, at least one amino acid residue can be substituted in the Fc region of the antibody, thereby changing the effector function of the antibody.

"ADCC" refers to antibody-dependent cell-mediated cytotoxicity. ADCC is mainly mediated by natural killer cells (NK cells) in the human body. In ADCC, the antibody binds to the antigen displayed on the surface of the target cell, and the FcγRIIIA on the surface of the NK cell recognizes the Fc region of the antibody, so that the NK cell is activated to release perforin and granulolytic enzyme, leading to the lysis and apoptosis of the target cell.

非放射性细胞毒性测定(Promega，Madison，WI))。适用于这些测定的效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。备选地或另外地，可以在体内评价目标分子的ADCC活性，例如，在如Clynes，R.等，Proc.Nat’lAcad.Sci.USA 95(1998)652-656中公开的动物模型中评价。 A non-limiting example of an in vitro assay to evaluate the ADCC activity of a target molecule is described in US 5,500,362 (see also, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063; And Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see also Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351- 1361). Alternatively, non-radioactive assay methods (for example, ^{ACTI TM} non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and

Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Suitable effector cells for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be evaluated in vivo, for example, in an animal model as disclosed in Clynes, R. et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656 .

"CDC" refers to complement dependent cytotoxicity. In CDC, the Fc region of the antibody binds to the complement molecule C1q, and then forms a membrane attack complex, leading to the elimination of target cells. See, for example, Liszewski and Atkinson, ch. 26, Fundamental immunology, 3rd edition, Paul ed., Raven Press, New York, 1993, pp917-940.

"ADCP" refers to antibody-dependent cell-mediated phagocytosis. In this process mediated by the Fc receptor, the target cells that bind to the antibody are swallowed by phagocytes such as macrophages, monocytes, neutrophils, and dendritic cells. A variety of Fc receptors can participate in this process. Richards et al., Mol. Cancer Ther. 7(8): 2517-2527 (2008) describe an in vitro test for ADCP.

"Label" or "label" herein refers to a detectable substance that can be used for the direct or indirect labeling purpose of the present invention. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/ Biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include Lu Minoan; Examples of suitable radioactive materials include 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.

"Cell membrane anchoring interval" and "membrane anchor" are used interchangeably herein, and refer to an amino acid sequence that enables the polypeptide or protein containing the region to span and anchor on the cell membrane. Preferably, the cell membrane anchoring region is a transmembrane domain or a transmembrane region.

"Multiple Infection" (MOI) refers to the number of virus particles infected per cell.

As used herein, "sequence identity" refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino-acid basis in the comparison window. The "percent sequence identity" can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and the same nucleic acid bases (for example, A, T, C, G, I, etc.) are present in the two sequences. ) Or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) The number of positions to get the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to produce the sequence identity percentage. The optimal alignment to determine the percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.

Each aspect of the screening platform of the present invention will be described separately below.

I. Construction of a mammalian cell Fc polypeptide display system

In one aspect, the present invention provides a mammalian cell display system, which is a cell library composed of a plurality of cell clones displaying Fc polypeptides on the surface; preferably, each cell clone displays a different Fc polypeptide; preferably The library capacity of the ground library reaches 1-5x10 ⁵ . However, the present invention can also be screened on display libraries containing more or fewer library members.

In order to construct the mammalian cell display library of the present invention, a pool of nucleic acids encoding a variety of different Fc polypeptides (herein, also referred to as Fc variant library) can be used to transform mammalian cells; The mammalian cell is cultured under the condition of expression and display on its surface.

As demonstrated in the examples, using the display system of the present invention, the steric hindrance of mammalian cell membrane components does not substantially affect the functional display of the Fc region and the binding of the Fc region to the target Fc receptor.

Fc polypeptide displayed on the surface of library cell members

In the present invention, the polypeptide expressed and displayed on mammalian cells is a polypeptide comprising an Fc region (herein, also referred to as an Fc polypeptide). In one embodiment, the Fc region is an Fc region derived from an immunoglobulin and includes at least one or more of a hinge region, a CH2 region, and a CH3 region. Sequence diversity can be introduced into the hinge region, CH2 region and/or CH3 region of the Fc region. Fc variants with altered Fcγ receptor affinity and/or specificity can be screened from the Fc region display library introduced with sequence diversity, and such Fc variants can confer, for example, altered effector functions. Alternatively, the display library can be screened for Fc variants that have increased or decreased interactions with FcRn, and such Fc variants can be used for fusion with, for example, antibody variable regions to change their half-life after administration.

For display on the cell surface, the Fc region variant polypeptide-encoding nucleic acid can be fused with DNA encoding the leader sequence to allow secretion through the endoplasmic reticulum (ER), and fused with membrane anchors to allow the Fc variant polypeptide to be immobilized on the cell surface .

In the present invention, therefore, in one embodiment, the Fc variant polypeptide of the present invention used for constructing the display library is an Fc fusion polypeptide, comprising from the N-terminus to the C-terminus:

-A secretory signal sequence, preferably the signal sequence is a signal peptide that can guide the Fc region polypeptide to secrete out of the cell, for example, the IL-2 protein signal peptide;

-Optionally, the tag sequence, preferably the tag sequence is an epitope tag sequence, such as HA epitope tag, Flag tag sequence, c-myc epitope tag;

-Fc region variant polypeptides, the Fc region is preferably derived from the Fc regions of IgG1, IgG2, IgG3, IgG4 and chimeras formed by different isotypes, more preferably human IgG1 Fc region, most preferably in SEQ ID No:1 A variant of the human IgG1 Fc region polypeptide shown, for example, the variant contains 1-10 mutations, such as 1, 2, 3, 4 or 5 mutations;

-Membrane anchors, preferably transmembrane domains; wherein the transmembrane domains anchor the Fc region on the cell membrane surface, preferably the transmembrane domains are derived from membrane-bound proteins expressed on the surface of mammalian cells, for example, the transmembrane domains of PDGFR protein Membrane region, such as the transmembrane region sequence described in SEQ ID NO: 4;

In the Fc polypeptide, preferably, the Fc region variant is operably linked to the tag sequence and the transmembrane domain, preferably using a linker, such as a short flexible linker sequence, to be covalently linked. In one embodiment, the tag sequence is attached to the N-terminus of the Fc region variant. In yet another embodiment, the tag sequence is attached to the C-terminus of the Fc region variant and at the N-terminus of the transmembrane region.

Fc region variants

In the display system of the present invention, in one embodiment, the Fc region used for display includes CH2 and CH3 regions, and preferably also includes (all or part of) hinge regions, or consists of them. In another embodiment, the displayed Fc polypeptide further comprises the VH-CH1 region or CH1 of the antibody heavy chain linked to the N-terminus of the Fc region. In yet another embodiment, the displayed Fc polypeptide preferably does not include the VH region of the antibody heavy chain, and does not include the CH1 region of the antibody heavy chain. In some embodiments, the hinge region can be derived from the same or different IgG type as the CH2 and CH3 regions, for example, both can be derived from IgG1; or the hinge region can be derived from IgG2 or IgG3, and the CH2 and CH3 regions can be derived from the IgG1 or IgG4 type.

In one embodiment, the mutation introduced in the Fc region may be one or more amino acid substitutions, deletions or additions, preferably amino acid substitutions. Mutations can be concentrated in one specific area, or multiple, for example, two specific areas. For example, mutations can be introduced in two or more mutation regions and/or mutation locations that are identified as having a significant impact on the binding of the target FcγR receptor by the method of the present invention; random mutations can also be introduced into a certain region on the Fc, for example, using 5 consecutive random amino acids replace 4 amino acids in Fc. Alternatively, mutations can be introduced by replacing one amino acid sequence in Fc with another longer or shorter amino acid sequence.

In the present invention, the Fc region variants of the present invention are constructed by introducing mutations in the parent Fc region. In some embodiments, the parent Fc polypeptide is a natural Fc polypeptide (i.e., a wild-type Fc polypeptide). In other embodiments, the parent Fc polypeptide is an Fc polypeptide that already contains a mutation relative to a wild-type Fc polypeptide.

In the present invention, in a preferred embodiment, an Fc variant library is constructed from a parent Fc polypeptide, wherein mutations can be introduced into predetermined mutation regions/mutation positions, or mutations can be randomly introduced into the parent Fc region to form Fc variants library. Therefore, in one aspect, the screening method of the present invention is performed on a clonal population of cells displaying variants of the "parent Fc polypeptide". Accordingly, in one aspect, the present invention provides a mammalian cell Fc display library, wherein members of the cell clone library display a variant of the "parent Fc polypeptide" on the cell surface, wherein the variant is relative to the parent Fc polypeptide Modified Fc polypeptide. In one embodiment, the cell clone is produced by introducing a mutation in the region/position of the Fc region of the parent Fc polypeptide sequence, and introducing the nucleic acid sequence encoding the variant into the cell, preferably by introducing a viral vector into the mammalian cell , So that the nucleic acid is integrated into the genome of the cell.

In order to construct a library of Fc variants, optionally, prior to introducing mutations, a bioinformatics evaluation of the parental Fc polypeptide sequence can be performed to provide mutation regions and mutations that are expected to affect the desired properties (for example, binding properties to a specific FcγR). / Or mutation location. After that, based on the biological information evaluation, a mutation strategy can be designed. It is also possible to use structural modeling to assist in the identification of amino acids to be mutagenized.

In some previous mutation analysis, it has been found that some amino acids on IgG that have a key role in the binding of FcγR are located in the lower hinge region and CH2 region (see, for example, Xinhua Wang et al., IgG Fc engineering to modulate antibody efficiency functions, Protein Cell 2018, 9 (1):63-73). In some embodiments, these amino acid positions can be selected as the Fc region mutation region where the mutation is to be introduced.

In addition, the co-crystal structure of human IgG1 Fc and various FcγRs has been reported (FcγRI (PDBID: 4W4O); FcγRIIA (PDBID: 3RY6); FcγRIIB (PDBID: 3WJJ); FcγRIIIA (PDBID: 5D6B)), which allows high resolution Map the binding interface of Fc and FcγR. According to the information of the binding interface, the Fc region where the mutation is to be introduced can be selected. For example, the amino acid position of the Fc region 5 angstroms away from the FcγR on the binding interface is selected as the position where the mutation is to be introduced.

In addition, it is known that Fc can be modified to increase or decrease its degree of glycosylation and/or change its glycosylation pattern, thereby changing the binding properties of Fc to receptors. The addition or deletion of Fc glycosylation sites can be conveniently achieved by changing the amino acid sequence so as to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions can be made to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site. Therefore, in one embodiment, the Fc region that may affect glycosylation modification can be selected as the location where the mutation is to be introduced.

In a preferred embodiment, in order to construct the diversity of the Fc variant library, the mutation region of the Fc region can be selected as follows:

-Based on the complex structure of the Fc region and the target Fc receptor, select the region close to FcR in the Fc, for example, the region within 5 angstroms as the region to be introduced for mutation;

-The region where known Fc variants with altered FcR binding capacity are located;

-Fc region that affects the orientation of glycosylation modification

Preferably, the mutation region is selected from the group consisting of positions 233-238 of EU numbering (ELLGGP) on IgG1 Fc, positions 265-271 of EU numbering (DVSHEDP), positions 295-300 of EU numbering (QYNSTY), and positions of EU numbering. 326-332 (KALPAPI).

Preferably, for improving FcγRIIIA binding or improving FcγRIIB binding, the mutation region is selected from: EU numbering 265-271 (DVSHEDP) and EU numbering 326-332 (KALPAPI); more preferably EU numbering 326-332 Bit.

One, two, three, or more mutations can be randomly introduced into the above-mentioned mutation regions; it is also possible to combine mutations of different mutation regions and introduce them into the Fc region to construct a mutant library.

After determining the region and/or location of the mutation to be introduced, any mutation introduction method known in the art, such as a nucleic acid mutagenesis method, can be used to generate an Fc variant encoding nucleic acid sequence. Generally, nucleic acid mutagenesis can be performed using methods known in the art, such as oligonucleotide-guided mutagenesis (Molecular Cloning: Laboratory Manual, 3rd Edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press). In one embodiment, for example, the identified amino acid positions/regions can be evaluated to bioinformatics, using degenerate primers including, for example, MNN (where M stands for A/C and N stands for A/G/C/T), by PCR, Random mutations are introduced at these specific positions/regions in the DNA encoding the parental Fc polypeptide.

In some preferred embodiments, after determining the region where the mutation is to be introduced, a mutation scan of a single amino acid or 1-3 amino acids in a specific region is performed. In a preferred embodiment, a random mutation library is constructed for use in the display library screening of the present invention. In yet another embodiment, the mutations that have been screened and determined by the method of the present invention or the mutations known in the art can be used as the basis for combinatorial mutagenesis, in which the sequence is changed at multiple positions simultaneously.

In some embodiments, by PCR assembly, a reference Fc polypeptide, such as a wild-type Fc polypeptide encoding nucleic acid, is used as a template to obtain an Fc variant polypeptide coding sequence with a mutation introduced.

Tag sequence

As understood by those skilled in the art, it is important to standardize the cell surface expression level of Fc when measuring the binding ability (dynamic binding and balanced binding) of the Fc variant with the target FcγR.

Therefore, preferably, the Fc polypeptide of the present invention displayed on the cell membrane surface contains a tag sequence fused to the Fc region. In one embodiment, the tag sequence fused to the Fc variant polypeptide can be used as a useful internal control to indicate the display of the Fc fusion polypeptide on the cell surface. For example, a label sequence binding molecule (such as an anti-tag sequence antibody) with a label (such as a fluorescent label) can be used to detect the presence of a label sequence on a cell, so as to quantify the Fc polypeptide on the cell surface independent of the binding activity of the Fc polypeptide and its receptor. exist. Therefore, in one embodiment, the method of the present invention includes: prior to the sorting step, by staining the tag sequence (for example, staining with a fluorescent stain coupled with an anti-tag sequence antibody) to show the cell surface display level of the Fc polypeptide .

The tag sequence can be placed at the N-terminus or C-terminus of the Fc region polypeptide, preferably the N-terminus.

The tag sequences that can be used include, but are not limited to, epitope tag sequences, such as HA epitope tags, Flag tag sequences, and c-myc epitope tags.

Signal sequence

In the context of the present invention, in order to produce an Fc polypeptide that is secreted from a cell, a nucleic acid encoding an Fc polypeptide may include a DNA segment encoding a "signal sequence" or a "leader peptide". As known in the art, the signal sequence can direct the newly synthesized polypeptide to reach and pass through the ER membrane, where the polypeptide enters the secretory route. When the protein crosses the ER membrane, the signal sequence is cleaved off by the signal peptide. As far as the function of the signal sequence is concerned, its recognition by the host cell secretion machinery is crucial.

In one embodiment, therefore, the present invention provides an Fc polypeptide with a signal peptide and a nucleic acid encoding the same, wherein the signal peptide directs the secretion of the Fc polypeptide from mammalian cells, and in particular, the signal peptide is located in the Fc region. At the N-terminus, preferably, a tag sequence and/or peptide linker can be inserted between the signal peptide and the Fc region polypeptide. In mammalian cells, the signal peptide is preferably cleaved from the Fc fusion polypeptide during processing and transportation.

Signal peptides that direct proteins to the secretory pathway of mammalian cells are generally known in the art and are disclosed, for example, in Nielsen et al., Protein Engineering 10 (1997) 1-6. In one embodiment, the signal peptide is derived from a secreted or type I transmembrane protein. In one embodiment, the signal peptide is derived from secreted proteins, such as cytokine family members (e.g. interleukin 2, IL-2), serum protein family members (albumin, transferrin, lipoprotein, immunoglobulin), cell Outer matrix proteins (collagen, fibronectin, proteoglycan), peptide hormones (insulin, glucagon, endorphins, enkephalin, ACTH), digestive enzymes (trypsin, chymotrypsin, amylase) , Ribonuclease, deoxyribonuclease) or milk protein (casein, lactalbumin). In one embodiment, the signal peptide is derived from an immunoglobulin, particularly an antibody heavy or light chain, such as an Igκ light chain signal peptide. In one embodiment, the signal peptide is a human IL-2 signal peptide, preferably the signal peptide sequence shown in SEQ ID NO: 3.

Membrane anchor

A feature of the display library is that the Fc protein stays on the cell surface, thereby establishing a physical connection between the Fc protein displayed by the cell and the coding DNA contained in the cell. Based on this physical connection, the cells expressing the Fc protein with the desired properties can be physically sorted and separated, and the corresponding coding DNA can be obtained.

In order to achieve the retention of Fc protein on the cell surface, various methods can be used, including but not limited to, direct fusion with membrane anchors, such as transmembrane domains such as the transmembrane domain of PDGF receptor, or direct fusion with GPI recognition sequence Fusion.

In one embodiment, in order to generate a mammalian display library of Fc variants, a membrane-bound form of the Fc region polypeptide is expressed.

In some embodiments, the membrane-bound form of the Fc region polypeptide is formed by fusing the Fc region polypeptide to the transmembrane domain at the C-terminus, and optionally, the connection is achieved through a linking sequence (for example, a flexible linker). The transmembrane domain can anchor the Fc region polypeptide on the cell membrane surface.

The transmembrane domain usually contains three different structural regions: the N-terminal extracellular region, the middle conserved transmembrane region and the C-terminal cytoplasmic region. In one embodiment, the transmembrane domain used to form the Fc polypeptide of the invention consists only of the transmembrane region. In one embodiment, the transmembrane domain includes an extracellular region and a transmembrane region in an N-terminal to C-terminal direction. In yet another embodiment, the transmembrane domain may additionally comprise an intracellular region or a cytoplasmic region.

In an Fc protein with a transmembrane region, preferably, the transmembrane region is located at the C-terminus of the Fc region, that is, the C-terminus of Fc CH3, and can cause the Fc to remain bound to the outer surface of the cell when the Fc protein is secreted out of the cell.

In one embodiment, the transmembrane region is derived from an integrated membrane protein.

In one embodiment, the transmembrane region is derived from a type I transmembrane protein (Do et al., Cell 85 (1996) 369-78; Mothes et al., Cell 89 (1997) 523-533) such as cell adhesion molecules (integrated Catenin, mucin, cadherin), lectin (sialoadhesin, CD22, CD33) or receptor tyrosine kinase (insulin receptor, EGF receptor, FGF receptor, PDGF receptor) intrinsic cessation Transfer membrane anchor sequence (internal stop-transfer membrane-anchor sequence).

In one embodiment, the transmembrane region is the transmembrane region of a human G class membrane-bound immunoglobulin.

In one embodiment, the transmembrane region is derived from receptor tyrosine kinases, more specifically from human platelet-derived growth factor receptor (hPDGFR), and most specifically from hPDGFR B chain (accession number NP002600).

In one embodiment, the transmembrane region is derived from a human PDGFR beta chain. In one embodiment, the transmembrane region comprises or consists of the sequence of SEQ ID NO: 4 or a substantially similar sequence (for example, a sequence with at least 90% or 95% or more than 99% identity).

It is also possible to achieve anchoring of the Fc polypeptide in the cell membrane through GPI connection (Moran and Caras, The Journal of Cell Biology 115 (1991) 1595-1600). Therefore, in other embodiments, the Fc polypeptide can also be fused with a GPI-anchor signal peptide to achieve cell surface display. "GPI-anchor" in this application refers to a post-translational modification attached to the C-terminus of a polypeptide or protein. The "GPI-anchor" has a core structure comprising at least one phosphoethanolamine residue, trimannoside, glucosamine residue and inositol phospholipid. The term "GPI-anchor signal peptide" refers to the C-terminal amino acid sequence of a polypeptide or protein. The C-terminal amino acid sequence consists of an amino acid to which the GPI-anchor can bind, an optional spacer peptide, and a hydrophobic peptide. Almost all of the signal peptide, that is, the optional spacer peptide and hydrophobic peptide, will be removed by the enzyme GPI-aminotransferase after translation, and the amino group of the phosphoethanolamine in the core of the GPI-anchor is bound to the GPI-anchor. A bond is formed between amino acids.

Linker sequence

In the Fc polypeptide of the present invention, the Fc region variant is operably linked to the tag sequence and the transmembrane domain. In a preferred embodiment, the Fc region variant is fused to the tag sequence and/or the transmembrane domain directly or preferably via a linker, such as a short flexible linker sequence.

The linker sequence that can be used in the present invention is preferably a flexible linker peptide or peptide linker composed of amino acid residues connected by peptide bonds. Such peptide linkers are usually rich in glycine, which exhibits flexibility, and serine or threonine, which exhibits solubility. For example, glycine and/or serine residues can be used alone or in combination. Non-limiting examples of flexible peptide linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008), WO2012/138475 and WO2014/087010, the contents of which are incorporated by reference in their entirety.

In some embodiments, the peptide linker consists of amino acid residues selected from twenty natural amino acids. In certain other embodiments, the one or more amino acids are selected from glycine, serine, threonine, alanine, proline, asparagine, glutamine, and lysine. In a preferred embodiment, the one or more amino acids are selected from Gly, Ser, Thr, Lys, Pro, and Glu.

In some embodiments, the length of the linker is about 1-30 amino acids, or about 10 to about 25 amino acids, about 15 to about 20 amino acids, or about 10 to about 20 amino acids, or any intermediate Amino acid length. In a preferred embodiment, the linker has a length of 15-25 amino acid residues, and in a more preferred embodiment, a length of 15-18 amino acid residues. In some embodiments, the length of the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. In one embodiment, the Fc region is connected to the tag sequence via a linker, and the linker is preferably 3-10 amino acids, more preferably 5 amino acids in length. In yet another embodiment, the Fc region is connected to the transmembrane region via a linker, and the linker is preferably 15-30 amino acids in length, more preferably 20-25, such as 23 amino acids in length.

Examples of peptide linkers that can be used in the present invention include: glycine polymer (G)n; glycine-serine polymer (G ₁ -5S _1-5 )n, where n is at least 1, 2, 3, 4, or 5. Integers; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Those skilled in the art can understand that, in some embodiments, the linker may be composed entirely of a flexible linking peptide, or the linker may be composed of a flexible linking peptide part and one or more parts that impart a smaller flexible structure. In one embodiment, the linker used to connect the Fc region and the tag sequence has the sequence GGGGS. In yet another embodiment, the linker used to connect the Fc region and the transmembrane region has the sequence GGGGSGSTSGSGKPGSGEGSTKG.

In one embodiment, the peptide linker is a Gly/Ser linking peptide. In one embodiment, the peptide linker is a (GxS)n linker, where G=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8. ), in one embodiment, x=4, n=6 or 7. In some embodiments, the linker may include the amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer from 1 to 7. In a preferred embodiment, x=4 and n=7. In one embodiment, the linker is (G4S)3. In one embodiment, the linker is (G4S)4. In one embodiment, the linker is (G4S)6G2.

Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS; TGEKP (see, for example, Liu et al., PNAS5525-5530 (1997)); GGRR (Pomerantz et al. 1995, supra); (GGGGS)n , Where n = 1, 2, 3, 4 or 5 (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070 ); KESGSVSSEQLAQFRSLD (Bird et al., 1988, Science242:423-426), GGRRGGGS; LRQRDGERP; LRQKDGGGSERP; LRQKD (GGGS) 2ERP. Alternatively, a computer program (Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS91:11099-11103 (1994)), or by phage or yeast display methods, rationally design flexible linkers.

Vector system for introducing Fc variant sequence library into mammals

After obtaining the Fc variant library, the Fc variant library encoding nucleic acid can be introduced into mammalian cells by any means known in the art, including but not limited to, transfection, electroporation, microinjection, etc., thereby forming a mammal. Animal cell library, each cell clone in the Chinese library displays Fc variant polypeptide on the surface.

Methods of introducing foreign nucleic acids into mammalian cells are known in the art. For example, vectors (including, but not limited to, viral vectors and plasmid vectors) can be used to transfect mammalian cells to achieve the introduction. In order to display the Fc variant polypeptide on the surface of mammalian cells and realize subsequent screening, it is preferable that the nucleic acid encoding the Fc variant polypeptide is integrated into the host cell genome after being introduced into the cell via a vector.

Viral vectors suitable for introducing Fc region variants into mammals can be any suitable viral vectors known in the art, for example including but not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., preferably lentiviral vectors.

The vector can usually contain restriction sites for insertion of foreign coding sequences. In one embodiment, standard molecular cloning methods can be used to introduce restriction sites through primers at both ends of the coding sequence to allow the coding sequence to be ligated into a viral (especially lentivirus) expression vector in a determined direction. In one embodiment, the restriction sites are different from each other, and at least one of them creates a single-stranded overhang ("sticky end"), thereby allowing directional cloning.

The vector can encode selectable marker genes, such as G418, hygromycin, puromycin and other antibody genes. Therefore, after the exogenous nucleic acid is introduced into the host cell, a selectable marker gene is used to select a stably transfected cell line.

In order to drive the expression of the inserted foreign nucleic acid in the host cell, the vector may include a promoter. In one embodiment, by selecting promoters of different strengths, the display level of the exogenous nucleic acid-encoded polypeptide on the cell surface can be adjusted to facilitate the screening of Fc variants based on affinity.

An illustrative example of a vector that can be used in the present invention is a lentiviral expression vector, for example, the pCDH vector as shown in FIG. 20.

Therefore, in one embodiment, the present invention provides a method for screening Fc region variants, the method includes constructing a mammalian cell Fc polypeptide display system, and the constructing includes the steps:

Provided is a library of mammalian cells displaying modified Fc region polypeptides on the cell surface, wherein the cell library comprises a collection of mammalian cells displaying a library of Fc variants, wherein each mammalian cell preferably displays a different modified Fc region Peptide

Wherein, a viral vector (preferably a lentiviral vector) is used to construct the mammalian cell display library, wherein the viral vector includes an expression cassette, the expression cassette includes a nucleic acid molecule, and the nucleic acid molecule encoding includes A peptide of the following sequence:

-Optionally, the secretory signal sequence, preferably the signal sequence is a signal peptide that can guide the Fc region polypeptide to be secreted out of the cell, for example, the IL-2 protein signal peptide;

-Optionally, the tag sequence, preferably the tag sequence is an epitope tag sequence, such as HA epitope tag, Flag tag sequence, c-myc epitope tag;

-Fc region variant polypeptide, preferably the Fc region of IgG1, IgG2, IgG3, IgG4, more preferably human IgG1 Fc region, most preferably the human IgG1 Fc region polypeptide variant shown in SEQ ID No:1, for example, comprising 1- 10 mutations, such as 1, 2, 3, 4 or 5 mutations;

-Membrane anchors (preferably transmembrane domains); wherein the transmembrane domain anchors Fc on the cell membrane surface, preferably the transmembrane region is derived from a protein expressed on the surface of mammalian cells, such as the transmembrane region of the PDGFR protein, for example The transmembrane region sequence described in SEQ ID NO: 4;

Preferably, the Fc region variant is connected to the tag sequence and the transmembrane domain through a linker;

Preferably, the expression cassette is under the control of a promoter, such as an inducible promoter;

Preferably, the diversity of the library is obtained by randomizing at least one codon of the nucleic acid encoding the Fc region polypeptide; in some embodiments, based on structural analysis, the mutation region to be introduced into the mutation is selected to construct a variant library.

In a preferred embodiment, a lentiviral vector system is used to form the mammalian display system of the present invention.

Various lentiviral vector systems are known in the art. In these systems, the cis-acting elements in the lentiviral genome (such as packaging signals, long terminal repeats) are separated from the sequence encoding the trans-acting protein. Thus, the carrier system includes packaging components and carrier components. The packaging component is constructed by removing the cis-acting sequence required for packaging, reverse transcription and integration from the viral genome, and can provide the protein required for the production of viral particles in trans; while the carrier component is complementary to the packaging component and contains packaging, reverse transcription and integration The required cis-acting sequence also has a multiple cloning site under the control of a heterologous promoter and a target gene inserted at this site.

In one embodiment, the lentiviral vector system used in the present invention may include a lentiviral expression vector and packaging helper virus components, wherein the lentiviral expression vector cannot replicate in cells. After constructing the Fc variant library, the expression cassette encoding the Fc variant can be inserted into the lentiviral expression vector. Then, the lentiviral expression vector is co-transfected into mammalian cells in the presence of the packaging helper virus component. Using this lentiviral expression system, the Fc expression cassette can be integrated into the mammalian cell nuclear genome.

In some embodiments of the present invention, a three-plasmid packaging system can be used in combination with the lentiviral expression vector of the present invention. The packaging system consists of expressing gag/pol, Rev, VSV-G (vesicular stomatitis virus G protein), respectively. It consists of three packaging plasmids. In addition, other lentivirus packaging systems can also be used, such as the five-plasmid system (gag-pro, vpr-pol, VSV-G, Tet-off, and tat-IRES-rev expression element plasmids are provided respectively). These lentiviral packaging plasmid systems can be purchased commercially, for example, from Invitrogen, Clontech, Didier Trono, etc.

In the present invention, in some embodiments, a lentiviral vector system consisting of the following 4 plasmids is used, which are:

(1) A lentiviral expression plasmid with the target gene, that is, the lentiviral expression vector expressing the Fc variant of the present invention as described above, which contains the genetic information required for virus packaging, transfection, and stable integration. In this vector, the Fc polypeptide-encoding nucleic acid is inserted between two LTRs (long terminal repeats), and the LTR sequence can promote the integration of the encoding nucleic acid into the genome of the mammalian host cell. In one embodiment, the 3'LTR of the expression plasmid has a partial sequence deleted, so that the integrated viral genome loses the ability to replicate itself.

(2) Two packaging plasmids, one of which encodes Rev and the other encodes Gag and Pol;

(3) A plasmid encoding an envelope protein, preferably the envelope protein is vesicular stomatitis virus G protein (VSV-G).

Before library screening, a lentiviral vector system such as the above-mentioned four plasmids can be used to co-transfect mammalian cells to assemble and produce infectious pseudoviral particles. Thereafter, the harvested infectious virus particles can be used, optionally after concentrating and/or detecting the virus titer, to infect mammalian cells, and to screen for Fc variants expressed on the cell surface.

Display of Fc polypeptide on the cell surface

In the present invention, using the display system of the present invention, the Fc protein of the present invention is expressed in cells, transported to the cell membrane, and stays on the cell surface as a membrane-bound protein.

Without being limited by theory, in mammalian cells, the Fc protein will be synthesized in the endoplasmic reticulum ER and enter the Golgi apparatus, and then transported to the cell surface in the intracellular vesicles. After the vesicles fuse with the cell surface, the Fc protein The C-terminal transmembrane domain or membrane anchor stays on the cell surface to achieve extracellular display. In this process, if the Fc region contains a hinge region, it can be assembled into a dimer through disulfide bonds in the hinge region and displayed on the cell surface in the form of an Fc dimer.

In some embodiments, the expression vector of the present invention expresses only one Fc polypeptide, thereby displaying Fc homodimers on the cell surface. However, it is also possible to use a viral expression vector containing a bicistronic to simultaneously express two Fc members on the same vector or to infect the same cell with two viruses encoding different Fc variants, thereby displaying an asymmetrical expression on the cell surface. Fc variant dimers (ie, one and the other of the dimerized Fc polypeptides have different sequences). In embodiments that display asymmetric Fc variant dimers, preferably, random mutations can be introduced into one of the two Fc polypeptide-encoding nucleic acids, while leaving the other polypeptide-encoding nucleic acid unchanged, for example, encoding a specific The wild-type Fc polypeptide, or encoding a specific mutation-containing Fc polypeptide, is used to construct an Fc variant display library to screen for dimers of Fc variants with asymmetric mutations.

Mammalian cell

Many pharmaceutical proteins containing Fc polypeptides, especially antibodies, are produced in mammalian cells and eventually applied to mammals. For example, CHO cells are used to produce Herceptin (an anti-HER-2 antibody approved for breast cancer treatment). In the screening process of these antibodies and therapeutic proteins, it is therefore advantageous to use mammalian cells that exhibit the same or as similar expression environment and post-translational modification mechanisms as possible. This can better simulate the drug production and application environment, and promote the acquisition of effective Fc variants in drug production and application. Unlike yeast cells, bacteria cannot reproduce the glycosylation, expression and secretion mechanisms of mammalian cells. Therefore, the display of the present invention on mammalian cells has advantages in drug development. In addition, the ability of mammalian cell surfaces to display large libraries also allows direct screening of the binding properties of tens of thousands of clones.

In the present invention, mammalian cells suitable for the screening method and display library construction of the present invention can be any cell or cell line derived from mammals. For example, the cells may be cells from mice, non-human primates, and humans. Alternatively, the cell may be a cell line, such as Chinese Hamster Ovary (CHO) cells and HEK293 cells, NS0 cells, Vero cells.

In one embodiment, the mammalian cells used to construct the display library comprise or specifically consist of cells selected from: (a) BHK cells, such as BHK21 cells, especially ATCC CCL-10; (b) Neuro -2a cells; (c) HEK-293T cells, especially ATCC CRL-11268; (d) CHO cells, such as CHO-K1 cells, especially ATCC CRL-62; and (e) HEK293 cells, such as 293FT cells. In a preferred embodiment, the mammalian cell is a cell suitable for producing high-titer lentivirus, such as 293FT cells.

In the present invention, screening can also be carried out in cells modified by glycosylation. For example, the FUT8 gene of mammalian cells is knocked out by genetic means. The antibody produced by this cell does not contain fucose residues and has a strong The ADCC activity of this cell can be screened for Fc variants with both glycosylation modification and amino acid modification by introducing an Fc variant library into the cell.

In one embodiment, therefore, the mammalian cells used to construct the display library have altered glycosylation machinery, thereby affecting the glycosylation of the Fc polypeptide expressed in the cell. In one embodiment, the present invention relates to the use of the mammalian cells to screen for Fc variants with altered binding affinity to the Fc receptor of interest based on the altered glycosylation. In one embodiment, the glycosylation alteration is low or no fucosylation. Cell lines capable of producing defucosylated or hypofucosylated Fc regions are known in the art. Examples of such cells include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986): 533-545; US 2003/0157108; and WO 2004/056312, Especially Example 11); and gene knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki, N., etc., Biotech. Bioeng. 87:614 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107). For another example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), so that they can be expressed in the Ms704, Ms705 and Ms709 cell lines lacking fucosyltransferase. Sugar antibodies. In addition, EP 1,176,195 also describes cell lines with a functionally disrupted FUT8 gene, and antibodies expressed in such cell lines exhibit hypofucosylation. Alternatively, fucosidase can also be used to cleave fucose residues of antibodies; for example, fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).

Library capacity

The cell display library of the present invention is a collection of a plurality of cell clones, wherein each cell clone contains recombinant DNA encoding an Fc polypeptide. The diversity of the library is a function of the number of different Fc polypeptides encoded by these clones. Each clone of the library can be produced by integrating the DNA encoding the Fc polypeptide into the cellular DNA to form a recombinant cell (for example, in the embodiment, by using a lentiviral vector, the DNA encoding the Fc polypeptide is integrated into the genome of a mammalian cell in). After the Fc polypeptide DNA is integrated into the cellular DNA, the resulting recombinant cells can be cultured to allow replication of the recombinant cells, thereby generating a cell clone from each of the initially formed recombinant cells. Therefore, each clone in the library is derived from an original cell that has integrated the donor DNA.

In drug development, it is advantageous to provide a large diversity library to maximize the possibility of identifying binding molecules that meet the requirements. In the present invention, mammalian display libraries of the invention may comprise at least ³ 100, 10, ^{104, 105, 106, 107} clones. In some preferred embodiments, the libraries of the invention have a high degree of diversity, coding and / or expression of at least ^103, at least ¹⁰⁴ or at least ¹⁰⁵ or at least ¹⁰⁶ different Fc polypeptide.

Sorting display library members

Once a library of transduced cells (ie, a mammalian cell population displaying Fc variants) is obtained, one or more cell clone subpopulations can be selected based on the binding ability of the Fc region displayed on the cell surface to the target Fc receptor.

Generally, selecting cell clones, or cell clone populations, involves physically isolating multiple cell clones from a library. Therefore, selection typically involves dividing library members into one group of cell clone populations to be collected, and another group of cell clone populations to be discarded. Thus, the enrichment of cell clone members with desired properties is achieved in the collected cell clone population. In this context, it should be understood that enrichment refers to an increase in the relative abundance of cell clone members with desired properties in a population. If necessary, the collected cell population can be selected again.

For example, in the screening method for displaying an Fc mutation library using mammalian cells of the present invention, in order to enrich cell clone library members with specific binding properties to the target FcγR from the library, the surface display of Fc variants and Fc variants can be used. Binding to the target FcγR, isolate cell clones from the library. In the embodiment of expressing and displaying the Fc variant polypeptide with the tag sequence, the anti-tag sequence antibody-FITC and the target FcγR-biotin/streptavidin-PE can be used to stain cells, and FACS based on FITC on the cell And PE staining intensity to sort cells to select cell clone subpopulations.

Select threshold

When selecting, you can set the percentage of "highest" or "best" clones to be selected. For example, in the selection based on affinity, a percentage or score of selected clones can be set, wherein the clones exhibit the highest target FcγR binding activity or the lowest target FcγR binding activity in the collected cell clone population.

The selection threshold may be determined by a person skilled in the art, or may be determined based on a preliminary analysis of samples of the selected population. For the positive selection of enriched cell clones, it can be set to select the clones with the highest signal level showing the desired properties, for example, the top 50% of the cells, the top 30%, the top 25%, the top 20%, the top 15%, the top 10% or top 5%, for example top 0.5-10% of cell clones. For negative selection, it can be set to select the clone with the lowest signal level that exhibits undesirable properties, for example, the top 50% of the cells, the top 30%, the top 25%, the top 20%, the top 15%, the top 10%, or the top 5%, such as the first 0.5-10% of cell clones.

In one embodiment, for example, from the collected cell population, the top 10% or top 5% clones with the highest or best value in the desired property are selected, for example, the top 0.5-10%. In one embodiment, the desired property is the ability to bind to a specific FcγR receptor, and the amount of FcγR receptor bound on the cell surface is a measure of the binding ability of the Fc polypeptide.

In yet another embodiment, for example, from the collected cell population, the top 10% or top 5% clones with the lowest value in terms of undesirable properties are selected, such as the top 0.5-10%. In one embodiment, the undesired property is the ability to bind to a specific FcγR receptor, and the amount of FcγR receptor bound on the cell surface is a measure of the binding ability of the Fc polypeptide.

In one embodiment, the amount of FcγR receptors that bind to the Fc displayed on the cell surface can be normalized relative to the display level of the Fc polypeptide on the cell, thereby quantifying the binding ability. For example, in a screening driven by a fluorescently labeled target receptor molecule, the binding activity can be measured as the amount of the fluorescent label bound to the cell clone relative to the value normalized to the Fc display level.

In one embodiment, in the sorting, a cell population displaying an Fc polypeptide with the target FcγR binding ability is collected, and the top 0.5-10% cell clones with the highest binding ability to the target FcγR receptor are selected from the collected population ( In positive selection), or select the top 0.5-10% cell clone with the lowest binding ability to the target FcγR receptor (in negative selection).

Method of choosing

The selection of the library usually includes the following steps: adding a specific target FcγR recombinant protein or FcRn recombinant protein to the library, so that the Fc polypeptide displayed on the cell surface contacts the target receptor molecule and forms a complex. By limiting the concentration of target molecules added to the library, Fc polypeptides with high affinity can be preferentially selected. When the Fc polypeptide displayed by the cell does not bind to the target molecule or has a weak binding ability, the cell will not bind to the target receptor molecule, or will bind fewer target receptor molecules on the cell. Thus, a cell population enriched with high-affinity Fc polypeptide display cells can be selected.

The selection process can optionally be repeated. For example, decreasing concentrations of target receptor molecules can be used to progressively increase the stringency of selection and increase the degree of enrichment of high-affinity clones. Alternatively, different target receptor molecules can be used to select combinations of Fc variants that have multiple different receptor molecule binding properties.

In a preferred embodiment, the selection includes sorting cell members into a population to be collected or a population to be discarded based on the level of target receptor molecules bound on the cell. The sorting gate can be set relative to the target receptor molecule binding level of control cells (for example, cells displaying wild-type Fc polypeptides) under the same experimental conditions. Alternatively, the sorting gate can be determined by those skilled in the art based on experience. In the positive selection, the cell components above the gate are collected, on the contrary, the cell members below the gate are discarded; on the contrary, in the negative selection, the cell members below the gate are collected. In a preferred embodiment, the target receptor molecule binding level on the cell is normalized relative to the Fc display level on the cell. The sorting gate is set according to the standardized combination level.

In one embodiment, double cell staining is used to detect the target receptor molecule binding level on the cell and the Fc display level on the cell. For example, the cell can be contacted with the tag sequence binding molecule and the target receptor molecule of interest for dual staining, wherein the tag sequence binding molecule and the target receptor molecule are labeled with different dyes (direct or indirect labeling). In one embodiment, the double dyeing can be performed simultaneously or sequentially, preferably simultaneously. After the staining, the Fc polypeptide display level on the cell can be quantified by measuring the amount of labeled tag sequence binding molecules present on the cell; and the amount of the labeled target receptor molecule present on the cell can be quantified. The level of binding of the Fc polypeptide displayed by the cell to the target receptor molecule. In a preferred embodiment, the cells are subjected to dual fluorescent staining, in which the cells are exposed to tag sequence binding molecules labeled with different fluorescent markers and the Fc receptor of interest.

In one embodiment, a plurality of different FcγRs can be used to select library cell members simultaneously or sequentially, wherein the selection can be positive selection, negative selection, or positive selection and pairing of one FcγR. Another combination of negative selection of FcγR to obtain an Fc variant with the desired binding affinity for a specific FcγR(s).

In a preferred embodiment, the present invention provides an Fc variant selected by the method of the present invention, which exhibits an improved affinity for one or more FcγRs relative to a wild-type Fc. In a further preferred embodiment, the Fc variant has an unaltered or reduced binding affinity on another FcγR or on another FcγR relative to a wild-type Fc.

Various sorting techniques can be used to screen the mammalian display library of the present invention. For example, positive selection can be performed to enrich library members, ie, cell clones expressing Fc variants with desired properties, or to increase the abundance or frequency of such members in the library; and/or negative selection can be performed from the library Remove specific library members, for example, cell clones expressing Fc variants with undesirable properties, or reduce the abundance or frequency of such members in the library.

The sorting method may be, for example, but not limited to, flow sorting, magnetic bead separation, and other separation methods that can achieve enrichment or elimination of library members. In the present invention, it is preferable to use fluorescent markers to stain the Fc polypeptide displayed on the cell surface, and to use a flow cytometry sorting method for positive and/or negative selection of library members.

Each selection will exert a certain evolutionary pressure on the cloned members of the library, which is conducive to the enrichment of library members that meet the selection criteria. Repeated selection on a single parameter property (such as the same FcγR receptor binding ability) can drive evolution in a direction that is conducive to the emergence of this property, but there is also the possibility of causing library members to change in other properties.

The present inventors found that in the method of the present invention, using FcγRIIIA-F158 to screen the mutation library, Fc variants with increased binding affinity to FcγRIIIA (F158) and FcγRIIIA (V158) can be obtained. Therefore, in one embodiment, the present invention provides a method for screening Fc variants with increased binding affinity to FcγRIIIA (F158) and FcγRIIIA (V158) simultaneously, wherein labeled FcγRIIIA (F158) is used to drive the screening of the mammalian cells of the present invention Fc mutant display library.

In addition, the inventors found that after one or two rounds of positive enrichment selection for a specific one or more FcγRs, negative selection for another or more FcγRs with significantly different structures can be obtained. Synergistic effect. For example, as shown in the examples, after positive selection using FcγRIIB, negative selection of FcγRIIIA on the sorted cells can synergistically increase the enrichment of Fc variants with dominant amino acids and desired binding properties in the library. set. (See, Figure 13-15)

When selecting cell library members, the target FcγR receptor molecule can be provided in a soluble form to drive the selection. The target receptor molecule can be labeled to facilitate selection. The target molecule can be labeled directly or indirectly. For example, a label such as a fluorescent dye can be directly fused to the target molecule. Alternatively, an avidin molecule with a label such as a fluorescent dye can be used to contact the biotinylated target molecule to indirectly label the target molecule. In the case of using a fluorescent dye, cells bound to a fluorescently labeled target molecule through the interaction of Fc/FcγR can be detected and separated by flow cytometry.

The method of solid phase separation can also be used to select library cell members. For example, the target receptor molecule or a second substance capable of binding to the target molecule can be immobilized on a solid support such as magnetic beads or agarose beads, so that the cells are in contact with the target receptor molecule and the solid support. Cells displaying the Fc variant that binds to the target receptor molecule will bind to the solid phase. By changing the stringency of the binding and/or washing conditions, it is possible to selectively remove cell members that have no or weak binding capacity of the displayed Fc variant.

Therefore, in one embodiment, the present invention provides a method of screening for Fc variants, the method comprising:

Using a mammalian cell Fc variant polypeptide display system, a cell population is selected from a mammalian cell library according to the characteristics of the Fc region polypeptide displayed on the cell surface; optionally, the sorted cells are proliferated and have the same binding properties or Different combination properties, repeat the selection;

Wherein, the options include:

-By using differently labeled (preferably fluorescently labeled) target Fc receptors and tag sequence binding molecules to stain the Fc region variant polypeptide displayed on the cell surface, wherein the target Fc receptor is labeled with a second label, and the first label is used Tag tag sequence binding molecule;

-(Preferably by flow cytometric sorting) Based on the staining intensity of the two markers on the cells, select cell clones that exhibit the desired FcR receptor binding capacity enhanced or reduced relative to the cells displaying the parental Fc.

In the described embodiment, preferably, based on the staining intensity of the first marker on the library cell clone member and comparing it with the negative control cell, the Fc-displaying positive cells in the library are determined, wherein the negative control cell is, for example, no Corresponding cells transfected with the viral expression vector of the present invention. In one embodiment, the first marker is a fluorescent dye with a first wavelength, and the second marker is a fluorescent dye with a second wavelength, wherein the threshold is set based on the emission fluorescence of the negative control at the first wavelength to determine the cell Whether it is positive for Fc display.

In this embodiment, preferably, among the cell subpopulations that are positive for Fc display, select the top 0.1 to the target Fc receptor binding activity value on the cell surface with the highest (in positive selection) or the lowest (in negative selection). 10% cell clone. Preferably, the Fc receptor binding activity value is a value normalized to the Fc display level. Preferably, the binding activity value can be measured as: the amount of the second marker bound on the cell clone is a value that is standardized with respect to the amount of the first marker bound on the cell clone.

For Fc region variants, the number of selection cycles required to achieve enrichment may be different depending on the region where the mutation is located and the specific target receptor molecule used to drive the selection. For example, for Fc variant screening driven by FcγRIIIA or FcγRIIB, at residues 326-332 (EU numbering) in the Fc region, a good enrichment of clones with increased affinity can be obtained through one or two rounds of screening. In some cases, for such mutation regions/locations where good clonal enrichment occurs relatively quickly in sorting, the probability of obtaining a positive Fc variant through the method of the present invention is significantly greater. Therefore, in some preferred embodiments, such mutation regions/locations are selected as candidate regions for Fc variant screening. However, those skilled in the art will also understand that, in some cases, in order to increase the diversity of the Fc variants screened out, and/or to fine-tune the binding properties of Fc variants to different Fc receptors (such as the ratio of binding capacity) It may be advantageous to combine mutations in different mutation regions/locations (including combining before sorting or after sorting).

Flow cytometric sorting (FACS sorting method)

In the present invention, it is preferable to isolate cells having desired binding properties to the target FcγR or FcRn by the FACS sorting method. In this regard, it is preferable to stain the cell by using a tag sequence labeled with a different fluorescent dye to bind the molecule and the target FcγR or FcRn to indicate the nature of the Fc variant displayed by the cell.

The fluorescent dyes that can be used for labeling in the present invention include, but are not limited to: (a) PerCP, allophycocyanin (APC), (b) Texas Red, (c) Rhodamine, (d) Cy3, (e) Cy5, (f) Cy5. 5, (f) Cy7, (g) Alexa Fluor dyes, especially Alexa 647nm or Alexa 546nm, (h) Phycoerythrin (PE), (i) Green fluorescent protein (GFP) ), (j) tandem dye (such as PE-Cy5) and (k) isothiocyanate fluorescent dye (FITC).

In one embodiment, the fluorescent dyes used to label the tag sequence binding molecule and FcγR or FcRn are FITC and PE, respectively.

The tag sequence binding molecule and the target FcγR molecule can be labeled with a fluorescent dye by any method known in the art. For example, the compound can be directly labeled by coupling a fluorescent dye to a compound such as an anti-tag sequence antibody or a target Fc receptor, wherein the coupling can be achieved by covalent as well as non-covalent binding. Alternatively, the compound may be indirectly labeled with a fluorescent dye by contacting the compound with a second compound containing a fluorescent dye that can bind to the compound.

In one embodiment, the fluorescent dye is covalently linked to a tag sequence binding molecule (e.g., an anti-tag sequence antibody). In one embodiment, the FcγR of interest is fluorescently labeled by direct or indirect means. Direct labeling can be the covalent binding of a fluorescent dye to the target FcγR. Indirect labeling can be achieved, for example, in one embodiment, by contacting a biotinylated target FcγR with avidin or streptavidin with a fluorescent dye.

Therefore, in one embodiment, the staining of the cell includes: exposing the cell to a fluorescent dye-labeled anti-tag sequence antibody, a biotinylated target FcγR, and a fluorescent dye-labeled avidin molecule.

After staining, the fluorescence level of the cells is detected. The presence and level of the labeled fluorescence of the tag sequence binding molecule indicates the display and level of Fc on the cell; the presence of the labeled fluorescence of the target Fc receptor indicates that the Fc change on the cell The binding of the body to the target receptor and its binding strength. Preferably, the detection is performed by a flow cytometer. The gating used for cell sorting can be set according to the fluorescent dye used and the binding intensity of the desired FcγR.

In a preferred embodiment, selecting a cell subpopulation or a single cell from the constructed Fc mutation library cell population includes the steps:

(a) The cells of the library cell population are contacted with the target FcγR, wherein the target FcγR is labeled with a second fluorescent dye, wherein the target FcγR is preferably labeled by an indirect method, more preferably the biological is indirectly labeled with a fluorescent dye-streptavidin molecule The target FcγR of vegetarianization, wherein the second fluorescent dye is especially PE;

(b) Contact the cells of the library cell population with an anti-tag sequence antibody, wherein the anti-tag sequence antibody is labeled with a first fluorescent dye, wherein the second fluorescent dye and the first fluorescent dye emit fluorescence of different wavelengths, and the first fluorescent dye is preferably Is FITC; and

(c) Identify the intensity of the first fluorescence and the second fluorescence on the cells by flow cytometry, and

(d) Sorting cells.

In the above embodiment, preferably, it further includes the steps:

(a') Construct a second mammalian cell population that expresses and displays the parent Fc polypeptide or wild-type Fc polypeptide on the cell surface;

(b') Stain the second cell population by labeling the target FcγR with a second fluorescent dye and labeling the tag sequence binding molecule with the first fluorescent dye;

(c') Identify the intensity and distribution of the first fluorescence and the second fluorescence of the cells of the second cell population by flow cytometry; and

(d') The first and second fluorescence intensities and distributions of the first cell population and the second cell population are compared to determine the gate used for FACS sorting of the first cell population.

In one embodiment, in FACS sorting, unstained cells without Fc introduction are included as blank controls to indicate autofluorescence on FITC channels and PE channels of the cells themselves. In a preferred embodiment, in positive screening, when sorting and setting gates, select the cell population (X-axis direction) whose FIFC direction is stronger than the blank control, and on this basis, select the top 0.5-1 with the strongest PE direction. % Of cells. In another preferred embodiment, in the negative screening, when sorting and setting gates, select the cell population whose FIFC direction is stronger than the blank control (X-axis direction), and on this basis select the weakest PE direction before 0.5- Around 1% of the cells.

In another embodiment, after the individual cells are separated by the FACS sorting method, the second, third and/or fourth FcγR can be further selected for positive or negative selection. Preferably, the positive or negative selection adopts flow cytometry. Cell sorting is performed.

In some embodiments, negative selection may include negative selection for binding to one or more unwanted FcγR activities in order to eliminate, for example, a pair of expressed Fc variants relative to a reference Fc polypeptide such as a wild-type Fc polypeptide This FcγR has an undesirably high binding activity to cells.

In one embodiment, the method of the present invention further includes the step: after each sorting, the obtained sorted cell population is cultured and proliferated, for example, the cells are propagated for, for example, 20 days before the next round of sorting.

Therefore, in one embodiment, the method of the present invention further includes:

-After sorting the enriched cells, proliferate the enriched cells,

-Subject the obtained cell population to one or more rounds of screening,

Wherein the screening is performed using the same target FcγR, optionally wherein the concentration of the target FcγR is progressively reduced to increase the stringency of the screening; and/or

Wherein, the screening is performed using different target FcγRs to select cell clones displaying Fc fusion polypeptides with different target FcγR binding levels as desired.

Preferably, after cell proliferation, before the next sorting, the display level of the Fc polypeptide on the cell and the binding ability to the target FcR are detected, and optionally compared with the original mutation library without sorting, or with the previous Round to compare to decide whether to implement the next sorting. In the technical solution using the fluorescence sorting method, the detection can be performed by fluorescence staining and flow cytometry. For example, when performing positive screening for the target Fc receptor, the tag sequence in the Fc polypeptide can be labeled with a first fluorescent dye and the target Fc receptor can be labeled with a second fluorescent dye. Cytometry detects the number of double fluorescent staining positive cell subpopulations and the migration trend toward the enhanced direction of the second fluorescent staining. Preferably, compared to the cell population before sorting, the cell population after sorting and proliferation is confirmed by fluorescent staining to have a significantly increased number of double-positive cell subpopulations, or the number of double-positive cell subpopulations reaches 50% of the entire stained cell population. % Or more, and more preferably has an overall migration to the second fluorescent dyeing enhancement direction.

In one embodiment, in the staining analysis of proliferating cells after sorting, unstained cells without Fc introduction are included as blank controls. When performing the staining analysis using flow cytometry, in one embodiment, the tag sequence in the Fc polypeptide is labeled with a first fluorescent dye (for example, FITC) and the target Fc receptor is labeled with a second fluorescent dye (for example, PE). . In one embodiment, the gate is set to determine the left border of the gate based on the background fluorescence value on the blank control, and in the positive screening, to display the fluorescence of the second fluorescent dye (for example, PE) on the cell population of WT Fc The value determines the lower boundary of the gate; while in negative screening, the fluorescence value of the second fluorescent dye (for example, PE) on the cell population displaying WT Fc is used to determine the upper boundary of the gate. In one embodiment, according to the number and distribution of the cell population in the gate, the cells proliferated after sorting are analyzed, for example, to determine the degree of enrichment of Fc variants in the cell bank and/or whether the next round of enrichment is required.

Deep sequencing and cluster analysis of sorting members

In conventional display library platforms and methods, the next common step after library screening is to isolate and identify the nucleic acid encoding the display molecule. For this reason, the nucleic acid encoding the display molecule is usually separated from the sorted cells and cloned into expression vectors and host cells to form a single clone. A limited number of single clones are picked, and the properties are tested and sequenced one by one.

Different from the conventional method, the method of the present invention performs deep sequencing and cluster analysis on the selected cell population after screening the display library. For cell populations sorted by deep sequencing, more than 100,000 readings can be obtained. Thus, the present invention can realize a thorough analysis of the sorted sequence variants.

Deep sequencing can sequence hundreds of thousands to millions of DNA molecules in parallel at a time. Compared with the first-generation sequencing technology, that is, the sanger sequencing method, deep sequencing is also called next-generation sequencing (NGS) or high-throughput sequencing. NGS sequencing includes second-generation sequencing (SGS) and third-generation sequencing (TGS). SGS technology is suitable for obtaining short read lengths, while third-generation sequencing (TGS) can obtain longer read lengths.

The methods used for second-generation sequencing include sequencing-by-synthesis (SBS) and sequencing-by-ligation (SBL). The SBS method includes pyrosequencing method, reversible terminator sequencing method (sequencing by reversible terminator), and sequencing method based on hydrogen ion detection (Sequencing by Detection of Hydrogen Ion). The SBL method includes sequencing methods based on hybridization and ligation. Methods used for third-generation sequencing include Single Molecule Real-Time Sequencing (SMRT) method and the like.

A variety of commercial deep sequencing platforms are available, including but not limited to various commercial sequencing platforms for next-generation sequencing, such as GS FLX sequencing platform (454 Life Sciences/Roche diagnostics), Genome Analyzer, HiSeq, MiSeq and NextSeq sequencing platforms (Illumina company), SOLiD sequencing platform (ABI company), and Ion Torrent PGM ^TM and Ion Torrent Proton ^TM sequencing platforms (Thermo Fisher); various commercial sequencing platforms that can be used for third-generation sequencing, Helicos ^TM Genetic Analysis System (SeqLL, LLC) ), SMRT Sequencing (Pacific Biosciences), Nanopore sequencing platform (Oxford Nanopore).

In some preferred embodiments of the present invention, SGS technology is used for deep sequencing. SGS includes two steps: template preparation step and sequencing step. In the template preparation step, it usually includes preparing a DNA library for sequencing, which includes end processing the DNA fragments and ligating with adaptors. Depending on the sequencing platform used, the length of the DNA fragments used for sequencing can be 150-800 bp. After the sequencing DNA library is prepared, the DNA fragments in the library are cloned and amplified, wherein the DNA fragments to be sequenced are individually bound to solid phases such as beads, ion surfaces, or flow cells. Depending on the sequencing platform, DNA fragments anchored on the solid phase can be amplified by emulsion PCR (emPCR) or bridge PCR (bridge PCR) to form millions of template fragments that are spatially separated. After the template library for sequencing is prepared, SBS sequencing or SBL sequencing can be performed.

For the deep sequencing method, you can refer to the literature: for example, US2019/0185933. Eid, J.et al. Real-time DNA sequencing from single polymerase molecules. Science 323,133-138, doi:10.1126/science.1162986 (2009); Giordano, F.et al. Denovo Yeast Genome Assemblies from MinlON, PacBio and MiSeq platforms.Scientific Reports 7,3935, doi: 10.1038/s41598-017-03996-z (2017); and Sheetal Ambardar, etc., High Throughput Sequencing: An overview of sequence Chemistry, 2016, 56(4):394-404. These documents are incorporated into this article as a reference

In the present invention, in some embodiments, before performing deep sequencing, the mutant region of the nucleic acid encoding the Fc polypeptide is amplified from the cell population obtained from the sorting. The amplification is preferably performed by polymerase chain reaction. The sequence and length of the amplification can be determined according to the deep sequencing method used and the mutation region to be analyzed. In short, two synthetic primers complementary to the target mutation region to be amplified are added to, for example, the lysate (purified or unpurified) of the sorted cell population, thereby amplifying the Fc between the two primers. DNA coding region. During the amplification process, barcodes can be added to the amplified target nucleic acid molecules, for example, barcode-carrying primers are used for amplification to achieve the addition of barcodes. In the case of merging sorted cell populations of multiple mutation libraries for sequencing, the barcode can be a mutation library barcode, that is, different mutation libraries correspond to different barcodes. In one embodiment, therefore, the primer used for amplification includes a first part at the 3'end that is complementary to the target amplification region and a second part at the 5'end that includes a mutant library barcode. Using such primers for amplification produces barcode-labeled nucleic acid molecules.

After the self-sorted cell population is amplified to generate a nucleic acid molecule library, methods known in the art can be used to perform deep sequencing (for example, next-generation sequencing). For example, the second-generation sequencing method of bridge PCR can be used. In addition, commercially available deep sequencing platforms, such as the Illumina MiSeq sequencing platform, can be used.

After deep sequencing, cluster analysis of reads can be performed to determine the degree of enrichment of the sorted population and the abundance of different sequences in the population. Optionally, it can be compared with the deep sequencing results of a reference population, such as the original population before sorting. Experiments have confirmed that by using the method of the present invention, deep sequencing and cluster analysis are used to select sequences with a high frequency of occurrence (ie, dominant amino acid sequences) in the sorted population, and preferably further combine with the detection of dominant amino acid residues. Quickly and efficiently obtain Fc variants with desired Fc receptor binding properties.

The present inventors further found that the use of FcγR receptors with different structures, such as FcγRIIB relative to FcγRIIIA, for sequential positive and negative display library member sorting, can synergistically increase the advantageous amino acid residues associated with the binding properties of the target FcγRIIB in deep sequencing and aggregation. The frequency of occurrence in the results of the class analysis facilitates the identification of Fc variants that meet the desired binding properties.

In the present invention, preferably, the cluster analysis includes converting the nucleic acid sequence obtained by sequencing into an amino acid sequence, and sorting according to the frequency of each mutant sequence from high to low.

In some embodiments, the cluster analysis further includes drawing a mutant accumulation curve to determine the degree of mutant enrichment in the mutant library (for example, after enrichment by flow sorting). In the drawing of the mutant accumulation curve, it is assumed that the total number of all the sequences is 100%. Starting from the largest number of mutants, the number of each type of mutant that is ranked lower in the order will continue to accumulate until the accumulation of all mutants is completed. . On the cumulative curve, generally, the X-axis represents the type of mutants, and the Y-axis represents the proportion of the cumulative number of mutants to the total mutants. Comparing the mutation library before and after sorting, the change of the curve slope can reflect the change of the enrichment degree of the mutation library after sorting.

In one embodiment, after amino acid sequence conversion is performed on the deep sequencing data, the dominant amino acid at a specific amino acid position and/or the preferred sequence motif in the measured sequence region are determined. In this context, the dominant amino acid residue is the residue that remarkably appears at a specific amino acid position in all amino acid sequences of the mutation library. In some embodiments, the dominant amino acid residue is the amino acid residue that appears frequently at a specific amino acid position on all amino acid sequence reads obtained by deep sequencing of the mutation library, preferably the first 1-5 amino acids with the highest frequency. , Preferably the first 1-3 amino acids, or the first 1-2 amino acids. However, those skilled in the art can also determine the range of dominant amino acid residues according to the specific needs of the screening, for example, to cover amino acids with relatively lower frequency rankings. In one embodiment, the predominant amino acid residue at a specific amino acid position in the selected mutation library is compared with that of the unscreened original mutation library to determine whether there is a predominant amino acid residue mutation at that position. In still another embodiment, based on the dominant amino acids at each position of the tested sequence, the preferred sequence motif of the tested sequence region can be determined. Multiple sequence alignments can be performed on the amino acid sequences obtained by sequencing the library to determine the dominant amino acid residues and preferred sequence motifs.

In some embodiments, the dominant amino acid residues are displayed by WebLogo mapping. Weblogo is based on multiple sequence alignment information, which can graphically display the conservative information of multiple sequences. Each logo is composed of a series of bases (amino acids). At each sequence position of the logo, the height of the letters of different bases (amino acids) indicates the frequency of its appearance. The frequency distribution can reflect the sequence conservation at this position. For WebLogo mapping methods, see, for example, Crooks, Gavin E., et al. "WebLogo: A Sequence Logo Generator." Genome Research 14.6 (2004): 1188-1190; and Schneider, TD and RMStephens, Sequence logos: a new way to display consensus sequences. Nucleic Acids Res, 1990.18(20): p.6097-100. These documents are incorporated herein as reference. You can use the WebLogo drawing tool provided by http://weblogo.threeplusone.com to draw the sequence logo.

In one embodiment, therefore, the present invention provides a method for screening Fc variant polypeptides, comprising:

The cell population obtained after the step of sorting and displaying the library is subjected to deep sequencing, such as next-generation sequencing (NGS), to identify Fc variants with desired FcγR binding properties, wherein the selection includes cluster analysis of the sequenced amino acid sequence ; Preferably, proceed as follows:

(c1) PCR amplify the mutation library enriched by the sorting step from the cell population, and optionally simultaneously amplify the original mutation library not enriched by the sorting as a control; preferably, the PCR amplification length is 50-200 bp Between, preferably 150bp;

(c2) Using next-generation sequencing, preferably next-generation sequencing based on bridge PCR, such as the HiSeq ^TM sequencing platform, to obtain the amino acid sequence of the mutation region of the mutation library;

(c3) Perform cluster analysis on the obtained sequencing results according to the type and frequency of the amino acid sequence of the mutation region;

(c4) Select the top 1-3% mutants with the highest frequency, such as the top 100 mutants with the highest frequency, preferably the top 50, the top 20, and the top 10 mutants. Preferably, the selected mutants contain dominant amino acid residues and/ Or preferred sequence motifs. In a preferred embodiment, WebLogo mapping is used to determine the dominant amino acid residues and preferred sequence motifs of the mutation library.

Fc variant binding properties detection

As pointed out above, unlike conventional platform screening methods, the present invention adopts deep sequencing (such as second-generation sequencing) and cluster analysis to directly obtain sequence information of Fc variants. Therefore, the present invention eliminates the steps of monoclonal isolation, detection and sequencing of the Fc variant population obtained by screening. In addition, the present inventors found that by using deep sequencing and cluster analysis, it is possible to select a limited number (for example, about 10 or 30 or no more than 100) of Fc variants with the highest frequency of occurrence and/or dominant mutations. In the body group, Fc variants with the expected improvement in binding properties (several, 10-fold, tens-fold, or even 100-fold improvement) are obtained. Thus, compared with conventional screening platforms, the method of the present invention significantly reduces the time and cost of identifying Fc variants.

In the present invention, it can be understood that identifying an Fc as having a certain property will include, based on the data obtained in the present invention, that the Fc is expected to have that property. Through the method of the present invention, a candidate population enriched with Fc variants with desired properties can be identified. Optionally, the properties of each Fc variant in the population can then be verified.

The detection/verification may include, for example, the expression, purification, and property detection of selected Fc variants or proteins (such as antibodies) containing Fc variants using methods of those skilled in the art. In the property detection, it is preferable to include the parent Fc (for example, wild-type Fc) from which the Fc variant is derived or the protein containing it as a reference.

In the present invention, after the Fc variants are selected through next-generation sequencing and cluster analysis, in one embodiment, the properties (one or more) of the selected Fc variants are further detected, and the properties include, for example, but Without limitation, the binding affinity of the Fc variant to the target FcγR or other (one or more) FcγRs corresponds to the effector function or other effector functions of the selected target FcγR.

In one embodiment, before performing the property analysis, according to the sequence information obtained by the aforementioned deep sequencing, the DNA encoding the Fc region of the selected Fc fusion polypeptide is expressed in vitro in mammalian cells, and optionally the soluble Fc region is expressed by secretion. Formal expression. After the Fc region polypeptide is expressed, it is optionally separated and purified.

In one embodiment, the binding ability between Fc and Fc receptor is detected. For example, the binding ability can be characterized by detecting the binding affinity, binding specificity, and/or effector function caused by binding between Fc and Fc receptor.

In some embodiments, the detection of the binding ability includes determining the binding affinity of the Fc variant polypeptide to the target FcγR and/or other (one or more) FcγR. For example, SPR surface plasmon resonance (SPR, Surface Plasmon Resonance) can be used for binding affinity analysis, in which the binding amount of Fc variants and FcγR immobilized on the chip is detected, and the affinity between the binding amount and Fc/FcγR is positive. Related. In other embodiments, the detection of the binding ability includes determining the effector function caused by the binding of the Fc variant polypeptide to the target FcγR and/or other (one or more) FcγR. In some embodiments, the Fc binding ability between the Fc receptor expressed by EC ₅₀ values initiator effector function.

In one embodiment, the properties of Fc variants selected from:

-Binding affinity with one or more of FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIB, FcγRIIA (H131), FcγRIIA (R131), such as two, or three, or all four FcγR receptors;

-Depending on the effector function of the target FcγR, including, for example, ADCC activity, ADCP activity, cellular immune activation, cellular immune suppression, tumor-killing effect, etc.

When performing effector function testing, in some embodiments, the Fc variant is converted into the form of a full-length antibody. For example, the Fc variant can be fused with the variable region of the heavy chain of the antibody to form a full-length antibody heavy chain by standard recombination methods, and combined with the corresponding light chain of the antibody to construct a full-length antibody. Preferably, the variable region of the antibody to be combined is selected according to the effector function to be detected. Afterwards, the effector function of the antibody with the selected Fc variant can be tested and compared with the antibody with the wild-type Fc segment.

In one embodiment, the Fc variants of the invention have increased FcγRIIIA binding capacity relative to wild-type Fc polypeptides. It is known in the art that the FcγRIIIA binding ability is related to the ADCC activity of antibodies. Therefore, it is possible to detect the increase in ADCC activity of the Fc variant of the present invention relative to the wild-type Fc polypeptide.

The ADCC efficacy of Fc variants can be tested in the following ways:

-Fuse the heavy chain variable region of an antibody (for example, an antibody that depends on ADCC to exert its main functional activity, such as Herceptin) with the Fc variant of the present invention to form the full-length heavy chain of the antibody;

-Combine the obtained heavy chain of the antibody with the corresponding light chain of the antibody to form a full-length antibody;

-In the presence of the target cell corresponding to the antibody, the killing effect of the antibody on the target cell after incubation with effector cells (such as peripheral blood mononuclear cells) is detected.

In one embodiment, the Fc variants of the present invention have increased FcγRIIB binding capacity relative to wild-type Fc polypeptides. Therefore, agonistic antibodies, such as CD40 agonistic antibodies, can be used to detect effector functions related to FcγRIIB binding ability by replacing the original heavy chain Fc of the antibody with the Fc variant of the present invention. In one embodiment, the antibody containing the Fc variant is incubated with mammalian cells expressing FcγRIIB and target cells expressing the antigen against which the antibody is surfaced, and the activating effect of the antibody on the target cells is detected. In one embodiment, the target cell contains a reporter gene system, and the degree of activation of the target cell is measured by detecting the expression level of the reporter gene system.

The method of the present invention can not only be used to exclude (or reduce abundance or frequency) Fc polypeptide members with undesirable binding properties from a library, but can also be used to facilitate selection from a library (by increasing frequency or abundance, or by Enrichment) Fc polypeptide members with desired binding properties. In the method of the present invention, the reference Fc polypeptide can be used to verify the change in the properties of the Fc variants screened by the method of the present invention.

The reference polypeptide to be compared with the Fc variant of the present invention may be, for example, the Fc parent from which the Fc variant is derived before screening using the method of the present invention. The Fc parent may be a wild-type Fc polypeptide, or an Fc polypeptide that already contains mutations, and the mutations may be located outside the mutant region to be introduced into the amino acid changes when the mutation library is established, in order to obtain performance through the method of the present invention. Further improvement, or a combination of multiple different desired properties.

The polypeptide to be compared with the Fc variant of the present invention may also be, for example, a known Fc variant that has improved or reduced properties for the altered properties selected in the screening of the Fc variant library.

In the present invention, it should be understood that when referring to desired properties, the expressions "larger" or "smaller", "higher level" or "lower level", "increased" or "decreased", etc., mean, Under relevant experimental conditions, compared with the control Fc or the cell clone displaying the control Fc, the Fc variant or the cell clone displaying the Fc variant has certain differences in the expected properties, and this difference allows the Fc variant or the cell clone to display it. Cell clones are distinguished from controls. The difference can be a statistically significant difference. The difference can optionally be quantified in %, for example a difference of at least 10%, or 20% or 25%. Alternatively, a multiple of difference can be used, for example at least 1 fold, 1.5 fold, 2 fold, 3 fold, 10 fold, or at least 100 fold.

Fc variants with improved FcγR binding ability

The population includes five main FcγR receptors, FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIB, FcγRIIA (H131), and FcγRIIA (R131). FcγRIIB is an immunosuppressive receptor; the remaining four are immunostimulatory receptors. The binding of Fc proteins to these different FcγR receptors triggers different effector functions.

Another aspect of the present invention provides Fc variants with improved FcγR binding ability that can be screened or identified by the method of the present invention.

In one embodiment, the Fc variant of the present invention has an altered FcγR receptor binding affinity(s) relative to a wild-type Fc polypeptide, wherein the altered affinity is increased or decreased by at least 1 Times, for example at least 5 times, at least 10 times, at least 50 times the binding affinity. In a preferred embodiment, the affinity is increased by at least 1-100 times, for example, at least 10-50 times. In a preferred embodiment, the affinity is reduced by at least 1-100 times, for example, at least 10-50 times.

In one embodiment, the present invention provides an Fc variant with increased binding capacity of FcyRIIIA (F158) and/or FcyRIIIA (V158). In another embodiment, the present invention provides an Fc variant with increased FcγRIIB binding ability. In yet another embodiment, the present invention provides an Fc variant in which the binding ability of FcγRIIB is increased, while the binding ability of FcγRIIIA (F158), FcγRIIIA (V158) and/or FcγRIIA (H131) is substantially unchanged or reduced. In one embodiment, the present invention provides an Fc variant with an increased ratio of FcγRIIB binding ability/FcγRIIIA binding ability. In one embodiment, the present invention provides an Fc variant with an increased ratio of FcγRIIB binding ability/FcγRIIA-R131 binding ability. In some embodiments, binding capacity is expressed by binding affinity. In other embodiments, the binding capacity is expressed by EC ₅₀ values initiator effector function.

In one embodiment, the Fc variant is an IgG1 Fc variant, which contains amino acid mutations in one or more of the following four regions, preferably mutations in one or more of the mutation regions 2-4.

-Mutation region 1: residues 233-238,

-Mutation region 2: residues 265-271,

-Mutation region 3: residues 295-300 and

-Mutation region 4: residues 326-332.

In a preferred embodiment, the Fc variant comprises a combination of mutations in mutated region 2 and mutations in mutated region 4.

In one embodiment, the present invention provides an Fc variant with increased FcγRIIIA binding ability, the Fc variant comprising one or more sequence motifs selected from:

Zone 2 (EU No. 265-271): DVX1X2X3X4P, where X1=S/A/D, preferably S/A; X2=E/G/H/D, preferably E/G/H, more preferably E/G; X3=E/D, preferably E; X4=E/D;

Region 3 (EU numbering positions 295-300): X1X2NX3TX4, where X1=C/Q/L, preferably C or Q; X2=Y/W, preferably Y; X3=S/A, preferably S; X4=any amino acid residue Group, preferably Y/L, more preferably Y;

Region 4 (EU numbering positions 326-332): X1ALPX2PX3, where X1=any amino acid, preferably K; X2=any amino acid, preferably A/M/L, more preferably A/M; X3=E/I/D, preferably E /D.

In one embodiment, the Fc variant of the present invention with increased FcγRIIIA binding ability preferably comprises one or more mutations selected from the following: S267A, H268E, H268G, D270E, Q295C, I332E, I332D. Preferably, the Fc variant comprises a mutation selected from: K326M/S/I/T+I332E/D; A330T/G/V/Y+I332E/D; K326T+A330M; H268E/D; H268E/D +S267A; H268G+D270E, I332D; Q295C; Q295L+Y296W

More preferably it contains a mutation selected from:

K326M+I332E A330T+I332E K326S+I332E A330G+I332D K326I+I332E A330V+I332D L328T+I332E A330Y+I332D K326T+A330M H268G+D270E H268E S267A+H268E To I332D Q295C Q295L+Y296W

More preferably, it contains the following mutations:

H268E+K326I+I332E H268E+K326M+I332E H268E+K326S+I332E H268E+A330Y+I332D H268E+A330T+I332E

In a preferred embodiment, relative to the wild-type Fc polypeptide, the Fc variants of the present invention have improved FcγRIIIA-F158 and V158 binding ability.

In still another preferred embodiment, the Fc variant of the present invention has an improved FcγRIIIA binding ability, and at the same time, has substantially unchanged FcγRIIB binding ability relative to a wild-type Fc polypeptide.

In yet another embodiment, the Fc variants of the present invention have increased FcγRIIIA binding and an increased FcγRIIIA binding ability/FcγRIIB binding ability ratio, such as a binding affinity ratio, relative to a wild-type Fc polypeptide.

In one embodiment, the Fc variants of the present invention have increased FcγRIIIA binding capacity relative to wild-type Fc polypeptides, and exhibit increased ADCC activity.

In one embodiment, the present invention provides an Fc variant with increased FcγRIIB binding ability, the Fc variant comprising one or more sequence motifs selected from:

Region 1 (EU numbering positions 233-238): ELX1X2GX3, where X1=any amino acid, preferably L; X2=G/N/D; X3=P/V/D/W/F, preferably P/V;

Zone 2 (EU No. 265-271): DX1X2X3X4DP, where X1=V/I/L, preferably V; X2=E/S/D, preferably E; X3=H/D; X4=E/G;

Zone 3 (EU number 295-300): QX1NSTX2, where X1=S/Y, preferably S; X2=K/Y, preferably K;

Region 4 (EU numbering positions 326-332): X1X2X3PAPI, where X1=any amino acid, preferably K; X2=A/E; X3=any amino acid, preferably L/S/T/W/A/F.

In one embodiment, preferably, the Fc variant of the present invention with increased FcγRIIB binding ability comprises one or more mutations selected from the following: G236N, P238V, S267E, Y296S, Y300K.

In one embodiment, the Fc variant of the invention with increased FcγRIIB binding capacity comprises one or more sequence motifs selected from:

Zone 2 (EU No. 265-271): DX1X2X3X4DP, where X1=V/I/L, preferably V; X2=E/S/Q, preferably E; X3=H/D/Q; X4=E/G/ P, preferably E/G;

Region 4 (EU numbering positions 326-332): X1X2X3PAPI, where X1=any amino acid, preferably K; X2=A/E; X3=any amino acid, preferably L/S/W/F/T/A/Y, where X3 It is more preferably L/F/W/Y, preferably L/F, and most preferably F.

In some preferred embodiments, the Fc variants also have substantially unchanged or reduced FcγRIIA-H131 binding ability.

In one embodiment, the Fc variant of the present invention with increased FcγRIIB binding ability preferably contains one or more mutations selected from the group consisting of S267E, A327E and L328F. The Fc variant preferably has substantially unchanged or reduced FcγRIIA-R131 or FcγRIIIA binding ability.

In some embodiments, preferably, the Fc variant of the present invention with increased FcγRIIB binding capacity comprises a mutation selected from:

P331N+I332M S267E+E269G K326V+L328A V266I+S267E A330R+I332D S267E+H268D L328F+A330S V266L+S267Q+H268Q A327E+L328F E269P K326D+P331S G236N+P238V A327E+L328W Y296S+Y300K K326P+L328F To

More preferably, it contains a mutation selected from:

K326V+L328A S267E+E269G A327E+L328F V266I+S267E S267E+H268D To

It most preferably contains a mutation selected from the group consisting of K326V+L328A, A327E+L328F, S267E+H268D.

In some preferred embodiments, the Fc variant comprises the following combination mutations:

S267E+E269G+K326V+L328A V266I+S267E+K326V+L328A S267E+H268D+K326V+L328A S267E+E269G+A327E+L328F V266I+S267E+A327E+L328F S267E+H268D+A327E+L328F G236D+V266I+S267E+K326V+L328A

P238D+V266I+S267E+K326V+L328A

Preferably, the antibody comprising the Fc region variant of the present invention with increased FcγRIIB binding capacity of the present invention exhibits an enhanced effect of activating target cells. More preferably, an antibody comprising a variant of the Fc region of the invention with an increased FcγRIIB binding ability of the invention exhibits an increased ratio of FcγRIIB to FcγRIIA or FcγRIIIA binding ability relative to an antibody containing a parent Fc region or a wild-type Fc region, especially The ratio of the binding capacity of FcγRIIB to FcγRIIA ^R131. Preferably, the binding capacity can be expressed by the EC ₅₀ value of the target cell activated by the antibody.

Preferably, the antibody is an anti-CD40 activated antibody, more preferably the antibody has the VH and VL sequences shown in SEQ ID NO: 10/11. Preferably, the antibody is an anti-4-1BB activated antibody, more preferably the antibody has the VH and VL sequences shown in SEQ ID NO: 12/13.

In some preferred embodiments, the Fc variant polypeptide provided according to the present invention has a defucosylated or hypofucosylated Fc region.

Proteins containing Fc variants

Another aspect of the present invention provides proteins comprising the Fc variants of the present invention, such as immune fusion proteins and antibodies.

In one embodiment, the present invention provides an antibody comprising an Fc variant, wherein the Fc variant has an altered FcγR receptor binding ability relative to a wild-type Fc, wherein the receptor is selected from: FcγRIIA (R131), FcγRIIA (H131 ), FcγRIIB, FcγRIIIA (F158) and FcγRIIIA (V158). In some embodiments, binding capacity is expressed by binding affinity. In other embodiments, the binding capacity is expressed by EC ₅₀ values initiator effector function.

In one embodiment, the present invention provides an antibody comprising an Fc variant, wherein the Fc variant has an increased FcγRIIIA ability (such as binding affinity) relative to a wild-type Fc, preferably the antibody is used to kill expression and antibody variable regions The target cell of the bound antigen. Preferably, the Fc variant of the antibody contains one or more mutations selected from: S267A, H268E, H268G, D270E, Q295C, Q295L, Y296W, I332E, I332D; preferably contains mutations: K326M/S/I/T+I332E /D; A330T/G/V/Y+I332E/D; K326T+A330M; H268E/D; H268E/D+S267A; H268G+D270E; Q295L+Y296W; more preferably a combination of mutations: H268E+K326I+I332E; H268E +K326M+I332E; H268E+K326S+I332E; H268E+A330Y+I332D; H268E+A330T+I332E. Preferably, the antibody has an enhanced ADCC activity relative to a corresponding antibody comprising a wild-type Fc polypeptide, and preferably, the ADCC activity is increased by at least 10-100 times. In a preferred embodiment, the antibody comprising the Fc variant also has increased FcγRIIA binding and exhibits enhanced ADCP activity. In one embodiment, the antibody is an anti-HER2 antibody, for example, comprising the VH/VL sequence shown in SEQ ID NO: 6/7 or having at least 85% or 90% or 95% identity with the VH/VL sequence and having the same CDR sequence. /VL sequence, preferably Herceptin antibody. In one embodiment, the antibody is an anti-CD20 antibody, for example, comprising the VH/VL sequence shown in SEQ ID NO: 8/9 or having at least 85% or 90% or 95% identity with the VH/VL sequence and having the same CDR sequence. /VL sequence, preferably Rituximab antibody. In one embodiment, the present invention also provides a method of using the antibody to treat a disease, wherein an effective antibody is administered to an individual in need, wherein the disease will benefit from the ADCC activity on target cells triggered by the antibody and / Or ADCP activity. The diseases are, for example, tumors such as breast cancer, lymphoma, leukemia.

In one embodiment, the present invention provides an antibody comprising an Fc variant, wherein the Fc variant has increased FcγRIIB binding ability (eg binding affinity) relative to wild-type Fc, preferably the antibody is used to activate expression and antibody variable The region binds to the target cell of the antigen. In a preferred embodiment, the antibody comprising the Fc variant exhibits an increased ratio of FcγRIIB to FcγRIIA or FcγRIIIA binding ability, especially FcγRIIB to FcγRIIA-R131 binding ability ratio. In a preferred embodiment, the Fc variant of the antibody contains one or more mutations selected from the group consisting of S267E, A327E and L328F. In a preferred embodiment, the Fc variant of the antibody contains mutations: P331N+I332M, S267E+E269G, K326V+L328A, V266I+S267E, A330R+I332D, S267E+H268D, L328F+A330S, V266L+S267Q+H268Q, A327E+ L328F, E269P, K326D+P331S, G236N+P238V, A327E+L328W, Y296S+Y300K, or K326P+L328F. In another preferred embodiment, the Fc variant of the antibody comprises mutations: K326V+L328A, A327E+L328F, S267E+E269G, V266I+S267E; and more preferably comprises a combination of mutations: S267E+E269G+K326V+L328A, V266I+S267E +K326V+L328A, S267E+H268D+K326V+L328A, S267E+E269G+A327E+L328F, V266I+S267E+A327E+L328F, S267E+H268D+A327E+L328F, G236D+V266I+S267E+K326V+LV328A, P238D +S267E+K326V+L328A. In one embodiment, the antibody is an agonistic antibody against the TNF receptor family. In still another embodiment, the antibody is an anti-CD40 agonistic antibody, preferably, the antibody has the VH and VL sequences shown in SEQ ID NO: 10/11 or is at least 85% or 90% or 95% identical to them VH/VL sequences that are sexual and have the same CDR sequence. In still another embodiment, the antibody is an anti-4-1BB agonistic antibody, preferably, the antibody has the VH and VL sequences shown in SEQ ID NO: 12/13 or at least 85% or 90% or 95% of the VH and VL sequences shown in SEQ ID NO: 12/13. VH/VL sequences that are% identical and have the same CDR sequence. In one embodiment, the present invention also provides a method of using the antibody to treat a disease, wherein an efficient antibody is administered to an individual in need, wherein the disease will benefit from the antibody's activating activity on target cells. The disease is, for example, a tumor, such as colon cancer.

In some preferred embodiments, the antibody comprising the Fc variant provided according to the present invention has defucosylation or hypofucosylation in the Fc region.

Composition of Fc variants

The Fc variant of the present invention may be in the form of an Fc polypeptide, or in the form of a fusion protein (preferably a full-length antibody or immune fusion protein) comprising the Fc variant, or a cell (preferably a mammalian) expressing the polypeptide or fusion protein. Animal cells) are included in the composition. Such a composition is an aspect of the invention. The composition may also include, for example, a pharmaceutically acceptable carrier.

The composition may include suitable carriers, excipients, and agents conventionally added to the formulation to provide improved delivery and other properties. Exemplary formulations can be found in Remington's Pharmaceutical Sciences. The Fc region-containing polypeptide intended for in vivo administration can be formulated for the desired route of administration, for example, injection of liquid. Various drug delivery systems are known in the art and can be used to administer the compositions of the present invention. For example, the route of administration includes, but is not limited to: intradermal, intramuscular, oral, parenteral, intraperitoneal, subcutaneous and the like. In addition, the composition of the present invention can be administered in combination with other biologically active agents. Administration can be systemic or local administration.

The present invention also relates to the application of the composition of the present invention, such as medical applications, including in the treatment of human or animal individuals. The treatment method may include: administering the composition of the present invention to an individual in need.

Example

Example 1

pCDH vector is used for cell surface display of antibody Fc segment

The lentiviral vector pCDH vector (System Biosciences, catalog number: CD510B-1) was used to display the antibody Fc segment on the cell surface.

Construct the pCDH expression cassette as shown in Figure 1, in which IL2 signal sequence (IL2 protein signal peptide (amino acid sequence: MYRMQLLSCIALSLALVTNS, SEQ ID NO: 3)) can guide the secretion of Fc protein out of the cell, and PDGFRTM (PDGFR protein transmembrane region ( Amino acid sequence: HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR, SEQ ID NO: 4)) Fc can be anchored on the cell membrane surface, and the FLAG tag at the nitrogen end of the Fc protein (amino acid sequence: N-DYKDDDDK-C, SEQ ID NO: 5) can be used with corresponding anti-FLAG fluorescence The antibody is stained. The hinge region and the Fc region in the expression frame represent the immunoglobulin hinge region and the immunoglobulin constant region located behind the hinge region, respectively. Figure 20 shows the pCDH vector used to insert the expression cassette, where the expression cassette will be inserted into the MCS multiple cloning site of the vector under the control of the CMV promoter.

1.1 Construction of lentiviral vector plasmid

Insert the expression cassette shown in Figure 1 into the pCDH vector digested with XbaI and NotI, where the expression cassette contains the nucleotide sequence encoding wild-type human IgG1Fc (wtFc) (as shown in SEQ ID NO: 2 nucleoside Acid sequence). After sequencing and verification, the constructed new plasmid was named pCDH2. This plasmid will express and produce the wild-type Fc protein containing the SEQ ID No:1 sequence shown in Figure 24. Use EcoRI and NheI to digest wild-type human IgG1Fc (wtFc) N297A mutant (Fc N297A) and V12 mutant (Fc V12) (mutation site of V12: E233D/G237D/P238D/H268D/P271G/A330R; About Fc V12 For mutants, see F. Mimoto et al., Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIaR131 and FcγRIIaH131, Protein Engineering, Design&Selection, Vol. 26, No. 10, pp589-598, 2013) for the coding nucleotide sequence , And digest pCDH2 by EcoRI and NheI at the same time. Using New England BioLabs' T4 DNA ligase (article number: M0202L), the digested fragments were ligated with the vector according to the molar ratio of vector: fragment = 1:7. After sequencing verification, two other expression plasmids expressing Fc N297A variant protein and Fc V12 variant protein were constructed.

1.2 Transfection of 293FT cells

Transfect the constructed plasmid into 293FT cells (purchased from ATCC) and express Fc protein.

In short, 293FT cells were plated in a 6-well plate 24 hours in advance, and 5×10 ⁵ per well were cultured in 2ml DMEM medium. After 24 hours, the cells could be used for transfection. Add 1ug pCDH2 plasmid, 1ug pMDLg/pRRE (purchased from Addgene, article number: 12251), 1ug pRSV-Rev (purchased from Addgene, article number: 12253), 1ug pCMV-VSV-G (purchased from Addgene, article number: 8454) into 100ul Opti-MEM (purchased from Thermo Fisher Scientific, article number: 31985088), and then add 20ul PEI (purchased from Polysciences, Inc. article number: 24885), mix well, incubate at room temperature for 10 minutes, and add dropwise to the well plate. After 6 hours of transfection, change to fresh DMEM medium and express for another 48 hours. The medium containing the virus supernatant was collected, centrifuged at 1000 rpm for 5 min to remove cell debris, the supernatant was taken, and 293FT cells (5×10 ⁵ /per well) plated in a 6-well plate 24 hours in advance were added. After 12 hours of infection, change to fresh medium and continue to express for 24 hours.

1.3 Cell staining and flow cytometry

After 24 hours, the cells were washed with 2ml PBS after removing the medium, and 1ml STEMPRO ACCUTASE (Thermo Fishier Scientific, catalog number: A1110501) was added, and placed in an incubator for 1 min. Blow down the cells with 1ml of fresh medium. Take 2×10 ⁵ cells and resuspend them in 200ul FACS buffer (FACS Buffer formula: 1% glucose (W/V) added to PBS) containing 5% fetal bovine serum FBS (purchased from BI, item number 04-001-1A) , 1mM Na ₂ EDTA, 1nM HEPES), blocked at 4 degrees Celsius for 30 min. After centrifugation at 1000 rpm for 5 minutes, the cells were resuspended in 200 ul of FACS Buffer containing 0.5 ug/ml biotinylated FcγRIIB (preparation method described in section 1.4 below) and incubated at 4 degrees Celsius for 30 minutes. After centrifugation at 1000 rpm for 5 minutes, the cells were washed with 500ul FACS buffer, and the washing step was repeated 3 times. Then add streptavidin-PE (diluted 1:1000, purchased from Thermo Fishier Scientific, item number 21627) and anti-FLAG-FITC (diluted 1:200, purchased from SIGMA, item number F4049-.2MG) containing 5% serum FACS Buffer, incubate at 4 degrees Celsius for 20 min. After that, centrifuge at 1000 rpm for 5 minutes, wash three times with 500 ul FACS buffer, and resuspend in 500 ul FACS Buffer. Detect the fluorescence of PE fluorescent dye and FITC fluorescent dye carried on the cell using LSR Fortessa flow cytometer of BD company.

As shown in Figure 2, compared with the control blank (293FT cells not infected with lentivirus and unstained), the three Fc are anchored and displayed on the cell membrane surface, and the cell population migrates to the direction of FITC fluorescence enhancement and can be FLAG antibody is detected. In addition, as shown in Figure 2, compared with the blank of the control group, the cell population transfected with wtFc also showed some migration to the direction of enhanced PE fluorescence staining. There were double-positive cells with FITC and PE fluorescence intensity higher than the control background fluorescence. Population (0.728%), which indicates that wtFc that can bind to the antigen FcγRIIB can be detected by streptavidin-PE after being displayed on the cell surface. While the Fc N297A variant with the FcγRIIB binding ability removed, although it can be displayed on the cell membrane surface, it cannot bind to the antigen FcγRIIB. As shown in Figure 2, there is almost no double-positive cell subgroup with enhanced FITC and PE fluorescence staining (0.352 %), FITC positive cell population does not migrate towards PE. The Fc V12 variants with enhanced FcγRIIB binding ability showed strong binding ability to the antigen FcγRIIB after being presented on the cell surface. As shown in Figure 2, compared with wild-type, FcV12 transfected cell population FTIC and PE double positive The number of sex cell subpopulations increased significantly (2.56%), and the migration range to the PE fluorescence enhancement direction was greater, that is to say, the migration of the cell population to the PE direction was more obvious than that of the wild type wt.

The above results indicate that the Fc protein can be effectively displayed on the surface of mammalian eukaryotic cells; and the Fc displayed on the cell surface can be detected by staining with fluorescein-labeled FLAG antibody, and further, the Fc protein displayed on the cell surface can be detected PE fluorescence staining. Compared with wtFc display cells, the intensity changes of PE fluorescence on Fc variant display cells can reflect the changes in the binding ability of Fc variants to Fc receptor antigens. However, wild-type Fc binds very weakly to FcγRIIB. In order to obtain Fc variants with enhanced binding, screening and enrichment are required.

1.4 Preparation of biotinylated FcγRIIB protein

The FcγRIIB gene was purchased from Sino Biological (Cat. No.; HG10259-M). The extracellular domain sequence was connected to the avitag (GLNDIFEAQKIEWHE) sequence, and the 6×his tag sequence was connected to the C-terminus, and then the EcoRI and NheI digested pFUSE expression vector (purchased from Invivogen, article number: pfuse-hg1fc1). After being expressed in 293F cells, it was purified with histrap FF chromatography column (GE Healthcare). The purified FcγRIIB protein was linked to avitag with Biotin Ligase (purchased from GeneCopoeia, catalog number BI001) to obtain biotinylated FcγRIIB.

Example 2

Construction of Fc mutation library

2.1 Determine the mutation area

It is generally believed that the region where Fc and its receptor are relatively close is likely to be the region where they interact. Therefore, in this example, the mutation region is set in such a region, but as those skilled in the art can understand after reading this specification , The mutation area can also be set outside these areas.

Specifically, according to the structure of the human IgG1/FcγRIIB complex (PDB NO: 3wjj) and the structure of the human IgG1/FcγRIIIA complex (PDB NO: 5d6d), the region within 5 angstroms from FcγR in IgG1 Fc is selected as the interaction region . From this, it is determined that the EU numbering 233-238 (ELLGGP), EU numbering 265-271 (DVSHEDP), EU numbering 295-300 (QYNSTY), EU numbering 326-332 on IgG1 Fc (KALPAPI) is the region where mutations are introduced, named mutation region 1, mutation region 2, mutation region 3, and mutation region 4, respectively. (The EU number on IgG1 Fc can be seen in Figure 21)

2.2 Preparation of Fc mutation library

The wild-type Fc region can be used as a template to construct an Fc mutation library. It is also possible to use a modified wild-type Fc region as a template. For example, using a wild-type Fc region with a stop codon introduced into the region to be mutated as a template can effectively reduce the number of cell populations expressing and displaying wild-type Fc protein in a cell display library constructed with a mutation library, and promote screening efficiency .

In this example, the wild-type IgG1 Fc sequence coding sequence (SEQ ID NO: 2) with a stop codon introduced into the region to be mutated was used as a template, and DNA fragment 1 and DNA fragment 2 were amplified by PCR. Build the library.

The primer combinations used for the amplification of DNA fragments 1 and 2 of different mutation regions are as follows:

Mutant region 1 fragment 1: forward primer TAA-5 (20μM), reverse primer 1-1 to 1-15 in equal molar amount (20μM)

Mutant region 1 fragment 2: forward primer fr1-F (20μM), reverse primer TAA-4 (20μM)

Mutant region 2 fragment 1: forward primer TAA-5 (20μM), reverse primers 2-1 to 2-21 mixed in equimolar amounts (20μM)

Mutant region 2 fragment 2: forward primer fr2-F (20μM), reverse primer TAA-4 (20μM)

Mutant region 3 fragment 1: forward primer TAA-5 (20μM), reverse primer 3-1 to 3-15 in equal molar amount (20μM)

Mutant region 3 fragment 2: forward primer fr3-F (20μM), reverse primer TAA-4 (20μM)

Mutant region 4 fragment 1: forward primer TAA-5 (20μM), reverse primer 4-1 to 4-15 mixed in equimolar amount (20μM)

Mutant region 4 fragment 2: forward primer fr4-F (20μM), reverse primer TAA-4 (20μM)

The sequences of the above-mentioned primers used to prepare the Fc mutation library are shown in FIG. 22.

The PCR system used for amplification is as follows:

The amplification procedure is as follows:

The amplified product was recovered with a DNA recovery kit, and fragment 1 and fragment 2 were subjected to overlap PCR to connect the two fragments into a complete Fc mutant.

The overlap PCR system is as follows:

Overlap PCR amplification program:

After ligating DNA fragment 1 and DNA fragment 2 by overlapping PCR, two amino acid mutations were randomly introduced into the above four mutation regions respectively. The overlapped fragments were digested with EcoRI and NheI, inserted into the pCDH2 vector digested with EcoRI and NheI, and T4 DNA ligase (New England BioLabs, catalog number: M0202L) was used to divide the two into the vector: Fragment = 1: 7 molar ratio, and connect according to the manufacturer's instructions. Take 10ul of the ligation product and transform Trans1-T1 Phage Resistant Chemically Competent Cell (full-form gold, item number: CD501-03), do 3 transformation reactions for each mutant library, and calculate the library capacity of each library based on the clones that are grown. Collect all the clones that grow out and extract the plasmid. Thus, four Fc mutation libraries were constructed: mutation library 1, mutation library 2, mutation library 3, and mutation library 4. After analysis, the capacity of the 4 libraries is shown in Table 1 below.

Table 1: Storage capacity of mutant libraries 1-4

To Mutation library 1 Mutation library 2 Mutation library 3 Mutation library 4 Storage capacity 2×10 ⁵ 1.4×10 ⁵ 2×10 ⁵ 1.6×10 ⁵

Example 3

Through flow sorting, a cell population with enhanced binding to FcγRIIIA (F158/V158) was screened out

3.1 The production of infectious virus

Inoculate 5×10 ⁶ 293FT cells in a 10 cm ² dish 24 hours in advance. Mutation library 1 to mutation library 4 and 3 lentivirus packaging plasmids: 4ug pMDLg/pRRE (purchased from Addgene, article number: 12251), 4ug pRSV-Rev (purchased from Addgene, article number: 12253), 4ug pCMV-VSV- G (purchased from Addgene, article number: 8454) mix, add 500ul Opti-MEM, and then add 80ul PEI. After mixing uniformly, incubate at room temperature for 10 minutes and add dropwise to the dish. After 6 hours of transfection, change to fresh medium and express again for 48 hours. Collect the virus-containing medium supernatant, centrifuge at 1000 rpm for 10 minutes, take the supernatant, pass through a 0.45um filter membrane (purchased from Sartorius, catalog number: 16533-K), add 1/4 volume of the filtrate to the obtained filtrate Lentivirus concentrate (purchased from BIOMIGA company, article number V2001-02), precipitate at 4 degrees Celsius for 12 hours. After that, centrifuge at 1500g for 45min at 4 degrees Celsius to precipitate the lentivirus to the bottom of the centrifuge tube, discard the supernatant, and resuspend in 2ml DMEM medium. The concentrated virus solution was tested for lentivirus titer with Lenti-X p24 Rapid Titer Kit (purchased from Clontech, article number: 632200) according to the manufacturer's instructions.

3.2 Preparation of biotinylated FcγRIIIA protein

The FcγRIIIA (F158) gene was purchased from Sino Biological, the catalog number is HG10389-G. The extracellular domain sequence was connected with the avitag (GLNDIFEAQKIEWHE) sequence, and the 6×his tag sequence was connected at the C end, and then the EcoRI and NheI digested pFUSE expression vector (purchased from Invivogen, catalog number: pfuse-hg1fc1) was inserted. After being expressed in 293F cells, it was purified with histrap FF chromatography column (GE Healthcare). The purified FcγRIIIA (F158) protein was linked to avitag with Biotin Ligase (purchased from GeneCopoeia, catalog number BI001) to obtain biotinylated FcγRIIIA (F158).

3.3 Screen the mutation library through cell staining and flow cytometric sorting

After detecting the virus titer, ^{1×10 7} 293FT cells were infected with a lentivirus amount of ^{4×10 5 pg.} After 12 hours of infection, the culture medium was changed to fresh DMEM medium, and the cells were stained after the culture was continued for 48 hours. The cells were digested with STEMPRO ACCUTASE (Thermo Fishier Scientific, catalog number: A1110501), and resuspended in 1ml of FACS Buffer containing 5% serum (FACS Buffer formula: 1% glucose (W/V), 1mM Na ₂ EDTA was added to PBS, 1nM HEPES), sealed at 4 degrees Celsius for 30 minutes. Centrifuge at 1000 rpm for 5 min, take the cell pellet, resuspend it in FACS Buffer containing 5% serum containing 1 ml of 250 nM biotinylated FcγRIIIA (F158), and incubate at 4 degrees Celsius for 30 min. After centrifugation at 1000 rpm for 5 minutes, the cells were washed with 500ul FACS buffer, and the washing step was repeated 3 times. Then add streptavidin-PE (diluted 1:1000, purchased from Thermo Fishier Scientific, item number 21627) and anti-FLAG-FITC (diluted 1:200, purchased from SIGMA, item number F4049-.2MG) containing 5% serum FACS Buffer, incubate at 4 degrees Celsius for 20 min. After centrifugation at 1000 rpm for 5 min, wash three times with 500 ul FACS buffer, and resuspend in 500 ul FACS Buffer. The PE+FITC+ double positive cell population was sorted by the BD company FACSAria III flow cytometer. Figure 3A shows the flow cytometry of the first round of sorting. The blank group is uninfected and unstained cells, which can be used to indicate the autofluorescence of the cells themselves on the FITC channel and the PE channel. When sorting and setting gating, select the cell population whose FITC direction is stronger than blank (X-axis direction), and on this basis select the first 0.5-1% of the cells with the strongest PE direction.

The sorted cells were successively cultured in 24-well plates, 6-well plates and T75 cell culture flasks. After cell proliferation for 20 days, before determining whether the second round of sorting is necessary, staining analysis should be performed on the proliferated cells after the first round of sorting. 2×10 ⁵ cells were stained with 125 nM biotinylated FcγRIIIA (F158) (the staining method is as described above), as shown in Figure 3B. In this figure, for each library, the circled gate in the flow cytometer uses the blank FITC background fluorescence value to determine the left boundary of the gate, and the WT PE fluorescence to determine the lower boundary of the gate. It can be seen from the figure that the double-positive cell populations in mutation bank 1-4 are stronger than wild-type WT; in mutation bank 4, the FITC-positive cell population is almost all PE positive, so the next round of enrichment is not required However, the enrichment of mutation library 1, 2, 3 is not obvious, and a second round of enrichment is required.

Biotinylated FcγRIIIA (F158) was used for the second round of sorting. The second round of sorting method and gate strategy were the same as the first round of sorting. After the second round of sorting mutation library 1, 2 and 3 were amplified, 2×10 ⁵ cells were taken, and 2× of the mutation library 4 after the first round of FcγRIIIA (F158) sorting and proliferation was taken. ¹⁰⁵ cells, these four kinds of cells were stained with biotinylated FcγRIIIA 125nM of (F158) and biotinylated FcγRIIIA (V158), shown in Figure 3C.

3.4 Screening results

The FcγRIIIA F158V single nucleotide polymorphism (SNP) is a naturally occurring mutation form in the population, and its allele frequency distribution in the population, for example, is about 66% F and 34% V in the Chinese Han population. (Zhang Yun et al., Chinese Han population FcγRIIIA F158V gene polymorphism is associated with systemic lupus erythematosus and lupus nephritis, Basic Medicine and Clinical Medicine, May 2017, Volume 37, Issue 5, 709-713). There is only one amino acid difference between FcγRIIIA F158 and V158, and it may be desirable to obtain antibodies with enhanced binding affinity to both FcγRIIIA F158 and V158 in some therapeutic situations where the ADCC effector function of the antibody is expected to be utilized. Therefore, in this example, the ability of the mutant library to bind to both F158 and V158 after screening and enrichment was tested.

As shown in Figure 3C, after two rounds of FcγRIIIA (F158) staining and enrichment for mutant library 1, compared to wild-type wtFc, the cell population did not show an overall shift in the direction of PE fluorescence enhancement. After two rounds of FcγRIIIA (F158) staining and enrichment for mutant library 2 and mutant library 3, the binding of FcγRIIIA (V158) and FcγRIIIA (F158) was significantly improved. Compared with wild-type wtFc, the cell population showed increased fluorescence toward PE After only one round of FcγRIIIA (F158) staining and enrichment in mutant library 4, compared with wild-type wtFc, the cell population has shown a significant overall shift to the direction of PE fluorescence enhancement, indicating that there is a difference in FcγRIIIA (V158) And the binding of FcγRIIIA (F158) has been significantly improved.

The above results indicate that through mammalian display and flow cytometric sorting, the enrichment of FcγRIIIA high-affinity Fc mutants can be achieved. Moreover, after sorting with F158, not only cells that had enhanced binding to F158 were sorted, but also cells that also showed strong binding to V158 were sorted. Based on this result, we believe that F158 can be used to screen clones that have enhanced effects on both F158 and V158.

Subsequent deep sequencing and SPR results confirmed that after two rounds of sorting in mutation area 2 and mutation area 3, and after one round of sorting in mutation area 4, these PE fluorescence-enhanced double-positive cell populations all contained A clone with stronger binding capacity than the wild type.

Example 4

Next-generation sequencing (NGS) identification of Fc mutants enriched for FcγRIIIA

4.1 Second-generation sequencing of the enriched mutation library

^{Take 5×10 5} cells from the mutant bank 2, mutant bank 3, and mutant bank 4 after the above-mentioned FcγRIIIA staining and enrichment, and add 100ul lysis buffer (500ul lysis buffer: 455ul deionized water, 25ul 1M KCl, 5ul 1M Tris-HCl (pH: 9.0), 5ul 10% TritonX-100 (purchased from Sigma Company, article number: X100-100ML), 10ul proteinase K (purchased from NEB Company article number: P8107S)) at 60 degrees Celsius After 60 minutes of lysis, it was inactivated at 94 degrees Celsius for 10 minutes (to remove proteinase K activity). Using the cell lysate as a template, the Fc mutation region integrated in the cell was amplified by PCR through the following procedure.

(1) Amplification of full-length Fc fragment

After lysing the cells in each bank, using the cell lysate as a template, the integrated Fc sequence in the cells was amplified with the forward primer FLAG-F and the reverse primer PDGFR-R.

The PCR system used for amplification is as follows:

Amplification procedure:

The amplified product was recovered with a DNA recovery kit (Company MACHEREY-NAGEL, NucleoSpin Gel and PCR Clean-up, catalog number 740609.50), and recovered in 30ul ddH ₂ O.

(2) Second-generation sequencing sample preparation

Nested PCR: Take the recovered aqueous solution containing Fc library double-stranded DNA as a template, and use barcode-containing primers to amplify the corresponding mutant region.

Amplification procedure:

The forward and reverse primers used for each post-enrichment mutation library amplification are shown in Table 2 below. As a control, mutation library 2 (original library 2), mutant library 3 (original library 3), and mutation library 4 (original library 4) that were not enriched were also used for the Fc mutation region using the primers in Table 3 below. PCR amplification.

Table 2: Primers used for the amplification of the enriched mutation library

Note: The primers used for amplification of different libraries carry different barcodes at the 5'end respectively

Table 3: Primers used for amplification of the mutation library (ie, the original library) before enrichment

Note: The primers used for amplification of different libraries carry different barcodes at the 5'end respectively

The amplified product was recovered with a DNA recovery kit (company MACHEREY-NAGEL, NucleoSpin Gel and PCR Clean-up, catalog number 740609.50). After the enrichment, 200 ng of the recovered products from the mutant libraries 2, 3, and 4 were taken for equal mass mixing, and then the next-generation sequencing was performed. At the same time, 200ng each of the PCR amplification products of mutation library 2 (original library 2), mutation library 3 (original library 3), and mutation library 4 (original library 4) that have not been enriched is taken and mixed for the second generation. Sequencing.

With second-generation sequencing, the DNA mixture, that is, all sequences in the mutation library can be combined and sequenced at one time. The second-generation sequencing of bridge PCR was completed by Jinweizhi using the HiSeq 2x150bp platform (the sequencing length of the platform is 150bp in forward direction and 150bp in reverse direction). Because the amplified sequences from the same library contain the same pair of barcodes at both ends, the sequencing results obtained after sequencing are merged and disassembled according to different barcodes in CLC Genomics Workbench V12 to obtain different mutation libraries. Fc variant sequence.

4.2 Analysis of sequencing results

Translate the DNA sequence obtained by sequencing into amino acid sequence, write JAVA code to count the frequency of different amino acid sequences before and after enrichment in different mutation libraries, and sort according to the frequency of each mutant sequence from high to low.

Comparing the cumulative curve of mutant types and their frequency of occurrence before and after enrichment of each mutant library. As shown in the cumulative graph of Figure 4, the X-axis represents the types of mutants, and the Y-axis represents the ratio of the cumulative number of mutants to the total mutants. It can be seen from Figure 4 that the accumulation curve of the three original libraries is close to 45 degrees before being enriched, that is, showing a uniform accumulation, indicating that the frequency of occurrence of each sequence in the library is basically the same. However, after the enrichment, the X-axis range was significantly reduced compared with that before the enrichment, indicating that the Fc variants in the mutant library were significantly reduced after the FcγRIIIA enrichment, and some mutants were enriched in a large amount. From the accumulation curve of mutant frequency, it can be judged that the mutant pool 2-4 is enriched in mutants after flow sorting.

4.3 WebLogo mapping shows dominant amino acid mutations

The next-generation sequencing results of the mutant library after FcγRIIIA enrichment and the corresponding original library before enrichment were mapped by WebLogo.

As shown in Figure 5, compare the changes of the dominant amino acids in the corresponding mutation regions before and after the enrichment: S267A, H268E, H268G, and D270E in the mutation library 2 are significantly enriched, and Q295C in the mutation library 3 is significantly enriched. I332E and I332D are obviously enriched. These enrichments indicate that these amino acid mutations are beneficial to the improvement of binding affinity.

According to the WebLogo diagram, after enrichment, the mutant regions 2-4 respectively show the following preferred sequence motifs:

Mutation region 2 (EU numbering positions 265-271): DVX1X2X3X4P, where X1=S/A/D, preferably S/A; X2=E/G/H/D, preferably E/G/H, more preferably E/G ; X3=E/D, preferably E; X4=E/D;

Mutant region 3 (EU numbering positions 295-300): X1X2NX3TX4, where X1=C/Q/L, preferably C or Q; X2=Y/W, preferably Y; X3=S/A, preferably S; X4=any amino acid Residue, preferably Y/L, more preferably Y;

Mutant region 4 (EU numbering positions 326-332): X1ALPX2PX3, where X1=any amino acid, preferably K; X2=any amino acid, preferably A/M/L, more preferably A/M; X3=E/I/D, preferably E/D.

Example 5

SPR analysis of Fc mutants bound by FcγRIIIA

5.1 Expression of Fc mutant

According to the analysis of the second-generation sequencing results in Example 4.2, the frequency of each mutant sequence in the mutation library is sorted from high to low.

For mutation area 2: select the first-ranked variant 2-4, the second-ranked variant 2-7, and the fourth-ranked variant 2-11; for the mutant area 3: select the first-ranked variant 3- 4. Ranked 3rd variant 3-14; for mutation area 4: Select 1st ranked variant 4-5, 2nd ranked variant 4-12, 3rd ranked variant 4-6, ranked No. 5th variant 4-14, 7th variant 4-30, 8th variant 4-10, 10th variant 4-25, 13th variant 4-11, ranked 15th 4-8, 16th variant 4-16, 20th variant 4-19.

According to the mutation site of the selected variant, using the wild-type Fc coding sequence (SEQ ID NO: 2) as a template, the Fc sequence containing the mutation was obtained by means of point mutations, and inserted into the EcoRI and NheI digested pFUSE vector ( Purchased from Invivogen, product number: pfuse-hg1fc1), the Fc variant protein expression plasmid was constructed. 293FT cells were transfected with plasmids, and the transfected cells were cultured in DMEM medium to express Fc protein. The Fc protein consisting of an Fc region with a hinge region is secreted into the culture medium. Aspirate the medium, centrifuge at 1000 RPM for 10 minutes, take the supernatant (about 2 ml), and purify it with proteinA beads (GE healthcare).

5.2 Determining the binding ability of mutants and receptors

Different Fc variant proteins were expressed and purified according to the method in Example 5.1. Compare the binding ability of Fc variant protein with human FcγRIIIA (F158), FcγRIIIA (V158) and FcγRIIB with wild-type Fc (WT).

The Fc protein solution to be tested was adjusted to 400 nM with PBS, and the volume was 500 μl. Preparation of his-tagged FcγR receptor protein. Adjust the concentration of his-tagged FcγRIIIA (F158), FcγRIIIA (V158), and FcγRIIB proteins to 400nM, with a volume of 1ml. Biacore T200 (GE Healthcare) was used to analyze the interaction of each Fc mutant with FcγRIIIA (F158), FcγRIIIA (V158), and FcγRIIB receptors. The following experiments are all carried out at 20 degrees Celsius. First, the anti-his tag antibody (GE Healthcare) is covalently bound to the S series sensor chip CM5 (GE Healthcare) by the amine coupling method. Using protein solution flow rate 10s/min and capture time 60s, chip 2 channels capture FcγRIIIA (F158); chip 3 channels capture FcγRIIIA (V158); chip 4 channels capture FcγRIIB. After capturing the protein in the three channels, the Fc protein solution flows through the 1-2-3-4 channels sequentially at 30μl/min for 90s. The RU value when the detection signal is stable reflects the Fc binding amount on FcγR, and the Fc binding amount is positively correlated with the affinity between Fc/FcγR. After each detection, it was eluted with 10mM Glycine-HCl (pH=1.5) to regenerate the sensor chip. The affinity between each Fc mutant and the FcγR receptor is shown in Figure 6, and the amino acid sequence of the mutant region of each Fc mutant tested is shown in Table 4 below.

Table 4: Sequences of Fc variants tested by SPR

As shown in Figure 6, all the tested clones have the same binding ability for F158 and V158. This result is consistent with the previous flow sorting result, which further confirms that in the method of the present invention, F158 can be used to screen clones that have enhanced effects on both F158 and V158.

Compared with the wild-type Fc, among the tested Fc mutants, except for variants 4-10, the other 15 have different degrees of increased affinity for FcγRIIIA V158 and F158, and the RU value is higher than that of the wild-type Fc. The sequences of these variants with increased affinity are consistent with the dominant amino acid residues and preferred sequence motifs obtained by weblogo analysis in Example 5.

In mutant region 4, most of the mutants with enhanced affinity have dominant residues I332E/I332D. This result is consistent with the previous report that I332E is an FcyRIIIA binding-enhancing mutation. Similarly, consistent with the literature report that H268D is an FcγRIIIA binding-enhancing mutation, in the mutation region 2, the Fc variant 2-11 containing H268E obtained by screening using the method of the present invention also showed binding to FcγRIIIA in SPR detection. Enhanced.

In addition, using the method of the present invention, a variety of new variants with improved performance have been screened, for example, variants 4-12 (with mutations K326T, A330M). In addition, it was found that variants 4-16, 4-25, 4-14, and 4-19 had further improved FcγRIIIA binding ability compared with variant 4-5 with only I332E mutation, indicating that K326M, K326S, K326I, A330T It can further enhance the binding of Fc and FcγRIIIA (F/V158) on the basis of I332E.

Example 6 Construction of Combination Mutants and Affinity Detection

The H268E single point mutations in the mutants 2-11 in the mutation library 2 screened in Example 4 and Example 5 were constructed to 4-14, 4-16, 4-25, 4- by means of point mutations. 30. Among the 4-19 mutants (combined mutants are prefixed with 2E- before the clone number). The combined mutants were tested for their ability to bind to FcγRIIIA (F158), FcγRIIIA (V158), and FcγRIIB, and compared with the uncombined mutant and wild-type Fc (WT).

The Fc protein solution to be tested was adjusted to 400 nM with PBS, and the volume was 500 μl. Preparation of his-tagged FcγR receptor protein. Adjust the protein concentration of his-tagged FcγRIIIA (F158), FcγRIIIA (V158), and FcγRIIB to 400nM, with a volume of 1ml. Biacore T200 (GE Healthcare) was used to analyze the interaction of each Fc protein with FcγRIIIA (F158), FcγRIIIA (V158), and FcγRIIB receptors. The analysis method is the same as in Example 5.2.

The affinity between each Fc mutant and the FcγR receptor, that is, the binding amount RU at the same Fc detection concentration, is shown in Figure 7A.

Furthermore, an activation test based on the Jurkat-Lucia ^™ NFAT-CD16 cell line was used to detect the binding and activation of the combined Fc mutant to the FcγRIIIA receptor expressed on the cell surface.

The Fc variants after the combination in Fig. 7A, namely 2E-4-16, 2E-4-14, 2E-4-25, 2E-4-19, 2E-4-30, were expressed and purified. According to the manufacturer’s instructions, 1ug of purified protein was added to a 96-well plate, coated at 4°C for 12 hours, and then 1×10 ⁵ Jurkat-Lucia ^TM NFAT-CD16 cell line (InvivoGen, catalog number: jktl-nfat-cd16) was added to stimulate After 24 hours, the cells were lysed with the lysate provided by the kit (Promega, catalog number E1500), and the luciferase expressed in the cells was detected. As shown in Figure 7B, the luciferase activity of all mutants is stronger than that of the wild type.

Example 7 ADCC activity detection of Fc variants

The Fc mutants: 2E-4-14, 2E-4-16, 2E-4-25, 2E-4-30, 2E-4-19 (composed of hinge region and CH2 and CH3 regions) and Herceptin The VH-CH1 segment was ligated and inserted into the EcoRI/NheI digested pFUSE expression vector to construct 5 kinds of Herceptin heavy chain mutant expression plasmids.

The sequence of Herceptin's VH-CH1: (SEQ ID NO: 6)

Light chain sequence of Herceptin: (SEQ ID NO: 7)

293F cells (purchased from Thermo Fisher Scientific, catalog number: R79007) were co-transfected with Herceptin heavy chain mutant expression plasmid and Herceptin light chain expression plasmid. After transfection, they were cultured in a 37°C cell incubator for 4 days. Afterwards, the proteinA (GE healthcare) column was used to purify the expressed antibody from the cell culture supernatant.

Non-Radioactive Cytotoxicity Assay试剂盒(购自Promega，货号：G1780)进行。根据检测到的上清中的乳酸脱氢酶含量，计算ADCC效应程度。结果如图8A所示。相对于野生型，检测的5个组合变体，在供体3中均导致了更高的ADCC％，且组合变体2E-4-16,2E-4-14和2E-4-25在5个供体中引起更高的平均ADCC％。 In RPMI 1640 medium (purchased from Thermo Fisher Scientific, catalog number: C11875500CP), the final concentration of 2ng/mL purified antibody, 1×10 ⁴ human breast cancer cells SKBR3 cells, and 1.5×10 ⁵ cells derived from 5 Peripheral blood mononuclear cells of a healthy person were incubated at 37°C for 4 hours. After that, the amount of lactate dehydrogenase (LDH) leaked from the cytoplasm of SKBR3 cells due to ADCC was measured, and CytoTox was used for this test.

Non-Radioactive Cytotoxicity Assay kit (purchased from Promega, article number: G1780) was performed. Based on the detected lactate dehydrogenase content in the supernatant, the degree of ADCC effect was calculated. The result is shown in Figure 8A. Compared with the wild type, the 5 combination variants tested resulted in a higher ADCC% in donor 3, and the combination variants 2E-4-16, 2E-4-14 and 2E-4-25 were 5 Each donor caused a higher average ADCC%.

For the selected combination variants, in different antibodies, draw the ADCC dose curve:

(1) According to the results in Example 7, the Herceptin combination variants of 2E-4-16, 2E-4-14 and 2E-4-25 were selected, and their ADCC effects were tested with different doses. (10-fold dilution, a concentration gradient of 8) to a final concentration of ¹⁰⁴ to 0.001ng / ml antibody variants with 1 × 10 ⁴ individual cells and SKBR3 breast cancer cells 2 × 10 ⁵ peripheral blood mononuclear cells in the individual Incubate at 37°C for 4 hours to detect the amount of lactate dehydrogenase (LDH) leaked from the cytoplasm of SKBR3 cells due to ADCC. As shown in Figure 8B, the ADCC effect of all variants is stronger than that of the wild type.

(2) According to the results in Example 7, 2E-4-16, 2E-4-14 and 2E-4-25 were selected and constructed in accordance with the above-mentioned construction method similar to the Herceptin antibody containing these combination variants. These combined mutant Rituximab antibodies. Use different antibody doses to detect the ADCC effect. (10-fold dilution, a concentration gradient of 7) to a final concentration of ¹⁰⁴ to 0.01ng / ml antibody variants with 1 × 10 ⁴ individual cells Ramos B lymphoma and 2 × 10 ⁵ individual peripheral blood mononuclear cells Incubate at 37°C for 4 hours to detect the amount of lactate dehydrogenase (LDH) leaked from the cytoplasm of Ramos cells due to ADCC. As shown in Figure 8C, the ADCC effect of all variants is stronger than that of the wild type.

The sequence of Rituximab's VH-CH1: (SEQ ID NO: 8)

Light chain sequence of Rituximab: (SEQ ID NO: 9)

Example 8

Through flow sorting, a cell population with enhanced binding to FcγRIIB was screened out

According to the same operation method as described in Examples 2 and 3, the construction of the mutation library and the screening of variants were carried out. In short, mutation pool 1 to mutation pool 4 were combined with three lentiviral packaging plasmids: pMDLg/pRRE, pRSV-Rev, and pCMV-VSV-G to co-transfect 293FT cells. After transfection, change to fresh medium and express again for 48 hours. Collect the lentivirus in the supernatant of the medium and concentrate. After detecting the lentivirus titer with Lenti-X p24 Rapid Titer Kit (purchased from Clontech, article number: 632200) ^{, 1×10 7} 293FT cells were infected with a lentivirus amount of ^{4×10 5 pg.} Afterwards, cells were stained with 250 nM biotinylated FcγRIIB, streptavidin-PE (1:1000 dilution) and anti-FLAG-FITC (1:200 dilution). Detect the fluorescence of PE fluorescent dye and FITC fluorescent dye carried on the cell using LSR Fortessa flow cytometer of BD company. The first 0.5%-1% PE+FITC+ double positive cell population was sorted by a flow cytometer sorter.

The sorted cells were successively cultured in 24-well plates, 6-well plates and T75 cell culture flasks. After cell proliferation for 20 days, proceed to the next round of sorting. The subsequent sorting method was the same as above, but the subsequent sorting was performed with 125nM biotinylated FcγRIIB. Before the next round of sorting, 2×10 ⁵ cells were taken from the proliferating cells for staining (the staining method was as described above), and compared with the wild type to observe whether the staining was enhanced.

Figure 9A shows the staining analysis after sorting and proliferation, in which the circle gate in each library flow chart uses the blank FITC background fluorescence value to determine the left boundary of the gate, and the PE fluorescence of WTFc to determine the lower boundary of the gate. It can be seen from the figure that after four rounds of FcγRIIB staining and enrichment for mutant library 1 and mutant library 3, compared with the wild type, a part of the double-positive cell population with enhanced PE fluorescence staining appeared. After two rounds of FcγRIIB staining and enrichment for mutant bank 2 and mutant bank 4, compared with the wild type, the number of double-positive cell populations increased significantly, and the cell populations showed overall migration in the direction of PE fluorescence enhancement, indicating that most of the cells paired The binding of FcγRIIB was significantly improved.

The above results indicate that the enrichment of FcγRIIB high-affinity Fc mutants can be achieved through mammalian display and flow sorting, especially the mutant libraries 2 and 4 achieve better enrichment effects.

Example 9

Negative screening of FcγRIIB binding positive cell population by flow sorting

As shown in Figure 9A, almost half of the cell populations of mutation pool 2 and mutation pool 4 after the second round of enrichment showed high affinity for FcγRIIB and showed positive staining. To confirm whether there are clones that do not or weakly bind to FcγRIIA (H131 or R131) and FcγRIIIA (F158 or V158) in the selected FcγRIIB positive group, that is, only highly bind to FcγRIIB, while maintaining the original wild-type binding ability with other receptors or Clones with weakened binding capacity. We stained the mutant library 2 and mutant library 4 enriched by FcγRIIB with FcγRIIA (H131), FcγRIIA (R131), FcγRIIIA (F158), and FcγRIIIA (V158), respectively.

Take 5×10 ⁵ FcγRIIB-positive cells of mutation pool 2 or mutation pool 4 after two rounds of enrichment, and use 250nM biotinylated FcγR (ie, FcγRIIA (H131) in the same manner as described in Example 3.3. ), FcγRIIA (R131), FcγRIIIA (F158), or FcγRIIIA (V158)), streptavidin-PE (1:1000 dilution) and anti-FLAG-FITC (1:200 dilution) for cell staining, and use BD's The LSR Fortessa flow cytometer detects the fluorescence of PE fluorescent dyes and FITC fluorescent dyes carried on the cells.

The result is shown in Figure 9B. In each of the library flow diagrams, the circle gate uses the blank FITC background fluorescence value to determine the left boundary of the gate. At the same time, because it is negatively stained, the WT PE fluorescence is used to determine the upper boundary of the gate. To show cell populations that are not stained by the corresponding receptor or are weakly stained. It can be seen from the figure that there are negative populations for FcγRIIIA (F158) and FcγRIIIA (V158) in mutation library 2 (see, gate cells in the figure), and mutation library 4 is for FcγRIIA (H131) and FcγRIIA respectively. (R131), FcyRIIIA (F158), FcyRIIIA (V158) negative population (see, the cells in the gate in the figure). However, the negative group for FcγRIIA (R131) in mutation library 4 is not obvious, which is consistent with the subsequent SPR results and reports in the prior art, that is, it is difficult to combine Fc clones with FcγRIIB and FcγRIIA (R131). Distinguish, this may be due to the extremely similar structure of the two receptors.

According to the results of the above-mentioned flow cytometry analysis, the above-mentioned mutation libraries 2 and 4 that have been positively screened by FcγRIIB were negatively screened respectively. Methods as below:

The cells sorted after the second round of positive screening were cultured in DMEM for proliferation, and then digested with STEMPRO ACCUTASE (Thermo Fisher Scientific, catalog number: A1110501), resuspended in 1ml FACS Buffer containing 5% serum, and blocked at 4 degrees Celsius 30min. Centrifuge at 1000 rpm for 5 min, take the cell pellet, resuspend it in FACS Buffer containing 5% serum containing 1 ml 250 nM biotinylated FcγR, and incubate at 4 degrees Celsius for 30 min. Centrifuge at 1000 rpm for 5 minutes, wash the cells with 500ul FACS buffer, and repeat the washing step 3 times. Then add streptavidin-PE (diluted 1:1000, purchased from Thermo Fisher Scientific, item number 21627) and anti-FLAG-FITC (diluted 1:200, purchased from SIGMA, item number F4049-.2MG) containing 5% serum FACS Buffer, incubate at 4 degrees Celsius for 20 min. Centrifuge at 1000 rpm for 5 minutes, wash three times with 500ul FACS buffer, and resuspend in 500ul FACS Buffer. The top 1% PE-FITC+ cell population was sorted using the FACSAria III flow cytometer of BD company. The sorted cells continue to proliferate in the 24-well plate, and transfer them to the 6-well plate as the cells proliferate, and continue to be cultured in the T75 cell culture flask.

For the cell population enriched in mutant bank 2 after two rounds of FcγRIIB staining, FcγRIIIA (F158) and FcγRIIIA (V158) were used for negative screening respectively. As shown in Figure 10, the cell populations with negative binding of FcγRIIIA (F158) or FcγRIIIA (V158) were selected from the sorting gate shown in the figure at a ratio of 1%.

For the cell population enriched in mutant library 4 after two rounds of FcγRIIB staining, negative screening was performed with FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA (H131), and FcγRIIA (R131). As shown in Figure 11, cell populations that are negative for FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA (H131), and FcγRIIA (R131) binding are selected from the sorting gate shown in the figure at a ratio of 1%.

Example 10

Next-generation sequencing (NGS) identification of Fc mutants enriched for FcγRIIB

(1) Comparison of the cell population after FcγR2B screening (mutant bank 1, 2, 3, 4) and the original bank cell population before screening (original bank 1, 2, 3, 4).

After the cells of mutation library 1, mutation library 2, mutation library 3, and mutation library 4 enriched with FcγRIIB were lysed by lysis buffer, the Fc mutant region sequence was amplified by PCR with forward and reverse primers with different barcodes. Come out and perform next-generation sequencing. The corresponding primers are shown in Table 6. At the same time, the non-enriched mutant library 1 (original library 1), mutant library 2 (original library 2), mutant library 3 (original library 3), and mutant library 4 (original library 4) were also sequenced for the next generation, with corresponding primers See Table 7. The sequencing method is the same as that described in Example 4.1.

Table 6: Primers used for the amplification of the enriched mutation library

Note: The primers used for amplification of different libraries carry different barcodes at the 5'end respectively

Table 7: Primers used for amplification of the mutation library (ie, the original library) before enrichment

Note: The primers used for amplification of different libraries carry different barcodes at the 5'end respectively

Comparing the cumulative curve of mutant types and their frequency of occurrence before and after enrichment of each mutant library. The result is shown in Figure 12. The Fc types of the mutant library after FcγRIIB enrichment were significantly reduced, and the high-affinity Fc mutants were enriched.

The second-generation sequencing results of the mutant library after FcγRIIB enrichment and the original library were subjected to WebLogo mapping in the same manner as described in Example 4, and the results are shown in FIG. 13. Comparing the changes of dominant amino acids in the corresponding mutation regions: G236N and P238V in mutation bank 1 were significantly enriched; S267E in mutation bank 2 was significantly enriched, and Y296S and Y300K in mutation bank 3 were significantly enriched; mutation bank 4 did not There was enrichment of specific amino acids, but the dominant amino acid type at position 328 changed significantly. In addition to the L residue, several other amino acid residues with a significantly increased frequency appeared.

According to the WebLogo diagram, after enrichment, the mutant regions 1-4 respectively show the following preferred sequence motifs:

Mutant region 1 (EU numbering positions 233-238): ELX1X2GX3, where X1=any amino acid, preferably L; X2=G/N/D; X3=P/V/D/W/F, preferably P/V;

Mutation region 2 (EU numbering positions 265-271): DX1X2X3X4DP, where X1=V/I/L, preferably V; X2=E/S/D, preferably E; X3=H/D; X4=E/G;

Mutation region 3 (EU number 295-300): QX1NSTX2, where X1=S/Y, preferably S; X2=K/Y, preferably K;

Mutant region 4 (EU numbering positions 326-332): X1X2X3PAPI, where X1=any amino acid, preferably K; X2=A/E; X3=any amino acid, preferably L/S/T/W/A/F.

(2) Comparison of the cell population after positive screening and negative screening of FcγRIIB with the original cell population before screening (mutation library 2, 4)

After two rounds of FcγRIIB staining and enrichment of mutation library 2 and negative screening of FcγRIIIA (F158) or FcγRIIIA (V158), the cells were lysed by lysis buffer, and the Fc mutant regions were separated by forward and reverse primers with different barcodes. The sequence was amplified by PCR and subjected to second-generation sequencing. The corresponding primers are shown in Table 8.

Table 8: Primers used for amplification of mutation library 2 after enrichment in positive and negative screening

Note: The primers used for amplification of different libraries carry different barcodes at the 5'end respectively

The mutant accumulation curves of the mutant library 2 after the above two screenings were compared with the original library 2, and the results are shown in FIG. 14.

The second-generation sequencing results of the two screened cell banks and the original bank 2 were subjected to WebLogo mapping, and the results are shown in FIG. 15. Comparing the changes of dominant amino acids in the corresponding mutation regions: S267E was significantly enriched.

According to the WebLogo diagram, the mutant region 2 after the negative sieve respectively shows the following preferred sequence motifs:

FcγRIIIA (V158) negative screening:

Mutation region 2 (EU numbering 265-271): DX1X2X3X4DP, where X1=V/L, preferably V; X2=E/S/Q; X3=H/Q; X4=E/G/P;

FcγRIIIA (F158) negative screening:

Mutation region 2 (EU numbering positions 265-271): DX1X2X3X4DP, where X1=V/I/L, preferably V; X2=E/S/Q, preferably E; X3=H/D/Q; X4=E/G .

The mutant library 4 was enriched by two rounds of FcγRIIB staining and negatively screened by FcγRIIIA (F158) or FcγRIIIA (V158) or FcγRIIA (H131) or FcγRIIA (R131). After the cells obtained were lysed by the lysis buffer, they were lysed with different barcodes. The forward and reverse primers amplify the Fc mutant sequence by PCR and perform second-generation sequencing. The corresponding primers are shown in Table 9 below.

Table 9: Primers used for amplification of mutation library 4 after enrichment in positive and negative screening

Note: The primers used for amplification of different libraries carry different barcodes at the 5'end respectively

The mutation accumulation curve of the mutant library 4 after the above four screenings was compared with the original library 4, and the result is shown in FIG. 14.

The second-generation sequencing results of the four screened cell banks and the original bank 4 were subjected to WebLogo mapping, and the results are shown in FIG. 16. Compare the changes of dominant amino acids in the corresponding mutation regions:

Compared with the weblogo map obtained after positive screening of mutation library 4, after negative screening of FcγRIIA (R131), the dominant amino acid residues at each position did not change much, which is different from the previous results-it is difficult to distinguish between Fc pair FcγRIIB and FcγRIIA (R131) The combination of-match.

However, compared with the weblogo map obtained after positive screening of mutation library 4, after negative screening of FcγRIIIA (F158), FcγRIIIA (V158) and FcγRIIA (H131), it can be seen that the frequency of A327E has further increased, especially in FcγRIIIA (V158) and FcγRIIA (H131) after the negative sieve. In addition, after passing FcγRIIIA (V158) or FcγRIIA (H131) negative sieve, the frequency of L328F was further significantly enriched.

According to the WebLogo diagram, after enrichment, the mutant region 4 respectively showed the following preferred sequence motifs:

FcγRIIIA (F158) negative screening:

Mutant region 4 (EU numbering positions 326-332): X1X2X3PAPI, where X1=any amino acid, preferably K; X2=A/E; X3=any amino acid, preferably L/S/W/F/T.

FcγRIIIA (V158) negative screening:

Mutant region 4 (EU numbering positions 326-332): X1X2X3PAPI, where X1=any amino acid, preferably K; X2=A/E; X3=F/L/W/Y, preferably F;

FcγRIIA(H131) negative screening:

Mutant region 4 (EU numbering positions 326-332): X1X2X3PAPI, where X1=any amino acid, preferably K; X2=A/E; X3=L/F/W/Y, preferably L/F.

Example 11

SPR analysis of Fc mutants bound by FcγRIIB

According to the results of deep sequencing:

(1) The top 4 mutants in mutation library 2 after two rounds of positive FcγRIIB screening and negative screening of FcγRIIIA (F158) are: 2F-10, 2F-3, 2V-3, 2V-9.

(2) The top 4 mutants in mutation library 2 after two rounds of FcγRIIB positive screening and FcγRIIIA (V158) negative screening are: 2F-10, 2V-9, D2-1, 2V-3.

(3) The top 4 mutants in mutation library 4 after two rounds of FcγRIIB positive screening and FcγRIIIA (F158) negative screening are: 4F-6, 4R-18, 4R-14, D4-1.

(4) The top 4 mutants in mutation library 4 after two rounds of FcγRIIB positive screening and FcγRIIIA (V158) negative screening are: 4F-6, D4-1, 4R-3, 4H-21.

(5) The top 4 mutants in mutation library 4 after two rounds of FcγRIIB positive screening and FcγRIIA (H131) negative screening are: 4F-6, 4R-3, D4-3, 4H-21.

(6) The top 4 mutants in mutation library 4 after two rounds of FcγRIIB positive screening and FcγRIIA (R131) negative screening are: 4R-18, 4R-14, 4V-14, 4R-3.

(7) The mutants with the highest enrichment degree after 4 rounds of FcγRIIB positive screening in mutant library 1 are: 1B-7.

(8) The mutants with the highest enrichment degree after 4 rounds of FcγRIIB positive screening in mutant library 3 are: 3B-4.

The sequences of the above mutants are shown in Table 10 below.

Table 10: Sequences of Fc variants tested

In the same manner as described in Example 5, the expression of the above-mentioned Fc variant and SPR analysis were performed. In short, the above-mentioned Fc mutant was expressed and purified in 293FT cells using the pFUSE vector. By using Biacore T200 (GE Healthcare), measure the binding ability of these Fc variants with FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIA (H131), FcγRIIA (R131), FcγRIIB, and compare with wild-type Fc (WT) .

The affinity between each Fc mutant and the FcγR receptor, that is, the binding amount RU detected at the same Fc measurement concentration, is shown in FIG. 17. It can be seen from Fig. 17 that most of the tested Fc variants showed higher binding affinity (RU value) for FcγRIIB than wild-type Fc. These high-affinity Fc variants are in sequence with the dominant amino acid residues and preferred sequence motifs obtained from previous weblogo analysis. In addition, it can be seen from the results that clones with strong binding ability to FcγRIIB are accompanied by enhanced binding ability to FcγRIIA (R131), that is, Fc cannot distinguish between the two FcRs. This result is similar to the flow chart of Figure 9B and the flow chart of Figure 16. The results of the weblogo diagram are consistent.

Example 12

Construction of combinatorial mutants and SPR analysis

Among the mutant Fc detected in Example 11, compared with wild-type WTFc, 2F-10, 2F-3, 2V-3 in mutant bank 2 and 4R-14, 4F-6 in mutant bank 4 showed resistance to FcγRIIB. High binding capacity. Combining 4R-14 with 2F-10, 2F-3, and 2V-3 respectively, the resulting combination variants Fc are named VAA-2F-10, VAA-2F-3, and VAA-2V-3, respectively. Combining 4F-6 with 2F-10, 2F-3, and 2V-3 respectively, the resulting combination variants Fc are named KEF-2F-10, KEF-2F-3, KEF-2V-3, respectively. The sequences of the combination variants are shown in the table below.

Table: Fc variants containing combined mutations of mutation regions 2 and 4

After these combined mutants were expressed and purified in 293FT cells by pFUSE vector, the binding ability to FcγRIIIA (F158), FcγRIIIA (V158), FcγRIIB, FcγRIIA (H131), and FcγRIIA (R131) was measured, and the binding ability to wild-type Fc was determined. (wt) and the corresponding clone before combination. The SPR analysis method is the same as that described in Example 6. The affinity between each Fc mutant and the FcγR receptor, that is, the binding amount RU at the same Fc measurement concentration, is shown in FIG. 18.

Example 13

Functional analysis of FcγRIIB binding to Fc variants

The targeted binding properties of the Fab domain in an agonist antibody are a key factor, but the interaction between the Fc domain of an antibody and FcγR (IgG Fc receptor) can also determine the potential of an agonist. Studies have shown that FcγRIIB is required for optimal agonist activation of costimulatory receptors such as CD28, CD40, OX40 and 4-1BB. For this reason, in this example, the Fc combination mutations screened in Example 12 were introduced into the agonistic antibody, and the cell activation effect mediated by it was detected.

Combine the Fc of Example 12 with mutations VAA-2F-10, VAA-2F-3, VAA-2V-3, KEF-2F-10, KEF-2F-3, KEF-2V-3 and mutation 2F-10, and introduce Wild-type hIgG1 Fc region (SEQ ID NO:1, consisting of hinge region and CH2 and CH3) sequence. The obtained variant Fc region was ligated with the VH-CH1 segment of the CD40 agonist antibody, and inserted into the EcoRI/NheI digested pFUSE expression vector to construct an expression plasmid for the heavy chain mutant.

Protein A(GE Healthcare,17-0402-01)操作说明书在GE公司纯化仪AKTA pure上对抗体进行纯化。 The density of 293F cells cultured in advance was adjusted to 5×10 ⁵ cells/ml, and a total of 50 ml of culture was used for the transfection of antibody expression plasmid. On the day of transfection, 25ug heavy chain mutant expression plasmid and 25ug light chain expression plasmid were mixed with 150ul PEI (purchased from Polysciences, Inc. catalog number: 24885). After incubating for 20 minutes at room temperature, the mixed solution was added dropwise to the 293F cell culture, transfected and expressed in a cell culture incubator at 37 degrees Celsius for 4 days. After 4 days, the cell culture was centrifuged at 3000rpm for 20min, the supernatant was taken, and passed through a 0.45um filter membrane (Jinlong, item number: JKLM50-0.45S), based on a 1ml affinity chromatography column

Protein A (GE Healthcare, 17-0402-01) operating instructions were used to purify antibodies on GE's purification instrument AKTA pure.

Amino acid sequence of CD40 agonistic antibody heavy chain (with wild-type hIgG1 Fc region): (SEQ ID NO: 10)

Amino acid sequence of CD40 agonistic antibody light chain: (SEQ ID NO: 11)

Spread 5×10 ⁶ 293FTs in a 10cm ² dish in advance, and cultivate them in 10ml DMEM medium for 24 hours. Incubate 16ug pCDH-FcγRIIB (the full-length FcγRIIB gene is inserted into the multiple cloning site of pCDH vector) plasmid and 80ul PEI in 1ml Opti-MEM (purchased from Thermo Fisher Scientific, catalog number: 31985088) for 10 min, and add dropwise to the cells. After 6 hours of transfection, change to fresh DMEM medium and continue to express for 24 hours. The transfected cells were named 293FT-FcγRIIB, and FcγRIIB was expressed on the cell surface.

293FT cells were co-transfected with pCDH-CD40 (CD40 expression plasmid), pMDLg/pRRE, pRSV-Rev, and pCMV-VSV-G, and the lentivirus was collected and infected with NF-κB/Jurkat/GFP cells (System Biosciences, catalog number TR850A-1 ), select monoclonal cells and construct the Jurkat-CD40-GFP cell line.

Plate 293FT-FcγR2B cells in a 48-well plate at a density of 1×10 ^{5 cells/well, add 1×10 5} Jurkat-CD40-GFP cell lines to each well, and incubate with different concentrations of antibodies for 24 hours, where The initial concentration is 3ug/ml, and it is diluted 3 times, and the final concentration is 0.0002ug/ml, with a total of 10 concentration gradients. After that, BD’s LSR Fortessa flow cytometer was used to detect the strength of GFP in Jurkat-CD40-GFP cells, and the MFI was calculated based on this to compare the degree of cell activation caused by each mutant and wild-type (wt). The result is shown in Figure 19A. The activating ability of 11-1 mutants after all combinations is stronger than that of wild-type 11-1. This is consistent with the higher FcγRIIB binding ability of these mutants in the previous SPR experiments.

From the non-linear regression curve (Figure 19A), calculate the EC50 value of the antibody, that is, the half activation concentration (obtained by GraphPad software). The EC50 of each Fc variant is listed in Table 10 below.

Table 10: Activation ability of CD40 antibodies containing Fc variants

mutant EC50(nM) 11-1 KEF-2F-3 0.1176 11-1 KEF-2F-10 0.08924 11-1 KEF-2V-3 0.09519 11-1 VAA-2F-3 0.1218 11-1 VAA-2F-10 0.2210 11-1 VAA-2V-3 0.1976 11-1 WT 5.462 11-1 2F-10 0.7209

Compared with wild-type Fc, the Fc variants obtained by screening have achieved an approximately 27-fold or 50-fold improvement in EC50 value. Moreover, the combined mutations in the mutation regions 2 and 4 further improved the activation effect of the antibody relative to the mutation in the single mutation region. For example, compared with 11-1 2F-10, 11-1 KEF-2F-10 and 11-1 VAA-2F-10 both show a lower EC50, indicating that it has a stronger activation effect. This indicates that KEF mutation and VAA mutation can enhance the ability to activate cell lines on the basis of the original mutation 2F-10, which is consistent with the higher FcγRIIB binding ability of these combination variants in SPR experiments.

Example 14 Functional analysis of FcγRIIB binding to Fc variants

In this example, the effect of the above-mentioned combined mutations in another agonistic antibody was tested. The Fc combination mutants VAA-2F-10, VAA-2F-3, VAA-2V-3, KEF-2F-10, KEF-2F-3, KEF-2V-3 and mutant 2F-10 of Example 12 were introduced into the wild Type hIgG1 Fc region (SEQ ID NO:1, consisting of hinge region and CH2 and CH3) sequence. The obtained variant Fc region was connected to the VH-CH1 segment of 4-1BB agonistic antibody (Utomilumab). The expression and purification method is as in Example 13.

293FT cells were co-transfected with pCDH-4-1BB (4-1BB expression plasmid), pMDLg/pRRE, pRSV-Rev, and pCMV-VSV-G, and the lentivirus was collected and infected with NF-κB/Jurkat/GFP cells (System Biosciences, Catalog No. TR850A-1), select monoclonal cells and construct the Jurkat-4-1BB-GFP cell line.

The 293FT-FcγR2B cells constructed as in Example 13 were plated in a 48-well plate at a density of 1×10 ^{5 cells/well, and 1×10 5} Jurkat-4-1BB-GFP cell lines were added to each well, and combined with 6.5 ug/ml antibody was incubated for 24h. Use BD’s LSR Fortessa flow cytometer to detect the strength of GFP in Jurkat-4-1BB-GFP cells, calculate the MFI based on this, and make a histogram to compare the degree of cell activation caused by each mutant and wild-type (wt) . The results show that all variants MFI are stronger than WT, as shown in Figure 19B.

Utomilumab agonistic antibody heavy chain amino acid sequence (with wild-type hIgG1 Fc region): (SEQ ID NO: 12)

Amino acid sequence of Utomilumab agonistic antibody light chain: (SEQ ID NO: 13)

Example 15 Functional analysis of FcγRIIB binding to Fc variants

The Fc variant VAA-2F-3 containing the combined mutation of mutation regions 2 and 4 in Example 12 is further combined with the dominant residue G236D or P238D in the mutation region 1 determined by the positive screening of FcγRIIB in Example 10 to form Combine mutations as follows:

G236D+V266I+S267E+K326V+L328A P238D+V266I+S267E+K326V+L328A

According to the method described in Example 13, these combined mutations were introduced into the CD40 agonist type antibody, and the cell activation effect mediated by it was examined.

(1) In vitro activation of 293FT cells overexpressing FcγRIIB, FcγRIIA (R131), and FcγRIIA (H131)

According to the method described in Example 13, 293FT cells overexpressing FcγRIIB, FcγRIIA (R131), and FcγRIIA (H131) were constructed, and the Jurkat-CD40-GFP cell line was used to detect the combination of mutant-introduced antibodies and wild-type antibodies in these 293FT cells. Mediated cell activation. The results are shown in Figure 25A and the table below.

Table 11: Cell activation effect mediated by CD40 antibody containing Fc variant polypeptide vs. wild-type Fc polypeptide

(2) Activation of human B cells in vitro

CD19 MicroBeads from Miltenyi Biotec were used to isolate B cells from healthy human peripheral blood mononuclear cells (PBMC). Resuspend 1×10 ⁵ B cells in 100μl RPMI medium and add them to a 96-well plate, then add 10-fold dilutions of CD40 antibody, and control antibody (Ctrl IgG, isotype irrelevant antibody) at the highest concentration. That is 100μg/ml. After 48 hours of stimulation, the cells were resuspended in FACS Buffer containing 5% serum and incubated at 4 degrees Celsius for 30 minutes. After centrifugation at 200g for 5 minutes, the cells were washed with 500ul FACS buffer, and the washing step was repeated 3 times. Then add anti-CD19-PE (1:200 dilution, purchased from BioLegend) and anti-CD86-APC (1:200 dilution, purchased from BioLegend) or anti-HLA-DR-FITC (1:200 dilution, purchased from BioLegend) FACS Buffer containing 5% serum, incubate at 4 degrees Celsius for 20 minutes. Centrifuge at 200g for 5min, wash three times with 500ul FACS buffer, and resuspend in 500ul FACS Buffer. Use BD's LSR Fortessa flow cytometer to detect CD86 or HLA-DR upregulated on the cell surface due to activation.

The results are shown in Figure 25B. Compared with the wild-type Fc polypeptide, the combined Fc variants increased the activation of B cells in vitro to varying degrees.

(3) Immune activation effect in vivo

Perform OVA-specific CD8+T cell activation experiments to detect the in vivo immune activation effects of CD40 antibodies with different Fc regions.

One day in advance, 2×10 ^{6 CD45.1 +} splenic OT-I cells were injected into FcγR/CD40 humanized mice through the tail vein. The OT-I cells were derived from OT-I mice (The Jackson Laboratory, catalog number 003831). ) The spleen was obtained after splitting red. One day later, 2 μg of DEC-OVA protein and 10 μg of control antibody (Ctrl IgG, an isotype irrelevant antibody) or CD40 antibody were injected intraperitoneally. On day 6, the mouse spleen was taken out and ground into single cells. After lysing the red blood cells, use anti-CD4 (ebioscience, clone RM4-5), anti-CD8 (Biolegend, clone 53-6.7), anti-CD45.1 (Biolegend, clone A20), anti-TCR-Vα2 (Biolegend, The clone B20.1) antibody stains OVA-specific CD8+T (ie CD45.1 ⁺ splenic OT-I) cells. DAPI (Invitrogen, D3571) was added to remove dead cells when the gate was closed. OVA-specific CD8 ⁺ T cells are defined as CD45.1 ⁺ CD8 ⁺ TCR-Vα2 ⁺ cells. ^{Detect and count the number of CD45.1 +} splenic OT-I cells and CD8 ⁺ T cells using the LSR Fortessa flow cytometer from BD.

The results show that in Figure 25C, the CD40 antibodies with different Fc region mutations showed different immune activation effects in vivo. Among them ^{, the Fc combination mutation P238D+V266I with the largest FcγRIIb/FcγRIIa R131} binding capacity ratio (Table 11, EC50 ratio=0.06) +S267E+K326V+L328A performed best.

Example 16 Use of CHO cells with proteofucosylation defects to display Fc polypeptides

In this example, the application of fucosyl-deficient mammalian cells as an Fc polypeptide display platform was studied.

Basically in accordance with Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87:614 (2004) 614-622, CHO cells produced α-1,6-fucosyltransferase gene FUT8 knockout, named It is clone9 cell.

According to the methods described in Examples 1.1 and 1.2, construct and express wild-type Fc protein (Fc region with SEQ ID NO:1) and Fc variant protein (with mutation combination 2E-4-16 (H268E/K326S/I332E) and 2E -4-25 (H268E/K326M/I332E)) pCDH2 plasmid, and transfect CHO-K1 cells or clone9 cells. After that, as described in Example 1.3, the cells were stained with biotinylated FcγRIIIA, streptavidin-PE and anti-FLAG-FITC, and the PE fluorescent dyes and PE fluorescent dyes carried on the cells were detected by the LSR Fortessa flow cytometer of BD. Fluorescence of FITC fluorescent dyes. The result is shown in Figure 26.

As shown in Figure 26, defucose-cell CHO is used as a display platform. Compared with the control CHO-K1 cell display platform, the binding fluorescence signal of the cell surface receptor FcγRIIIA of the wild-type and variant Fc polypeptides is enhanced, which is beneficial Inspection of fluorescence sorting signal.

Moreover, as shown in Figure 26, the combination of defucose and the effective FcγRIIIA enhanced variants 2E-4-16 and 2E-4-25 showed a synergistic increase in the binding of fluorescent signals to receptors displayed on the cell surface. Efficacious. Compared with the CHO-K1 display platform, the defucose cell display platform is more fluorescent sorting enrichment of FcγRIIIA enhanced variants and subsequent acquisition of effective variants.

Some embodiments involved in the present invention are as follows:

1. A method for screening and/or identifying Fc partners, preferably Fc variants with altered Fc receptor binding properties, wherein a mammalian cell library displaying Fc polypeptides on the cell surface is used.

2. The method of the foregoing embodiment, wherein the cell library is a plurality of cell clones expressing diverse Fc variant sequences, preferably, based on the Fc and FcγR complex structure, the Fc region for introducing sequence diversity is selected.

3. The method of the preceding embodiment, wherein the cell library contains at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , or 10 ⁷ cell clones.

4. The method of the foregoing embodiment, wherein the Fc polypeptide is a fusion protein comprising an Fc region, which comprises from N-terminus to C-section:

Signal sequence; tag sequence; Fc region; transmembrane domain, wherein each part is optionally connected by a linker.

5. The method of the preceding embodiment, wherein the Fc region comprises a CH2 region and a CH3 region, and optionally a hinge region.

6. The method of the foregoing embodiment, wherein the Fc polypeptide may further comprise a CH1 region, which is connected in front of the CH2 region by a hinge region.

7. The method of the preceding embodiment, wherein the screening and/or identification comprises:

-Contact the labeled Fc receptor with the cell library, thereby allowing the labeled Fc receptor to bind to the Fc polypeptide displayed by the cell;

-Detect the amount of Fc receptors bound to the cell surface;

-Based on the binding amount, cell clones are selected from the library.

8. The method of embodiment 7, wherein the screening and/or identification further comprises the step of determining the display level of the Fc polypeptide on the cell clone, preferably comprising:

-Contact the labeled tag sequence binding molecule with the cell library, thereby allowing the tag sequence binding molecule to bind to the Fc polypeptide displayed by the cell;

-Detect the amount of tag sequence binding molecules bound to the cell surface to determine the display level of the Fc polypeptide on the cell;

Preferably, wherein said screening and/or identification further comprises the following steps:

-Based on the display level, standardize the measured Fc receptor binding amount on the cell,

-Based on the standardized binding amount, cell clones are selected from the library to enrich or remove Fc polypeptides with high affinity to the Fc receptor.

9. The method of embodiment 7, wherein the Fc receptor is biotinylated, and the receptor is labeled with a labeled avidin molecule.

10. The method of embodiment 8, wherein the cell library is contacted with the Fc receptor and the tag sequence binding molecule at the same time.

11. The method of embodiment 8, wherein the display level of the Fc polypeptide on the cell and the Fc receptor binding ability of the Fc polypeptide are detected at the same time.

12. The method of embodiment 7-11, wherein the label is a fluorescent label, wherein the fluorescent dye used to label the tag sequence binding molecule and the fluorescent dye used to label the Fc receptor have different wavelengths.

13. The method of embodiment 8, wherein based on the standardized binding amount, the top 5%, or the top 1%, or the top 0.5% of the cell clone members with the highest binding amount in the library are selected; or based on the standardized binding For the binding amount, select the cell clone members with the lowest binding amount in the top 5%, or the top 1%, or the top 0.5% in the library.

14. The method of embodiment 1-13, wherein flow cytometric sorting is used for selection of cell clone members.

15. The method of embodiment 1-14, wherein the Fc receptor is one or more selected from the group consisting of FcyRIIIA F158/V158, FcyRIIA R131/H131 and FcyRIIB.

16. The method of embodiment 1-15, wherein the selection is a forward selection to enrich cell clones whose displayed Fc polypeptide has a relatively high affinity for Fc receptors.

17. The method of embodiment 1-15, wherein the selection is negative selection to select and remove cell clones whose displayed Fc polypeptide has a relatively high affinity for Fc receptors.

18. The method of embodiment 1-16, wherein after positive selection is performed for one or more Fc receptors, negative selection is performed for another or more Fc receptors,

19. The method of embodiment 18, wherein the screening/identification comprises: positive selection of the library using FcγRIIB, collecting cell clone populations showing relatively high binding of FcγRIIB, and negative selection using FcγRIIIA on the collected cell population .

20. The method of embodiment 1, wherein the Fc variant polypeptide-encoding nucleic acid in the cell clone population obtained by screening/identification is subjected to deep sequencing, and the Fc variant polypeptide-encoding nucleic acid is selected based on the sequencing result.

21. The method of embodiment 20, wherein second-generation sequencing, preferably a second-generation sequencing method based on bridge PCR, is used for deep sequencing, such as the HiSeq ^TM sequencing platform.

22. The method of embodiment 20, wherein the sequence obtained by deep sequencing is transformed into the encoded amino acid sequence, the frequency of occurrence of each amino acid sequence is counted, and sequence ranking is performed based on the frequency, thereby determining the dominant amino acid sequence;

Wherein, optionally, the multiple sequence alignment of the amino acid sequence is further used to determine one or more dominant amino acid residues at the specified amino acid position,

Preferably, the dominant amino acid residues are determined by using WebLogo mapping;

Preferably, the dominant amino acid residue is the first 1-20, the first 1-15, or the first 1-10 amino acids with the highest frequency in a designated amino acid position in the sequence reads obtained by deep sequencing, for example, the 1-9, first 1-7, first 1-6, preferably first 1-5, first 1-4, first 1-3, more preferably first 1-2 amino acids;

Preferably, the deep sequencing result of the sorted cell population is compared with the deep sequencing result of the reference cell population to determine the dominant amino acid residues. Preferably, the reference cell population may be, for example, the library cell population before screening, or In the case of multiple rounds of screening, a cell population collected after one or more rounds of screening.

23. The method of embodiment 22, wherein the Fc variant polypeptide-encoding nucleic acid is selected based on the ranking of sequence types and/or the presence or absence of dominant amino acids.

24. A method of producing an Fc variant polypeptide, comprising:

-Construction of an Fc variant polypeptide display library, in which a plurality of Fc variant encoding nucleic acid sequences are introduced into mammalian cells and expressed to form the display library, and each mammalian cell clone in the Chinese library displays the Fc variant on its surface Peptide

-Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the FcγR receptor;

-Perform deep sequencing of the nucleic acid encoding the Fc variant polypeptide on the selected cell clone population with the binding properties;

-Based on the results of deep sequencing, Fc variant polypeptides are selected.

25. A method for identifying dominant amino acid positions in the Fc region, comprising:

-Construction of an Fc variant polypeptide display library, in which a plurality of Fc variant encoding nucleic acid sequences are introduced into mammalian cells and expressed to form the display library, and each mammalian cell clone in the Chinese library displays the Fc variant on its surface Peptide

-Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the FcγR receptor;

-Perform deep sequencing of the nucleic acid encoding the Fc variant polypeptide on the selected cell clone population with the binding properties;

-Based on the results of deep sequencing, determine the amino acid positions in the Fc polypeptide sequence that facilitate the introduction of mutations to change the FcγR binding properties of the Fc polypeptide.

26. A method for identifying dominant mutation regions or dominant amino acid sequences in the Fc region, comprising:

-Construction of an Fc variant polypeptide display library, in which a plurality of Fc variant encoding nucleic acid sequences are introduced into mammalian cells and expressed to form the display library, and each mammalian cell clone in the Chinese library displays the Fc variant on its surface Peptide

-Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the FcγR receptor;

-Perform deep sequencing of the nucleic acid encoding the Fc variant polypeptide on the selected cell clone population with the binding properties;

-Based on the results of deep sequencing, determine the advantageous mutation regions in the Fc polypeptide sequence that are conducive to introducing mutations to change the FcγR binding properties of the Fc polypeptide, wherein the determination optionally includes comparison with a reference cell clone population, such as Unsorted display library.

27. The method of embodiments 25 and 26, wherein the sequence of the cell clone population obtained by deep sequencing is sorted according to the amino acid sequence it encodes, and/or the dominant amino acid at the specified amino acid position is determined, preferably Dominant amino acids are the first 1-20, 1-15, or 1-10 amino acids with the highest frequency in a designated amino acid position on the sequence obtained after deep sequencing of the selected cell clone population, for example, 1-9, first 1-7, first 1-6, preferably first 1-5, first 1-4, first 1-3, more preferably first 1-2 amino acids.

28. The method of embodiment 27, wherein the dominant amino acid and/or the dominant mutation region in the Fc variant polypeptide are selected based on the ranking of the sequence types and/or the presence or absence of dominant amino acids.

29. The method of embodiment 1-28, wherein mutations are introduced into one or more of the following four regions of the IgG1 Fc region, preferably mutations are introduced into one or more of the mutation regions 2-4 to produce The plurality of Fc variant polynucleotides constructed by the library:

-Mutation region 1: residues 233-238,

-Mutation region 2: residues 265-271,

-Mutation region 3: residues 295-300 and

-Mutation region 4: residues 326-332.

30. The method of embodiment 29, wherein, relative to wild-type Fc, an Fc variant with enhanced FcγRIIIA receptor binding ability is selected, preferably the Fc variant has a selected from the following:

-Basically unchanged or reduced FcγRIIB binding capacity;

-Increased ratio of FcγRIIIA/FcγRIIB receptor binding capacity;

-Simultaneously increased FcγRIIIA-F158 and V158 binding capacity.

31. The method of embodiment 29, wherein relative to wild-type Fc, an Fc variant with enhanced FcγRIIB binding ability, preferably unchanged or reduced FcγRIIIA or FcγRIIA receptor binding ability is selected, wherein said Fc variant more preferably has Increased ratio of FcγRIIB to FcγRIIA or FcγRIIIA binding ability, especially FcγRIIB to FcγRIIIA-R131 binding ability ratio.

32. Fc variant polypeptides with altered FcγR binding properties produced by the method of embodiments 1-31.

33. An Fc variant polypeptide comprising a mutation selected from the following in the IgG Fc sequence, preferably in the sequence of SEQ ID NO:1:

S267A, H268E, H268G, D270E, Q295C, Q295L, Y296W, I332E, I332D;

K326M/S/I/T+I332E/D; A330T/G/V/Y+I332E/D; K326T+A330M; H268E/D; H268E/D+S267A; H268G+D270E; Q295L+Y296W;

Wherein, the Fc variant has at least 95% sequence identity with SEQ ID NO:1, and the residue positions are positions numbered according to EU.

34. The Fc variant polypeptide according to embodiment 33, wherein the mutation is selected from:

K326M+I332E A330T+I332E K326S+I332E A330G+I332D K326I+I332E A330V+I332D L328T+I332E A330Y+I332D K326T+A330M H268G+D270E H268E S267A+H268E To I332D Q295C Q295L+Y296W

35. The Fc variant of embodiment 34, comprising mutation: H268E,

And contains a mutation selected from the following:

K326M+I332E, K326S+I332E, K326I+I332E, L328T+I332E, A330T+I332E, A330G+I332D, A330V+I332D, A330Y+I332D, K326T+A330M.

36. The Fc variant of embodiment 34, comprising mutation: H268E,

And contains a mutation selected from the following:

K326M+I332E, K326S+I332E, K326I+I332E, A330T+I332E, A330Y+I332D,

Preferably, the Fc variant causes increased ADCC activity relative to a wild-type Fc polypeptide.

37. An Fc variant polypeptide comprising a mutation selected from the following in the IgG Fc sequence, preferably in the sequence of SEQ ID NO:1:

P331N+I332M S267E+E269G K326V+L328A V266I+S267E A330R+I332D S267E+H268D L328F+A330S V266L+S267Q+H268Q A327E+L328F E269P K326D+P331S G236N+P238V A327E+L328W Y296S+Y300K K326P+L328F To

More preferably, it contains a mutation selected from:

K326V+L328A S267E+E269G A327E+L328F V266I+S267E S267E+H268D To

Most preferably contains a mutation selected from: K326V+L328A, A327E+L328F, S267E+H268D,

Wherein, the Fc variant has at least 95% sequence identity with SEQ ID NO:1, and the residue position is the position numbered according to EU.

38. The Fc variant polypeptide of embodiment 36, which comprises a combination mutation selected from:

Among them, the antibody comprising the Fc variant preferably has an enhanced cell activation effect compared to the antibody comprising the wild-type Fc variant.

39. A fusion protein comprising the Fc variant sequence of embodiments 32-38, preferably the fusion protein is an antibody.

40. A composition comprising the fusion protein of embodiment 39, such as an antibody.

41. A method for producing Fc variants, the method comprising the steps:

(a) Constructing a mammalian cell Fc polypeptide display system;

(b) Sorting (especially sorting by flow cytometry) to display library members;

(c) Perform deep sequencing and cluster analysis on the library members obtained by sorting to obtain Fc variants with altered binding properties relative to the reference Fc polypeptide; and optionally

(d) The binding properties of the obtained Fc variants are tested.

42. The method of embodiment 41, wherein the construction of the display system in step (a) includes:

Provide a library of mammalian cells displaying modified Fc region polypeptides on the cell surface, wherein the cell library comprises at least two kinds of mammalian cells, wherein each mammal comprises a different modified Fc region polypeptide;

Among them, a viral vector (preferably a lentiviral vector) is used to construct the mammalian cell display library,

Wherein the viral vector contains an expression cassette, and the expression cassette contains the 5'to 3'direction of the polypeptide translation

-Optionally, a nucleic acid encoding a secretory signal sequence, preferably the signal sequence is a signal peptide that can guide the Fc region polypeptide to be secreted out of the cell, for example, an IL-2 protein signal peptide;

-A nucleic acid encoding a tag sequence, preferably the tag sequence is an epitope tag sequence, such as HA epitope tag, Flag tag sequence, c-myc epitope tag;

-Nucleic acids encoding Fc region variant polypeptides, preferably IgG1, IgG2, IgG3, IgG4 Fc regions, more preferably human IgG1 Fc regions, most preferably human IgG1 Fc region polypeptide variants shown in SEQ ID No:1, for example Contains 1-10 mutations, such as 1, 2, 3, 4 or 5 mutations;

-A nucleic acid encoding a transmembrane domain; wherein the transmembrane domain anchors Fc on the cell membrane surface, preferably the transmembrane region is derived from a protein expressed on the surface of mammalian cells, such as the transmembrane region of the PDGFR protein, such as SEQ ID NO: 4 the transmembrane region sequence;

Preferably, the Fc region variant is connected to the tag sequence and the transmembrane domain through a linker;

Preferably, the expression cassette is under the control of a promoter, such as an inducible promoter;

Preferably, the diversity of the library is obtained by randomizing at least one codon of the nucleic acid encoding the Fc region polypeptide; preferably, before introducing the diversity, based on structural analysis, the mutation region to be introduced is selected.

43. The method according to embodiment 41, wherein the sorting of display library members in step (b) comprises:

(b1) Using a mammalian cell Fc polypeptide display system, select a cell population from a mammalian cell library according to the characteristics of the Fc region polypeptide displayed on the cell surface;

(b2) Optionally, proliferate the sorted cells, and repeat the selection for the same binding properties or different binding properties;

Wherein, the options include:

-By using differently labeled (preferably fluorescently labeled) target Fc receptors and tag sequences to stain the Fc region variant polypeptides displayed on the cell surface;

-(Preferably by flow cytometric sorting), the cells are selected based on the staining intensity of the two markers on the cells.

44. The method according to embodiment 41, wherein the deep sequencing and cluster analysis of step (c) include:

Perform deep sequencing, such as next-generation sequencing (NGS), on the cell population obtained after sorting step (b), and perform cluster analysis on the amino acid sequence obtained by sequencing to identify Fc variants with desired FcγR binding properties;

Preferably, the identification is performed in the following manner:

(c1) PCR amplify the mutation library enriched in the sorting step from the cell population, optionally in parallel with the non-enriched mutation library of step (a) as a control; preferably, the PCR amplification length is 50 Between -100bp, preferably 150bp;

(c2) Using second-generation sequencing, preferably bridge PCR, second-generation sequencing, to obtain the amino acid sequence of the mutation region of the mutation library;

(c3) Perform cluster analysis on the obtained sequencing results according to the type and frequency of the amino acid sequence of the mutation region;

(c4) The top 1-3% mutants with the highest frequency are selected. Preferably, the selected mutants contain dominant amino acid residues, wherein the dominant amino acid residues are prominently present in all amino acid sequences in the mutation library. The residues of the amino acid positions can be mapped by WebLogo, for example, to show the dominant amino acid residues of the mutation library.

45. A method for identifying a dominant amino acid sequence in an Fc region, wherein the dominant amino acid sequence is conducive to improving the binding properties of the Fc region to a target Fc receptor, the method comprising:

-Construction of an Fc variant polypeptide display library, in which a plurality of Fc variant encoding nucleic acid sequences are introduced into mammalian cells and expressed to form the display library, and each mammalian cell clone in the Chinese library displays the Fc variant on its surface Peptide

-Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the target Fc receptor;

-Perform deep sequencing of the nucleic acid encoding the Fc variant polypeptide on the selected cell clone population with the binding properties;

-Based on the results of deep sequencing, sort according to the type and frequency of the amino acid sequence of the mutation region,

Among them, the first 1-100, the first 1-90, the first 1-80, the first 1-70 or the first 1-60 mutant amino acid sequences with the highest frequency, preferably the first 1-50 and the first 1-40 , Or the first 1-30, more preferably the first 1-20 mutated amino acid sequence, more preferably the first 1-10, 1-9, 1-8, 1-7, 1-6 or 1-5 One, more preferably the first 1-4 mutant amino acid sequences, are identified as the dominant amino acid sequence of the Fc region;

Alternatively, the top 1-5%, 1-4%, top 1-3%, and top 1-2% of the mutant amino acid sequences with the highest frequency are identified as the dominant amino acid sequences of the Fc region.

Claims (21)

A method for screening Fc variant polypeptides with altered Fc receptor binding properties, wherein a mammalian cell library displaying Fc polypeptides on the cell surface is used, and the method includes: (a) Constructing a library of mammalian cells displaying Fc polypeptides, wherein a plurality of Fc variant polypeptide-encoding nucleic acid sequences containing mutations in one or more regions of the Fc region are introduced into mammalian cells and expressed to form the The display library, in which each mammalian cell clone in the Chinese library displays an Fc variant polypeptide on its surface; (b) Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the target Fc receptor by flow cytometry; (c) Perform deep sequencing and cluster analysis of the mutation region in the Fc variant polypeptide encoding nucleic acid on the cell population obtained by sorting, and select Fc variant polypeptides with altered Fc receptor binding properties; The cluster analysis includes: -Transform the sequence obtained by deep sequencing into the encoded amino acid sequence, and perform sequence sorting according to the frequency of each amino acid sequence, thereby determining the dominant amino acid sequence; optionally, further multiplying the converted amino acid sequence Sequence alignment to determine the dominant amino acid residues at one or more specified amino acid positions, -Selecting an Fc variant polypeptide comprising a dominant amino acid sequence, optionally, the Fc variant polypeptide further comprising one or more dominant amino acid residues. The method of claim 1, wherein the dominant amino acid sequence is identified based on the frequency ranking of the sequence, wherein the first 1-100 mutant amino acid sequences with the highest frequency are preferably the first 1-50 mutant amino acid sequences, more preferably the first 1- 10, the first 1-5 mutant amino acid sequences were identified as the dominant amino acid sequence. The method of claim 1, wherein the dominant amino acid residue is determined by using WebLogo mapping; preferably, the dominant amino acid residue is, in the WebLogo diagram, the most frequently occurring amino acid position at a specified amino acid position 1-10 kinds, or the first 1-5 kinds, or the first 1-3 kinds, or the first 1-2 kinds of amino acids. The method of claim 1, wherein the cluster analysis of step (c) further comprises: Compare the deep sequencing results of the sorted cell population with the deep sequencing results of the reference cell population, and show the changes of dominant amino acid residues to determine the Fc polypeptide sequence that facilitates the introduction of mutations to change the Fc receptor binding of the Fc polypeptide The position of the dominant amino acid of the nature, and/or the determination of preferred sequence motifs that are beneficial to alter the Fc receptor binding properties of the Fc polypeptide, Preferably, the reference cell population is an unsorted library cell population, or in the case of multiple rounds of sorting, a library cell population that has undergone one or more rounds of sorting. The method of claim 1, wherein the cluster analysis in step (c) further comprises: drawing a mutant accumulation curve to determine the degree of enrichment of the variants after sorting. The method of claim 1, wherein in step (a), one or more mutations are introduced into one or more regions of the Fc region of the Fc polypeptide to generate the plurality of Fc variant codes for library construction Nucleic acid sequence: Preferably, the Fc region is an IgG1 Fc region, and the introduction of mutations includes introducing one or more mutations in one or more of the following four regions, -Mutation region 1: residues 233-238, -Mutation region 2: residues 265-271, -Mutation region 3: residues 295-300, and -Mutation region 4: residues 326-332, Preferably, the Fc region mutations of different mutation regions selected in step (c) are combined to further improve the receptor binding properties of the Fc variant. The method of claim 1, wherein the Fc polypeptide displayed on the cell surface is a fusion protein comprising an Fc region, which (preferably from N-terminal to C-terminal) comprises: a signal sequence; a tag sequence; an Fc region; a transmembrane domain, Optionally, the Fc region is connected to the tag sequence and the transmembrane domain via a linker, Optionally, the tag sequence is an epitope tag sequence, such as HA epitope tag, Flag tag sequence, c-myc epitope tag; Further optionally, the transmembrane region is derived from a protein expressed on the surface of mammalian cells, such as the transmembrane region of the PDGFR protein. The method of claim 7, wherein, in step (b), the sorting comprises -Contact the first fluorescently labeled tag sequence binding molecule with the library cell clone to determine the display level of the Fc polypeptide on the cell clone; -Contact the second fluorescently labeled target Fc receptor with the library cell clone, and detect the amount of the target Fc receptor bound to the surface of the cell clone; -Based on the display level, standardize the measured Fc receptor binding amount on the cell, -Sorting cell clones from the library based on the standardized binding amount to enrich Fc polypeptides with altered Fc receptor binding properties relative to wild-type Fc polypeptides, Wherein, the first fluorescent dye used to label the tag sequence binding molecule and the second fluorescent dye used to label the Fc receptor have different wavelengths. The method of claim 1, wherein the method is used to select an Fc variant polypeptide having an increased first Fc receptor binding capacity and a substantially unchanged or weakened second Fc receptor binding capacity relative to a parent Fc polypeptide, Wherein the first and second Fc receptors are different. More preferably, the first Fc receptor is an inhibitory Fc receptor and the second Fc receptor is an agonistic Fc receptor, or the first Fc receptor is an agonistic Fc receptor. Body and the second Fc receptor is an inhibitory Fc receptor, Wherein, the method includes: -The first Fc receptor driven positive selection and the second Fc receptor driven negative selection are performed in the sorting combination of step (b), and -Optionally, compare the dominant amino acid residues between the cell population sorted by positive selection and the cell population sorted by negative selection, Preferably, the sorting combination results in an increased enrichment in the sorted library of Fc variant polypeptides with dominant amino acids related to the binding properties of the first Fc receptor. The method of claim 1, wherein the method is used to select an Fc variant polypeptide having the following changes in binding properties relative to a parent Fc polypeptide: -Have enhanced FcγRIIB binding ability; or -Having an increased ratio of FcγRIIB to FcγRIIA or FcγRIIIA binding ability, especially FcγRIIB to FcγRIIA ^R131 binding ability ratio. The method of claim 1, wherein the method is used to select an Fc variant polypeptide having the following changes in binding properties relative to a parent Fc polypeptide: -Simultaneously enhanced FcγRIIIA ^F158 and FcγRIIIA ^V158 binding ability; or -Increased FcγRIIIA binding capacity, but basically unchanged or reduced FcγRIIB binding capacity; or -Increased ratio of FcγRIIIA/FcγRIIB receptor binding capacity. The method of claim 10, wherein the sorting step (b) comprises: -Use FcγRIIB to contact the library for positive selection, -Use FcγRIIIA or FcγRIIA to contact the library for negative selection, -Sorting Fc variant polypeptides with the altered binding properties relative to wild-type Fc polypeptides. The method of claim 11, wherein, in the sorting step (b), FcγRIIIA ^F158 and FcγRIIIA ^{V158 are used to} contact the library for forward selection, and the Fc variant polypeptides having the binding properties changed relative to the wild-type Fc polypeptide are sorted . The method of claim 1, wherein the mammalian cell is a cell having a defect in protein fucosylation, such as a FUT8 knockout CHO cell. A method for identifying a dominant amino acid sequence in an Fc region, wherein the dominant amino acid sequence is beneficial to improve the binding properties of the Fc region to a target Fc receptor, and the method includes: (a) Constructing a mammalian cell library displaying Fc variant polypeptides, wherein a plurality of Fc variant polypeptide-encoding nucleic acid sequences are introduced into mammalian cells and expressed to form the display library, and each mammalian cell in the Chinese library is cloned in The Fc variant polypeptide is displayed on its surface; (b) Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the target Fc receptor; (c) Perform deep sequencing of the nucleic acid encoding the Fc variant polypeptide on the cell clone population obtained by sorting; (d) Based on the results of deep sequencing, sort the occurrence frequency according to the type of amino acid sequence of the mutation region, Among them, the first 1-100 mutant amino acid sequences with the highest frequency, preferably the first 1-20 mutant amino acid sequences, more preferably the first 1-10, and more preferably the first 1-4 mutant amino acid sequences, are identified as the dominant amino acids in the Fc region sequence. A method for identifying advantageous amino acid positions and/or amino acid types in the Fc polypeptide sequence that facilitate the introduction of mutations to change the Fc receptor binding properties of the Fc polypeptide, and/or for determining the Fc receptor binding properties of the Fc polypeptide The preferred sequence motif method, the method comprises: (a) Constructing a mammalian cell library displaying Fc variant polypeptides, wherein a plurality of Fc variant polypeptide-encoding nucleic acid sequences are introduced into mammalian cells and expressed to form the display library, and each mammalian cell in the Chinese library is cloned in The Fc variant polypeptide is displayed on its surface; (b) Sorting cell clones in the library based on the binding properties of the Fc variant polypeptide on the cell surface and the target Fc receptor; (c) Perform deep sequencing of the nucleic acid encoding the Fc variant polypeptide on the cell clone population obtained by sorting; (d) Perform cluster analysis based on deep sequencing results, The cluster analysis includes: Compare the deep sequencing results of the sorted cell population with the deep sequencing results of the reference cell population, and show the changes of dominant amino acid residues to determine the Fc polypeptide sequence that facilitates the introduction of mutations to change the Fc receptor binding of the Fc polypeptide The amino acid position and/or amino acid type of the nature, and/or determine the preferred sequence motifs that are beneficial to alter the Fc receptor binding properties of the Fc polypeptide, Preferably, the reference cell population is an unsorted library cell population, or in the case of multiple rounds of sorting, a library cell population that has undergone one or more rounds of sorting. An Fc variant polypeptide, which in the IgG Fc sequence, preferably in the sequence of SEQ ID NO:1, contains mutations selected from the following: K326M/S/I/T+I332E/D; A330T/G/V/Y+I332E/D; K326T+A330M; H268E; H268E/D+S267A; H268G+D270E; K326M/S/I/T+I332E/ D; A330T/G/V/Y+I332E/D; K326T+A330M; H268E/D; H268E/D+S267A; H268G+D270E; I332D; Q295C; Q295L+Y296W; L328T+I332E; Wherein, the Fc variant has at least 95% sequence identity with SEQ ID NO:1, wherein the residue position is the position numbered according to EU, Preferably, the Fc variant includes a mutation selected from the following in the sequence of SEQ ID NO:1: P331N+I332M S267E+E269G K326V+L328A V266I+S267E A330R+I332D S267E+H268D L328F+A330S V266L+S267Q+H268Q A327E+L328F E269P K326D+P331S G236N+P238V A327E+L328W Y296S+Y300K K326P+L328F To More preferably, it contains a mutation selected from: K326V+L328A S267E+E269G A327E+L328F V266I+S267E S267E+H268D To Most preferably comprise a combination of mutations selected from: S267E+E269G+K326V+L328A V266I+S267E+K326V+L328A S267E+H268D+K326V+L328A S267E+E269G+A327E+L328F V266I+S267E+A327E+L328F S267E+H268D+A327E+L328F G236D+V266I+S267E+K326V+L328A P238D+V266I+S267E+K326V+L328A Wherein, the Fc variant has at least 95% sequence identity with SEQ ID NO:1, and the residue positions are positions numbered according to EU. The Fc variant polypeptide of claim 17, which is in an afucosylated form or a hypofucosylated form. A nucleic acid molecule encoding an Fc variant polypeptide comprising claim 17 or 18. A host cell comprising the nucleic acid molecule of claim 17 or 18. A composition comprising the Fc variant polypeptide of claim 17, wherein the Fc variant polypeptide is in the form of an antibody or immune fusion.

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