JP2008154468A - Human cd16 transgenic non-human animal and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To evaluate biological activities and pharmacological activities of antibody drugs by using non-human animal models. <P>SOLUTION: The non-human animal in which the genome is modified so that human CD16 and a human accessory molecule thereof are co-expressed is obtained; consequently, the non-human animal or an offspring thereof co-expressing with the human CD16 and the human accessory molecule thereof is provided, and a method for utilizing the non-human animal or the offspring is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule and use thereof.

Recent advances in antibody engineering have made it possible to produce recombinant antibodies that have low antigenicity to humans and are clinically applicable. Specifically, development of a humanized antibody production technique such as a human chimeric antibody or a humanized antibody (also referred to as a human complementarity determining region (CDR) transplanted antibody) (see Non-Patent Documents 1-5) In addition, the development of fully human antibody production technology using human antibody-producing transgenic mice and phage libraries (see Non-Patent Document 6-12) has made it possible to use monoclonal antibody therapy as a medicine. As a result, monoclonal antibody drugs were approved one after another from the latter half of 1990. Most antibodies developed as pharmaceuticals are human IgG having a molecular weight of about 150 kDa, and are glycoproteins in which two N-glycoside-linked complex sugar chains are bound to the Fc region. The Fc region is a region where Fc receptors and complements bind, and through this part, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) Effector activity is exhibited. In recent clinical results of antibody drugs, effects such as prolongation of life and prolongation of the pathological condition have been observed in many human malignant tumors such as breast cancer, colon cancer, and blood cancer (see Non-Patent Documents 13-16). . From the polymorphism analysis of Fc receptors in cancer patients, ADCC activity is one of the main anti-tumor mechanisms of anti-CD20 antibody Rituxan ^R (rituximab) for non-Hodgkin lymphoma and breast cancer drug anti-Her2 antibody Herceptin ^R (trastuzumab). It has been clarified (see Non-Patent Documents 17-21), and the development of ADCC activity enhancement technology applicable to pharmaceutical development has attracted attention as a next-generation antibody technology. Today, when the genome information of humans as antigens has been completely deciphered, antibody drugs have entered a fierce competition era as representatives of molecular target drugs (see Non-Patent Document 22). Currently, more than 100 clinical trials in the world It is said that more than 300 preclinical studies are being conducted.

  However, the clinical effects of current antibody drugs are not always sufficient, and there is a problem that the drug cost is high due to the large dose. One of the causes is that sufficient pharmacological evaluation of antibody drugs has not been performed in non-clinical experiments using disease model animals. This is because, due to the difference in species, the human antibody that is the test drug does not bind well with the Fc receptor of the disease model animal and it is difficult to sufficiently evaluate the effector activity, and the expression distribution of the Fc receptor is not necessarily consistent with humans. This is due to the fact that human effects cannot be assumed.

  In order to solve this problem, it is indispensable to create a disease model animal that reproduces the human immune system. As a model animal that expresses human CD16, the production of a transgenic mouse in which human CD16 is expressed on NK cells or macrophages using a human CD16 expression vector containing a human CD16 promoter of about 4.6 kb has been reported (non-native). (See Patent Document 23). However, there is no report on the effector activity evaluation or pharmacological evaluation of human antibodies using this transgenic mouse, and sufficient functional evaluation of human antibodies has not been successful. Then, using this transgenic mouse, backcrossing with C57BL / 6JNIcr mouse and C.B17 / Icr-scid mouse (see Non-Patent Document 24), or mating with CD16 knockout mouse or human CD20 expression mouse (patent) Reference 1) is performed.

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  In humans, highly differentiated immune systems have been constructed for biological defense, and antibody drugs using this immune system can be expected to have high pharmacological effects. Evaluation using an appropriate human disease model is indispensable for screening and selecting which human antibodies exhibit high pharmacological effects. Development of a non-human animal capable of reconstructing the human immune system across the barriers between species that can construct such a human disease model is desired from the viewpoint of effective antibody drug development. An object of the present invention is to provide a non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule, and a method for using the same.

The present invention relates to the following (1) to (16).
(1) A non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule.
(2) The non-human animal or progeny thereof according to (1), wherein the human accessory molecule of human CD16 is a molecule selected from the group consisting of the following (a) to (c).
(a) the human CD3ζ chain;
(b) the human Fcε receptor Iγ chain;
(c) Human CD3ζ chain and human Fcε receptor Iγ chain.
(3) The non-human animal or progeny thereof according to (1) or (2), wherein the genome is modified so that expression of human CD16 or a human accessory molecule thereof is controlled by a human CD16 promoter region.
(4) The non-human animal of any one of (1) to (3) or a human CD16, wherein the human CD16 is a protein encoded by a DNA selected from the group consisting of the following (a) to (d): progeny.
(a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b) DNA consisting of the base sequence represented by SEQ ID NO: 2;
(c) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having an activity of binding to the Fc region of a human antibody;
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 2 under a stringent condition and encodes a protein having an activity of binding to the Fc region of a human antibody.
(5) The non-human animal or progeny thereof according to any one of (1) to (3), wherein human CD16 is a protein selected from the group consisting of the following (a) to (f):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4;
(c) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 3 and having an activity of binding to the Fc region of a human antibody;
(d) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4 and having an activity of binding to the Fc region of a human antibody;
(e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 and having an activity of binding to the Fc region of a human antibody;
(f) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having an activity of binding to the Fc region of a human antibody.
(6) The non-human animal or progeny thereof according to any one of (2) to (5), wherein the human CD3ζ chain is a protein encoded by the following DNA (a) or (b):
(a) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(b) A DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a protein that associates with human CD16 and has an intracellular signal transduction function.
(7) The non-human animal or progeny thereof according to any one of (2) to (5), wherein the human CD3ζ chain is a protein selected from the group consisting of the following (a) to (c).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 6;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6, and which is associated with human CD16 and has an intracellular signal transduction function ;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6, and being associated with human CD16 and having an intracellular information transmission function.
(8) The non-human animal according to any one of (2) to (7), wherein the human Fcε receptor Iγ chain is a protein encoded by a DNA selected from the following (a) or (b): Its descendants.
(a) DNA consisting of the base sequence represented by SEQ ID NO: 7;
(b) A DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions and encodes a protein that associates with human CD16 and has an intracellular signal transduction function.
(9) The non-human animal according to any one of (2) to (7), wherein the human Fcε receptor Iγ chain is a protein selected from the group consisting of the following (a) to (c): progeny.
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 8, and is associated with human CD16 and has an intracellular signal transduction function ;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and being associated with human CD16 and having an intracellular information transmission function.
(10) The non-human animal or progeny thereof according to any one of (3) to (9), wherein the human CD16 promoter region is DNA selected from the following (a) or (b):
(a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b) hybridizes with DNA comprising the nucleotide sequence represented by SEQ ID NO: 9 under stringent conditions and expresses human CD16 or its human accessory molecule in natural killer cells, macrophages, monocytes or dendritic cells Active DNA;
(11) The nonhuman animal is an animal selected from the group consisting of cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys and rabbits, according to any one of (1) to (10) The described non-human animal or its progeny.
(12) A method for measuring antibody-dependent cytotoxic activity of an antibody, comprising the following steps (1) to (3).
(1) A step of isolating effector cells from the non-human animal or progeny thereof according to any one of (1) to (11);
(2) An isolated effector cell is mixed with an antibody and a cell expressing an antigen targeted by the antibody, and the effector cell, the antibody and the antigen targeted by the antibody are expressed in the reaction solution. Contacting with living cells;
(3) A step of quantifying the survival rate of cells expressing the target antigen in the reaction solution.
(13) A method for evaluating pharmacological activity based on antibody-dependent cytotoxic activity of an antibody, comprising the following steps (1) and (2).
(1) A step of administering an antibody as a test substance to the non-human animal according to any one of (1) to (11) or a descendant thereof;
(2) A step of evaluating the pharmacological activity based on the antibody-dependent cytotoxic activity of the antibody as the test substance in the non-human animal or its offspring.
(14) A method for screening an antibody, comprising the following steps (1) to (3).
(1) A step of administering an antibody as a test substance to the non-human animal according to any one of (1) to (11) or a descendant thereof;
(2) a step of evaluating pharmacological activity based on antibody-dependent cytotoxic activity of an antibody as a test substance in the non-human animal or its offspring;
(3) A step of selecting an antibody having the pharmacological activity.
(15) The method according to any one of (12) to (14), wherein the antibody is a protein selected from the group consisting of the following (a) to (e).
(a) a human antibody;
(b) a humanized antibody;
(c) Human chimeric antibody
(d) an antibody fragment comprising the Fc region of (a), (b) or (c);
(e) A fusion protein having the Fc region of (a), (b) or (c).
(16) The method according to (15), wherein the antibody class is IgG.

  INDUSTRIAL APPLICABILITY According to the present invention, there are provided a non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule, and a method for using the same. The non-human animal and its progeny of the present invention are useful for biological activity and pharmacological evaluation of human antibody drugs.

The non-human animal of the present invention or its progeny is any non-human animal or its progeny as long as it is a non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule. Is included.
In the present invention, human CD16 means a protein having an activity of binding to the Fc region of a human antibody. It is known that there is a genetic polymorphism in human CD16. Specifically, the amino acid residue at position 176 from the N-terminal methionine of the human CD16 signal sequence is phenylalanine (hereinafter referred to as human CD16 Phe). The existence of a protein and a protein that is valine (hereinafter referred to as human CD16 Val) is known. Therefore, in humans, there are three types of phenotypes: human CD16 Phe / human CD16 Phe or human CD16 Val / human CD16 Val homotype and human CD16 Phe / human CD16 Val heterotype. In the present invention, human CD16 encompasses any polymorphism.

As human CD16 in the present invention, specifically, a protein encoded by the following DNA (a), (b), (c) or (d), or the following (e), (f), (g), Examples include the protein (h), (i) or (j).
(a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b) DNA consisting of the base sequence represented by SEQ ID NO: 2;
(c) a DNA that hybridizes with a DNA comprising the base sequence represented by SEQ ID NO: 1 under a stringent condition and encodes a protein having an activity of binding to the Fc region of a human antibody;
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and encodes a protein having an activity of binding to the Fc region of a human antibody;
Or
(e) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(f) a protein comprising the amino acid sequence represented by SEQ ID NO: 4;
(g) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 3 and having an activity of binding to the Fc region of a human antibody;
(h) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4 and having an activity of binding to the Fc region of a human antibody;
(i) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 and having an activity of binding to the Fc region of a human antibody;
(j) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having an activity of binding to the Fc region of a human antibody.

  In the present invention, the DNA that hybridizes under stringent conditions is, for example, a colony hybridization method using, as a probe, DNA such as DNA having a base sequence represented by SEQ ID NO: 1 or 2, or a partial fragment thereof. , Means a DNA obtained by using a plaque hybridization method or a Southern blot hybridization method, and specifically, 0.7 to 1. After hybridization at 65 ° C. in the presence of 0 M sodium chloride, a 0.1 to 2 fold concentration SSC solution (composition of a 1 fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate) Use and wash the filter at 65 ° C It is possible to increase the DNA more can be identified. Hybridization is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning Second Edition), Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997 And abbreviated as Current Protocols in Molecular Biology), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995) and the like. Specifically, the DNA capable of hybridizing is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 1 or 2, preferably 70% or more, more preferably 80% or more, still more preferably A DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more can be mentioned.

  In the present invention, the amino acid sequence represented by SEQ ID NO: 3 or 4 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and has an activity of binding to the Fc region of a human antibody. Proteins include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982 ), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. For example, it means a protein that can be obtained by introducing a site-specific mutation into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 3 or 4. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. However, deletion, substitution or addition may be performed by a known technique such as the above-described site-directed mutagenesis. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

  In the present invention, a protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 or 4 and having an activity of binding to the Fc region of a human antibody is BLAST [J. Mol. Biol., 215, 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)], and the like, a protein having the amino acid sequence of SEQ ID NO: 3 or 4 The protein is at least 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.

  In the present invention, the accessory molecule of human CD16 means a protein capable of transmitting a signal from human CD16 bound with the Fc region of a human antibody into a cell. Any molecule can be used as long as it is a protein capable of transmitting a signal from human CD16 into a cell. Specific examples include human CD3ζ chain, human Fcε receptor Iγ chain, or a combination thereof. It is done.

Specific examples of the human CD3ζ chain in the present invention include a protein encoded by the following DNA (a) or (b), or a protein (c), (d) or (e) below.
(a) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(b) a DNA that hybridizes with a DNA comprising the base sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a protein that associates with human CD16 and has an intracellular signal transduction function;
Or
(c) a protein comprising the amino acid sequence represented by SEQ ID NO: 6;
(d) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6, and which is associated with human CD16 and has an intracellular signal transduction function ;
(e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6, and being associated with human CD16 and having an intracellular information transmission function.

Specific examples of the human Fcε receptor Iγ chain in the present invention include a protein encoded by the following DNA (a) or (b), or a protein (c), (d) or (e) below. .
(a) DNA consisting of the base sequence represented by SEQ ID NO: 7;
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions and encodes a protein that associates with human CD16 and has an intracellular signal transduction function;
Or
(c) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(d) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 8, and is associated with human CD16 and has an intracellular signal transduction function ;
(e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and being associated with human CD16 and having an intracellular information transmission function.

  In the present invention, the DNA that hybridizes under stringent conditions is, for example, a colony hybridization method using, as a probe, DNA such as DNA having a base sequence represented by SEQ ID NO: 5 or 7 or a fragment thereof. , Means a DNA obtained by using a plaque hybridization method or a Southern blot hybridization method, and specifically, 0.7 to 1. After hybridization at 65 ° C. in the presence of 0 M sodium chloride, a 0.1 to 2 fold concentration SSC solution (composition of a 1 fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate) Use and wash the filter at 65 ° C It is possible to increase the DNA more can be identified. Hybridization is performed according to the method described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be done according to this. Specifically, the hybridizable DNA is DNA having at least 60% or more homology with the base sequence represented by SEQ ID NO: 5 or 7, preferably 70% or more, more preferably 80% or more, still more preferably A DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more can be mentioned.

  In the present invention, the amino acid sequence represented by SEQ ID NO: 6 or 8 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and associates with human CD16 to function in intracellular signal transduction. The protein possessed by Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 ( 1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. For example, it means a protein that can be obtained by introducing a site-specific mutation into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 6 or 8. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. However, deletion, substitution or addition may be performed by a known technique such as the above-described site-directed mutagenesis. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

  In the present invention, a protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 or 8 and associated with human CD16 and having an intracellular signal transduction function is BLAST [J. Mol. Biol., 215, 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)], etc., and a protein having the amino acid sequence set forth in SEQ ID NO: 6 or 8 And at least 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.

The non-human animal or its progeny of the present invention includes a non-human animal or its progeny whose genome has been modified so that expression of human CD16 is controlled by the human CD16 promoter region.
Here, the genome has been modified so that the expression of human CD16 is controlled by the human CD16 promoter region. The gene that encodes human CD16 is under the control of the genomic region that controls the expression of the human CD16 gene on the human chromosome. It means that it modifies so that it may be located. Specifically, modification to be located under the control of the genomic region that controls the expression of the human CD16 gene specifically encodes human CD16 downstream of the genomic region that controls the expression of the human CD16 gene on the human chromosome An example of modification so that the gene is located is given. As a method for obtaining such a non-human animal or its progeny, any method can be used as long as the target genome can be modified. Specifically, a method for producing an individual by introducing a DNA to be subjected to a desired genetic modification into a fertilized egg or embryonic stem cell, a method for producing a cloned individual using the nucleus of a cell subjected to the intended genetic modification, etc. Can be given.

In the present invention, the human CD16 promoter region includes any region as long as it is a region involved in tissue-specific expression control of the human CD16 gene on the human chromosome. Human CD16 has been observed to be expressed in natural killer cells, macrophages, monocytes and dendritic cells [Nat. Rev. Immunol., 4 , 89 (2004); Nat. Rev. Immunol., 2 , 580 ( 2002)], the human CD16 promoter region has at least an activity to express a gene under control in these cells.

Specific examples of the human CD16 promoter region in the present invention include the following DNA (a), (b) or (c).
(a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 9 under stringent conditions and has an activity capable of expressing a gene under control in human natural killer cells;
(c) DNA having an activity capable of expressing a gene under the control of natural killer cells, macrophages, monocytes and dendritic cells.

In the present invention, the antibody includes any molecule as long as it contains the Fc region of the antibody, but a human-derived antibody is preferred. Specifically, human antibodies, humanized antibodies, human chimeric antibodies, antibody fragments containing the Fc region of those antibodies, and fusion proteins having the Fc region obtained by fusing these antibody Fc regions with other proteins Etc.
As human antibodies, in addition to antibodies secreted by hybridoma cells prepared from human spleen cells sensitized to antigen, human antibodies prepared by genetic recombination technology, that is, antibody expression vectors into which antibody genes are inserted, Examples thereof include human antibodies obtained by introduction into host cells.

Humanized antibodies Human chimeric antibodies are non-human animal antibody H chain V regions (hereinafter also referred to as “HV” or “VH”) and antibody L chain V regions (hereinafter also referred to as “LV” or “VL”). ) And the H chain C region of a human antibody (hereinafter also referred to as “CH”) and the L chain C region of a human antibody (hereinafter also referred to as “CL”). Any animal other than humans can be used as long as it can produce a hybridoma such as a mouse, rat, hamster, or rabbit.

  The human chimeric antibody had a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing mammals other than humans with myeloma cells derived from mice, rats, etc. CDNAs encoding VH and VL are obtained from hybridomas producing monoclonal antibodies and inserted into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively, to construct human chimeric antibody expression vectors And can be expressed and produced by introduction into a host cell.

  The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as “hIg”), but is preferably of the hIgG class, and more preferably hIgG1, hIgG2, Any of the subclasses such as hIgG3 and hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

The humanized antibody is also referred to as a human CDR-grafted antibody, and refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies at appropriate positions of the human antibody VH and VL.
Humanized antibody constructs cDNA encoding V region by grafting VH and VL CDR sequences of non-human animal antibodies to VH and VL CDR sequences of any human antibody, and human antibody CH and human antibody A humanized antibody expression vector can be constructed by inserting it into an expression vector for a host cell having a gene encoding CL, and the humanized antibody can be expressed and produced by introducing the expression vector into a host cell. .

The CH of the humanized antibody may be any as long as it belongs to hIg, but the hIgG class is preferable, and any of the subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class can be used. The CL of the humanized antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
A human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody transgenic produced by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Antibodies obtained from animals or human antibody transgenic plants are also included.

The antibody present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing and cloning EB virus and the like, and culturing lymphocytes producing the antibody. Can be purified.
The human antibody phage library is a library in which antibody fragments such as Fab and single chain antibody are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. From the library, phages expressing antibody fragments having a desired antigen-binding activity can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.

  A human antibody transgenic non-human animal refers to an animal in which a human antibody gene is incorporated into cells. Specifically, a transgenic non-human animal that produces human antibodies can be produced by introducing a human antibody gene into embryonic stem cells, transplanting the embryonic stem cells to other early embryos, and generating them. It is also possible to produce a transgenic non-human animal that produces a human antibody by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg. A method for producing a human antibody from a transgenic non-human animal that produces a human antibody is obtained by culturing a human antibody hybridoma by a hybridoma production method performed in a mammal other than a normal human and culturing the human antibody in the culture. Antibodies can be accumulated.

Examples of transgenic non-human animals include cows, sheep, goats, pigs, horses, mice, rats, chickens, monkeys, and rabbits.
Further, in the present invention, the antibody recognizes an antibody recognizing a tumor-related antigen, an antibody recognizing an antigen related to allergy or inflammation, an antibody recognizing an antigen related to cardiovascular disease, and an antigen related to an autoimmune disease. Or an antibody that recognizes an antigen associated with viral or bacterial infection, and the antibody class is preferably IgG.

The antibody fragment refers to a fragment containing the Fc region of the antibody. The antibody fragment may be any fragment containing the Fc region of the above antibody, and specific examples include H chain monomers and H chain dimers.
The fusion protein having an Fc region may be any substance as long as it is a substance obtained by fusing an antibody or antibody fragment containing an Fc region of an antibody with a protein such as an enzyme or cytokine.

Hereinafter, the production method and utilization method of the non-human animal and its progeny of the present invention will be described in detail.
The nonhuman animal or its progeny whose genome has been modified so that human CD16 and its human accessory molecule are co-expressed according to the present invention is 1) a method for producing a transgenic animal in which a gene of interest is introduced into an individual animal, 2) A method for producing a gene-replacement animal in which a target gene and a target transgene are exchanged. 3) A method for producing a clone individual using a nucleus of a cell subjected to a target gene modification. Can do. Each will be specifically described below.
1. Production of non-human animal of the present invention using a method for producing a transgenic animal in which a gene of interest is introduced into an animal individual (1) Acquisition of DNA encoding human CD16 As a DNA encoding human CD16, human CD16 CDNA and genomic DNA encoding can be mentioned. Genomic DNA may contain introns. The codons in the coding region are not limited to those in cDNA and genomic DNA, and any codon combination may be used as long as it is a codon encoding the amino acid sequence of human CD16. Examples of DNA encoding human CD16 include human CD16 cDNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or 2.

  A human CD16 cDNA sequence or a human CD16 gene genomic sequence is searched from the base sequence database, and the base sequence information is obtained. Examples of the genomic sequence of the human CD16 gene that can be obtained from the base sequence database include, for example, the base sequence registered with GenBank accession numbers NC_000001 and AL_590385, and the sequence of the human CD16 cDNA includes, for example, the base registered with GenBank accession number NM_000569. An array can be given.

  Based on the nucleotide sequence information of the obtained DNA encoding human CD16, a cDNA sequence or a genomic sequence region including the region encoding human CD16 is appropriately selected, and the 5 ′ end 20-40 bp sequence of this region At the 3 ′ end and a DNA containing a sequence complementary to the 3 ′ end 20-40 bp sequence of this region at the 3 ′ end. A genomic DNA encoding human CD16 can be isolated by PCR using human DNA as a template by PCR using human cDNA as a template and PCR using human genomic DNA as a template.

Human cDNA can be made from RNA prepared from human tissue or cells. Methods for preparing total RNA from tissues or cells include guanidine thiocyanate-cesium trifluoroacetate method (Methods in Enzymology, 154 , 3, 1987), acidic guanidine thiocyanate / phenol / chloroform method (Analytical Biochemistry, 162 , 156). , 1987; Experimental Medicine, 9 , 1937, 1991). Also, by using kits such as Absolute RNA RT-PCR Miniprep Kit (Absolutely RNA RT-PCR Miniprep Kit, Stratagene), RN Easy Mini Kit (RNeasy MIni Kit, Qiagen) Can be prepared. As a method of preparing mRNA as poly (A) ⁺ RNA from total RNA, oligo (dT) immobilized cellulose column method [Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001 (hereinafter referred to as Molecular Abbreviated as the third version of cloning))] and the like. Furthermore, by using kits such as FastTrack 2.0 mRNA Isolation Kit (FastTrack 2.0 mRNA Isolation Kit, manufactured by Invitrogen) and Quickprep mRNA Purification Kit (QuickPrep mRNA Purification Kit, manufactured by Amersham Biosciences) mRNA can be prepared. Methods for synthesizing cDNA from total RNA or mRNA include those described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, etc., or commercially available kits such as Superscript for RT-PCR. Single strand synthesis system (SUPERSCRIPT First-Strand Synthesis System for RT-PCR, manufactured by Invitrogen), Prostar first strand RT-PCR kit (ProSTAR First Strand RT-PCR Kit, manufactured by Stratagene), PCR-select cDNA subtraction Examples thereof include a method using a kit (PCR-Select cDNA Subtraction Kit, manufactured by Clontech). Moreover, cDNAs of various human tissues commercially available from Clontech and the like can also be used. Examples of the DNA encoding human CD16 thus obtained include DNA having the base sequence shown in SEQ ID NO: 1 or 2.

Human genomic DNA can be prepared from human tissues or cells by the method described in Molecular Cloning 3rd edition. Alternatively, it can be prepared by using a kit such as an easy DNA kit (Easy-DNA Kit, manufactured by Invitrogen), a DNA extraction kit (DNA Extraction Kit, manufactured by Stratagene) or the like. Human genomic DNA commercially available from Clontech and the like can also be used.
(2) Acquisition of DNA encoding human CD3ζ chain Examples of DNA encoding human CD3ζ chain include cDNA and genomic DNA encoding human CD3ζ chain. Genomic DNA may contain introns. The codons in the coding region are not limited to those in cDNA and genomic DNA, and any codon combination may be used as long as it is a codon encoding the amino acid sequence of human CD3ζ chain. As an example of DNA encoding human CD3ζ chain, cDNA of human CD3ζ chain consisting of the base sequence shown in SEQ ID NO: 5 can be mentioned.

  A human CD3ζ chain cDNA sequence or a human CD3ζ chain gene genomic sequence is searched from the base sequence database, and the base sequence information is obtained. The genomic sequence of the human CD3ζ chain gene that can be obtained from the base sequence database is, for example, the base sequence registered with GenBank registration number NC_000001, and the sequence of the human CD3ζ chain cDNA is registered, for example, with GenBank registration number NM_000734. The base sequence can be raised.

  Based on the nucleotide sequence information of the DNA encoding the obtained human CD3ζ chain, a cDNA sequence or a genomic sequence region including the region encoding the human CD3ζ chain is appropriately selected, and the 5 ′ end of this region is 20-40 bp. And a DNA containing a sequence complementary to the 3 'end 20-40 bp sequence of this region at the 3' end. Using these DNAs as primers, the human CD3ζ chain can be isolated by PCR using human cDNA as a template, and the genomic DNA encoding human CD3ζ chain can be isolated by PCR using human genomic DNA as a template .

Human cDNA and human genomic DNA are the above-mentioned 1. Can be prepared according to the method described in (1). As an example of the DNA encoding the human CD3ζ chain thus obtained, there can be mentioned DNA having the base sequence shown in SEQ ID NO: 5.
(3) Acquisition of DNA encoding human Fcε receptor Iγ chain Examples of DNA encoding human Fcε receptor Iγ chain include cDNA encoding human Fcε receptor Iγ chain and genomic DNA. Genomic DNA may contain introns. The codons in the coding region are not limited to those in cDNA and genomic DNA, and any codon combination may be used as long as it is a codon encoding the amino acid sequence of human Fcε receptor Iγ chain. As an example of DNA encoding the human Fcε receptor Iγ chain, cDNA of the human Fcε receptor Iγ chain consisting of the base sequence shown in SEQ ID NO: 7 can be mentioned.

  The sequence of the human Fcε receptor Iγ chain cDNA or the genome sequence of the human Fcε receptor Iγ chain gene is searched from the base sequence database, and the base sequence information is obtained. The genomic sequence of the human Fcε receptor Iγ chain gene that can be obtained from the base sequence database is, for example, the base sequence registered under GenBank accession number NC_000001, and the sequence of the human Fcε receptor Iγ chain cDNA is, for example, the GenBank accession number The base sequences registered in NM_004106 can be listed.

  Based on the nucleotide sequence information of the DNA encoding the obtained human Fcε receptor Iγ chain, a cDNA sequence or a genomic sequence region containing the region encoding the human Fcε receptor Iγ chain is appropriately selected, and A DNA containing a 5′-end 20-40 bp sequence at the 3 ′ end and a DNA containing a 3′-end 20-40 bp sequence complementary to this region at the 3 ′ end are prepared. Using these DNAs as primers, PCR encoding human Fcε receptor Iγ chain by PCR using human cDNA as a template, and genomic DNA encoding human Fcε receptor Iγ chain by PCR using human genomic DNA as a template It can be isolated.

Human cDNA and human genomic DNA are the above-mentioned 1. Can be prepared according to the method described in (1). As an example of the DNA encoding the human Fcε receptor Iγ chain thus obtained, a DNA having the base sequence shown in SEQ ID NO: 7 can be mentioned.
(4) Acquisition of human CD16 promoter region Examples of the human CD16 promoter region include genomic DNA involved in tissue-specific expression control of the human CD16 gene on the human chromosome. Specifically, genomic DNA in a region within 10 kb upstream from the start codon of the human CD16 gene having an activity responsible for tissue-specific expression control of the human CD16 gene can be mentioned. As an example of the human CD16 promoter region, a human CD16 promoter region consisting of the base sequence shown in SEQ ID NO: 9 can be mentioned.

  A human CD16 cDNA sequence or a human CD16 gene genomic sequence is searched from the base sequence database, and the base sequence information is obtained. Examples of the genomic sequence of the human CD16 gene that can be obtained from the base sequence database include, for example, the base sequence registered with GenBank accession numbers NC_000001 and AL_590385, and the sequence of the human CD16 cDNA includes, for example, the base registered with GenBank accession number NM_000569. An array can be given.

  Based on the nucleotide sequence information of the obtained DNA encoding human CD16, a cDNA sequence or a genomic sequence region including the region encoding human CD16 is appropriately selected, and the 5 ′ end 20-40 bp sequence of this region At the 3 ′ end and a DNA containing a sequence complementary to the 3 ′ end 20-40 bp sequence of this region at the 3 ′ end. By using these DNAs as primers and PCR using human genomic DNA as a template, genomic DNA in a region within 10 kb upstream from the start codon of the human CD16 gene can be amplified and isolated in units of several kb.

The human genomic DNA is the above-mentioned 1. Can be prepared according to the method described in (1). The human CD16 promoter region can also be isolated by using a genomic DNA library screening system (Genome Systems) or Universal Genome Walker ^™ Kits (CLONTECH). As an example of the human CD16 promoter region thus obtained, DNA having the base sequence shown in SEQ ID NO: 9 can be mentioned.
(5) Production of a transgene containing DNA encoding human CD16 and its human accessory molecule DNA encoding human CD16 obtained in (1) above, and 1. (2) and 1. When the DNA encoding the human accessory molecule of human CD16 obtained in (3) is introduced into a non-human animal, an expression vector in which the DNA is linked downstream of an appropriate promoter is preferably used as the transgene. . Specifically, an expression vector in which each of human CD16 and its human accessory molecule is linked downstream of an appropriate promoter may be used as a transgene, or human CD16 and its human accessory molecule may be used as an in-sequence ribosome entry site (internal An expression vector linked by a ribosome entry site (IRES) and linked downstream of one promoter may be used as a transgene.

  As the promoter used in the expression vector, any promoter can be used as long as it can initiate transcription in a non-human animal cell into which DNA encoding human CD16 and its human accessory molecule is introduced. For example, promoters of genes derived from viruses (cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, simian virus, retrovirus, etc.), various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) And avian (such as chicken) genes [eg, albumin, insulin II, erythropoietin, endothelin, osteocalcin, muscle creatine kinase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen types I and II, atrial natriuretic Factor, dopamine β-hydroxylase, endothelial receptor tyrosine kinase (Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 Tissue inhibitor (TIMP1), MHC class I antigen (H-2L), smooth muscle α-actin, polypeptide chain elongation factor 1α (EF-1α), β-actin, α and β myosin heavy chain, myosin light chain 1 and 2, Fusion of myelin basic protein, serum amyloid P component, myoglobin, renin, glycolytic enzymes such as phosphoglycerate kinase (PGK), genes such as heat shock proteins, etc., enhancer sequences and promoter sequences of the above genes Promoters [for example, the promoter of the early gene of simian virus 40 (SV40) and the SRα promoter consisting of a part of the long terminal repeat of human T cell leukemia virus 1, the immediate early (IE) gene enhancer of cytomegalovirus, CAG promoter consisting of chicken β-actin promoter And the like, but it is preferable to use a promoter with the same transcriptional regulatory activity and tissue-specific expression control of the human CD16 gene, it is particularly preferable to use its own promoter of the human CD16 gene. Specific examples thereof include the above-described 1. Examples include the human CD16 promoter region obtained in (4). By using a promoter showing human CD16 gene tissue-specific expression, it can be expressed in a specific tissue of a transgenic non-human animal into which DNA encoding human CD16 and its human accessory molecule has been introduced. In addition, the expression site, expression time or expression level of the introduced foreign gene can be controlled by combination with the Cre-loxP system. A DNA encoding a structural gene in the expression vector is inserted between two loxP sequences, and a transgenic non-human animal is produced by the method described later in (6) using the expression vector. Then, if the transgenic non-human animal is allowed to express Cre recombinase using an adenovirus vector, human CD16 and its human accessory molecule specifically at the site or time of infection with the adenovirus for expressing Cre recombinase Is deleted, and the expression of the transgene to be expressed can be suppressed. In addition to the expression vector for human CD16 and its human accessory molecule in the method described later in (6), for example, a Cre recombinase expression vector using a promoter exhibiting tissue-specific expression is introduced to introduce a transgenic non-human animal. As a result, a transgenic non-human animal in which the expression of human CD16 and its human accessory molecule is suppressed can be produced only in tissues where Cre recombinase is expressed.

  The expression vector preferably has a polyadenylation signal necessary for polyadenylation of the 3 ′ end of mRNA downstream of DNA encoding human CD16 and its human accessory molecule. Examples of polyadenylation signals include polyadenylation signals contained in the genes derived from the above viruses, various animals and birds, for example, late or early genes of SV40, rabbit β globin gene, bovine growth hormone gene, human CD16 gene, etc. The polyadenylation signal can be raised.

  In addition, in order to further express human CD16 and its human accessory molecules, splicing signals, enhancer regions, and introns of each gene are partly 5 'upstream of the promoter region, between the promoter region and the translation region, or 3' of the translation region. You may connect downstream. Hereinafter, a DNA construct comprising a promoter, human CD16, DNA encoding the human accessory molecule and a polyadenylation signal is referred to as human CD16 and the human accessory molecule expression unit.

More expression vectors, E. coli (Escherichia coli, the following E. Abbreviated coli) for the preparation of a vector using, the E. Coli replicable in a replication origin and E. Coli selectable marker of transformants of A drug resistance gene containing an endogenous promoter is required. Examples of drug resistance genes include E. coli- derived ampicillin resistance gene (β-lactamase gene), tetracycline resistance gene, kanamycin resistance gene, and the like. Examples of replication origins capable of replication in E. coli include the replication origin of pBR322 and the replication origin of colE1.

When a retrovirus is used for gene introduction, a recombinant retrovirus vector is prepared by inserting human CD16 and its human accessory molecule expression unit into a retrovirus vector. A recombinant retrovirus can be produced by introducing the recombinant retrovirus vector into an appropriate packaging cell. A viral vector can be introduced by infecting a fertilized egg or the like with the obtained recombinant virus. For introduction of recombinant retroviral vectors into packaging cells, known gene transfer methods (eg, calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc.) ) Can be used.
(6) Introduction of expression vector into fertilized egg and selection of transgenic non-human animal The non-human animal of the present invention comprises the above-mentioned 1. It can be produced by introducing the human CD16 produced in (5) and the gene containing the human accessory molecule expression unit into a target non-human animal. Specifically, the gene is introduced into a fertilized egg, embryonic stem cell (hereinafter abbreviated as ES cell), sperm or unfertilized egg of a target non-human animal, and from an individual generated using these cells. The non-human animal of the present invention can be produced by selecting an individual in which a gene containing DNA encoding human CD16 and its human accessory molecule is integrated on the chromosomes of all cells including germ cells. The presence of the transgene in the generated transgenic non-human animal germ cells can be confirmed by the fact that the offspring of the generated animal has the transgene in all of the germ cells and somatic cells. The selection of an individual is carried out on genomic DNA prepared from a part of the tissue constituting the individual, for example, blood tissue, epithelial tissue, connective tissue, cartilage tissue, bone tissue, muscle tissue, oral tissue or skeletal tissue. This is done by confirming the presence of the gene at the DNA level. Individuals selected in this way are usually heterozygotes with the transgene on one side of the homologous chromosome, so by crossing heterozygous individuals, the transgene can be found on both homologous chromosomes from the offspring. Homozygous animals can be obtained. By mating the homozygous male and female animals, all the offspring become homozygotes that stably hold the gene, so that the non-human animal of the present invention can be bred and passaged in a normal breeding environment. Can do.
(I) Production of transgenic non-human animals by gene transfer to fertilized eggs Production of transgenic non-human animals by gene transfer to fertilized eggs is performed by Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press ( 1994) (hereinafter abbreviated as Manipulating Mouse Embryo 2nd edition), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Mutation mouse using ES cells It can be produced by the method described in Production, Yodosha (1995), Developmental Engineering Experiment Manual, How to Make Transgenic Mice, Kodansha (1987), etc.

  A fertilized egg into which a gene containing human CD16 and its human accessory molecule expression unit is introduced can be obtained by mating male non-human animals and female non-human animals of the same species. For example, by mating male and female mice, preferably inbred male and female mice, fertilized eggs of mice can be obtained. Male rats and female rats, preferably Wistar male and Wistar females By mating the rat, a fertilized egg of the rat is obtained. Although fertilized eggs can be obtained by natural mating, a method of mating with male non-human animals after artificially adjusting the sexual cycle of female non-human animals is preferred. As a method for artificially adjusting the sexual cycle of a female non-human animal, for example, a method of first administering follicle stimulating hormone and then luteinizing hormone by, for example, intraperitoneal injection is preferable. It depends on the type of non-human animal. When the non-human animal is a mouse, it is preferable to administer luteinizing hormone about 48 hours after administration of follicle stimulating hormone to female mice, and to obtain fertilized eggs by mating with male mice. Is 2-20 IU / individual, preferably about 5 IU / individual, and the dose of luteinizing hormone is 0-10 IU / individual, preferably about 5 IU / individual. When the non-human animal is a rat, a method of obtaining a fertilized egg by administering a luteinizing hormone to a female rat after about 48 hours and mating with a male rat is preferable. Is 20-50 IU / individual, preferably about 30 IU / individual, and the dose of luteinizing hormone is 0-10 IU / individual, preferably about 5 IU / individual. In addition, when using an inbred mouse or a rat of the Wister strain, it is preferable to use a mouse of 8 weeks of age or more that has been raised for about 1 week under a light condition (for example, 7: 00-19: 00) for about 12 hours. .

The resulting fertilized egg was microinjected (WJ Gordon et al .; Proc. Natl. Acad. Sci. USA, 77 , 7380, 1980, JJ Mullins et al .; Nature, 344 , 541, 1990, RE Hammer et al .; Nature, 315 , 680, 1985, VG Pursel et al .; Immunol. Immunopathol., 17 , 303, 1987) and retrovirus-based methods (manipulating mouse embryo 2nd edition), etc., including human CD16 and its human accessory molecule expression unit A non-human having a chromosomal DNA incorporating a gene containing the introduced human CD16 and its human accessory molecule expression unit by artificially transplanting and implanting the fertilized egg into a female non-human animal after the gene has been introduced Animals are obtained.

  When the gene is introduced by the microinjection method, it is preferable to use a fertilized egg in which a male pronucleus appears about 12 to about 24 hours after fertilization. The gene containing human CD16 and its human accessory molecule expression unit introduced by microinjection can be used in either a circularized state or a linearized state, but the structural gene region and promoter of human CD16 and its human accessory molecule expression unit It is preferable to introduce the expression regulatory region such as a straight line without destroying it. When a gene is introduced by a method using a retrovirus, it is preferable to use a fertilized egg at a developmental stage before the morula (generally before the 8-cell stage).

When a fertilized egg is transplanted to a female non-human animal, the fertilized egg obtained by artificial pregnancy is transplanted to a pseudopregnant female non-human animal in which fertilization ability is induced by mating with a sperm ligated male non-human animal. And a method of implantation is preferred. Although pseudopregnant female non-human animals can be obtained by natural mating, fertility is induced by mating with male non-human animals after administration of luteinizing hormone-releasing hormone (hereinafter abbreviated as LHRH) or its analogs. Pseudo-pregnant female non-human animals can also be obtained. For example, [3,5-Dil-Tyr5] -LHRH, [Gln8] -LHRH, [D-Ala6] -LHRH, des-Gly10- [D-His (Bzl) 6] -LHRH ethylamide Etc. The dose of LHRH or its analog and the timing of mating with male non-human animals after the administration vary depending on the type of non-human animal. When the non-human animal is a female rat, it is usually preferable to mate with a male rat about 4 days after administration of LHRH or an analog thereof. The dose of LHRH or an analog thereof is usually 10 to 60 μg. / Individual, preferably about 40 μg / individual.
(Ii) Production of transgenic non-human animals by gene transfer into ES cells
Transgenic non-human animals by gene transfer into ES cells are described in Manipulating Mouse Embryo 2nd Edition, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of mutant mice using ES cells, Yodosha (1995), Developmental Engineering Experiment Manual, How to Make Transgenic Mice, Kodansha (1987), etc.

  After introducing a gene containing human CD16 and its human accessory molecule expression unit into non-human animal ES cells, the obtained ES cells are incorporated into fertilized eggs of non-human animals using the assembly chimera method or the injection chimera method, A non-human chimeric animal partially having cells having a chromosomal DNA incorporating a gene containing human CD16 and a human accessory molecule expression unit introduced by artificially transplanting and implanting the fertilized egg into a female non-human animal can get.

Non-human animal ES cells include mouse ES cells (MJ Evans et al .; Nature, 292 , 154, 1981; GR Martin; Proc. Natl. Acad. Sci. USA, 78 , 7634, 1981), rat ES cells ( PM Iannaccone et al .; Dev. Biol., 163 , 288, 1994) porcine ES cells (MB Wheeler; Reprod. Fertil. Dev., 6 , 563, 1994), monkey ES cells (JA Thomson et al .; Proc. Natl. Acad. Sci. USA, 92 , 7844, 1996), and the ES cell establishment method described in US5453357 or US5670372. AB2.2 (manufactured by Lexicon Genetics) or the like can be used as mouse ES cells.

  For introduction of exogenous genes into ES cells, known gene transfer methods (eg, calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, viral vector method) Etc.) can be used. The gene containing human CD16 and its human accessory molecule expression unit to be introduced can be used in either a circularized state or a linearized state, but the region and promoter encoding the structural gene of human CD16 and its human accessory molecule expression unit It is preferable to introduce the expression regulatory region such as a straight line without destroying it.

The fertilized egg is the above-mentioned 1. It can be obtained by the method described in (i) of (6).
When ES cells are incorporated into fertilized eggs of non-human animals using the assembly chimera method, it is generally preferable to use fertilized eggs at the developmental stage prior to the 8-cell stage. When ES cells are incorporated into fertilized eggs of non-human animals using the injection chimera method, it is generally preferable to use fertilized eggs at the blastocyst development stage from the 8-cell stage.

When a fertilized egg is transplanted to a female non-human animal, the fertilized egg obtained by artificial pregnancy is transplanted to a pseudopregnant female non-human animal in which fertilization ability is induced by mating with a sperm ligated male non-human animal. And the method of implantation is preferred, and pseudopregnant female non-human animals are It is obtained by using the method described in (i) of (6).
If non-human animals of ES cells and non-human animals of fertilized eggs are of the same species but have different phenotypes such as body color, individuals that are non-human chimeric animals and chimera ratio (ES Individuals with a high ratio of cells derived from cells, that is, the ratio of cells having chromosomal DNA into which an introduced foreign gene is incorporated can be easily selected.

Among non-human chimeric animals partially having cells having chromosomal DNA incorporating a gene containing human CD16 and its human accessory molecule expression unit, individuals having a high chimera rate (preferably male) and non-human animals (preferably , Female). Human CD16 and its human introduced on genomic DNA prepared from tissues of born pups, for example, blood tissue, epithelial tissue, connective tissue, cartilage tissue, bone tissue, muscle tissue, oral tissue or skeletal tissue When the presence of a gene containing an accessory molecule expression unit is detected by Southern hybridization, PCR, etc., and human CD16 introduced into the genomic DNA of all born babies and a gene containing the human accessory molecule expression unit are present The parent non-human chimeric animal is selected as the germline chimera. By mating a germline chimera and a non-human animal individual, an individual having a gene containing human CD16 introduced onto the chromosome of a whole-body cell and its human accessory molecule expression unit can be obtained as a offspring. This individual is usually a heterozygote having a gene containing the introduced human CD16 and its human accessory molecule expression unit only on one side of the homologous chromosome. Further, by mating males and females of the individual, a homozygous transgenic non-human animal containing a human CD16 introduced into both homologous chromosomes and a gene containing the human accessory molecule expression unit can be obtained.
(Iii) Production of transgenic non-human animals by gene introduction into sperm Production of transgenic non-human animals by gene introduction into sperm is performed by Perry et al. (Science, 284 , 1180, 1999) and Wakayama et al. (Nature Biotechnology, 16 , 639, 1998).

  As described in 1. After incorporating the human CD16 produced in (5) and the gene containing the human accessory molecule expression unit, the resulting sperm is fertilized into an unfertilized egg using a micro-fertilization method, By artificially transplanting and implanting a female non-human animal, a non-human animal in which a gene containing human CD16 incorporated into sperm and its human accessory molecule expression unit is integrated on the chromosome can be obtained.

When the foreign gene is incorporated into the sperm, the sperm whose cell membrane has been disrupted is treated with a medium containing the foreign gene at a concentration of about 0.5 to about 100 ng / mL (preferably 5 to 10 ng / mL) (for example, CZB medium). It is preferable to use a method of incubation for several minutes (preferably about 1 minute) in a NIM medium or the like. Methods for disrupting the sperm cell membrane include treatment with a low concentration (eg, 0.05%) surfactant (eg, TritonX-100), 10% fetal calf serum (hereinafter abbreviated as FCS) Either a method of freeze-drying sperm suspended in a medium containing EDTA but not containing EDTA, or a method of freeze-thawing the sperm suspension is preferred. Sperm that has destroyed the cell membrane by such treatment no longer has the ability to spontaneously fertilize, but promotes embryo development by fertilization using a micro-fertilization method using a micromanipulator or the like. In the case of microinsemination, a method of injecting the head of a sperm into which an exogenous gene has been incorporated into the cytoplasm of an unfertilized female non-human animal in the second stage of meiosis is preferred.
(Iv) Transgenic non-human animal production by gene transfer and in vitro fertilization to unfertilized eggs Chan et al. (Proc. Natl. Acad. Sci. USA, 95 , 14028, 1998).

  Infecting non-fertilized eggs of non-human animals with a retrovirus incorporating a gene containing human CD16 and its human accessory molecule expression unit, the gene is introduced, and the resulting egg is generated using in vitro fertilization methods A non-human animal in which a gene containing the introduced human CD16 and its human accessory molecule expression unit is integrated on the chromosome by artificially transplanting and implanting a fertilized egg that has started development into a female non-human animal Is obtained.

A retrovirus incorporating a gene containing human CD16 and its human accessory molecule expression unit can be prepared by the method described in Manipulating Mouse Embryo Second Edition. When infecting unfertilized eggs with a recombinant retrovirus, it is preferable to use unfertilized eggs excluding the zona pellucida. As a method of removing the zona pellucida of unfertilized eggs, a method of physically removing the membrane using a micromanipulator or the like, a method of chemically dissolving and removing the membrane using a chemical substance (for example, acidic tyrode) Either of these is preferable.
2. Production of a non-human animal of the present invention using a method for producing a gene-replaced animal in which a target gene and a target transgene are exchanged (1) Acquisition of DNA encoding a non-animal target gene The DNA encoding the target gene of the non-human animal is obtained by the method described in (1). To add the non-coding region of the target gene of the non-human animal to the 5 ′ end and 3 ′ end of the gene region containing human CD16 and its human accessory molecule expression unit, as described below, the 5 ′ end and 3 ′ end An appropriate restriction enzyme site is added in the vicinity of using synthetic DNA.
(2) Acquisition of genomic DNA containing a target gene of a non-human animal Obtain genomic DNA containing a target gene of a non-human animal. In addition to the region encoding the target gene and the intron, the genomic DNA must also include regions 5 ′ and 3 ′ from the region encoding the target gene. The length is preferably at least 500 bp, preferably 1 kbp or more, more preferably 3 kbp or more, and most preferably 5 kbp or more to the 5 ′ side and 3 ′ side of the region encoding the target gene.
Examples of a method for preparing genomic DNA containing a target gene include a method of preparing a genomic DNA library of the non-human animal and isolating a genomic DNA clone containing the gene by plaque hybridization or colony hybridization.

A genomic DNA library is obtained by extracting genomic DNA from the cells and tissues of the non-human animal, partially cleaving with a restriction enzyme, inserting it into an appropriate vector, and inserting it into an appropriate host such as E. coli according to the vector. It can produce by introducing. Preparation of genomic DNA and genomic DNA library can be prepared by the methods described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology and the like. As vectors, λ phage vectors such as λ EMBL3 (Stratagene), λ DASH II (Stratagene), λ FIX II (Stratagene), bacterial artificial chromosome (BAC) vectors such as BeloBACII, pAd10sacBII And bacteriophage P1 vectors such as pCYPAC1, P1 artificial chromosome (PAC) vectors such as pCYPAC1, and cosmid vectors such as SuperCos I (Stratagene).

  As a probe used for plaque hybridization or colony hybridization, a fragment of a genomic gene partial fragment that is a target of the non-human animal or a cDNA partial fragment of the target gene can be used. These partial fragments can be obtained by searching the genomic sequence or cDNA sequence of the target gene of the non-human animal from the base sequence database and using PCR using primers prepared based on this base sequence.

Plaque hybridization, colony hybridization, and probe labeling and preparation can be performed by the methods described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology and the like.
In addition, when the genomic sequence of the target gene of the non-human animal is obtained by searching a nucleotide sequence database, the region of the genomic sequence to be amplified is appropriately selected, and the 5 ′ end 20-40 bp sequence of this region is 3 ′ end. And DNA containing a sequence complementary to the 3 'end 20-40 bp sequence of this region at the 3' end, and using this DNA as a primer and PCR using the genomic DNA of the non-human animal as a template The genomic DNA containing the target gene of the non-human animal can be isolated.

In addition, by using a kit such as a genomic DNA library screening system (Genome Systems) or a universal genome walker kit (Universal Genome Walker ^™ Kit, Clontech), genomic DNA containing the target gene of the non-human animal can be obtained. It can also be isolated.
As genomic DNA containing the target gene of the non-human animal obtained by the above method, for example, mouse genomic DNA containing hypoxanthine phosphoribosyltransferase (hprt) gene on X chromosome or mouse containing Rosa26 gene on chromosome 6 Genomic DNA can be raised.
(3) Preparation of DNA in which 5 ′ non-coding region and 3 ′ non-coding region of the genomic sequence of a target gene of a non-human animal are added to both ends of a gene containing human CD16 and its human accessory molecule expression unit . Of the gene containing human CD16 and its human accessory molecule expression unit prepared in (5) above in (5) above. A non-coding region having an appropriate length 5 ′ from the start codon of the target gene genomic sequence of the non-human animal prepared in (2) of (2) above is added, and 3 ′ from the stop codon of the genomic sequence is added to the 3 ′ end. DNA added with a non-coding region of an appropriate length (hereinafter also referred to as DNA for homologous recombination) is used for the construction of a targeting vector necessary for homologous recombination of the target gene. This DNA can be produced by various methods using ordinary genetic engineering techniques. As a specific example, for example, each gene fragment can be prepared and obtained by performing PCR using as a primer a synthetic DNA containing an appropriate restriction enzyme site for ligation. The length of the non-coding region to be added is desirably 500 bp or more on both the 3 ′ side and the 5 ′ side, more preferably 1 kbp or more, further preferably 3 kbp or more, and most preferably 5 kbp or more.
(4) Preparation of target vectors and introduction into ES cells Target vectors for homologous recombination of non-human animal target genes are Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), BioManual Series 8 Gene targeting, production of mutant mice using ES cells (Yodosha) (1995) and the like can be used. The target vector can be used in either a replacement type or an insertion type. The target vector is the above-mentioned 2. (3) for homologous recombination DNA was prepared, the gene required for selection of homologous recombinants, and, E. Coli necessary for the preparation of vectors with E. Autonomously replicable origin of replication in E. coli and Has a drug resistance gene. As genes required for selection of homologous recombinants, drug selection genes such as hprt gene and neomycin resistance gene used for positive selection (method for selecting recombinants containing the gene used for selection), negative selection (selection) And a diphtheria toxin (DT) gene used in a method for screening a recombinant not containing a gene used in (1). The gene used for positive selection is inserted into a non-coding region of DNA for homologous recombination, and the gene used for negative selection is inserted at a position different from that for DNA for homologous recombination. The gene used for positive selection may be linked to a promoter necessary for the expression of the gene, or is not linked to a promoter and is expressed by the target gene promoter on the ES cell chromosome when homologous recombination occurs. You may make it make it. When two loxP sequences are added and inserted at both ends of the gene used for positive selection, after selecting the homologous recombination, the Cre recombinase expression vector is introduced into the homologous recombination. Genes used for positive selection can be removed from the chromosome.

As a specific target vector, a vector in which DNA for homologous recombination is inserted into a GenOway plasmid ready-to-use Quick knock-in ^TM vector, for example, a mouse in a ready-to-use Quick knock-in ^TM vector Examples of the plasmid SAT1-HR include a non-coding region of genomic DNA containing a target receptor gene and an expression unit of DNA for homologous recombination consisting of a gene containing human CD16 and its human accessory molecule expression unit.

  For introduction of a target vector into ES cells, known gene transfer methods such as calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, virus vector method, etc. Can be used. The target vector to be introduced can be used in either a circular or linear form, but it is preferably linearized and introduced without disrupting the genes necessary for selection of DNA for homologous recombination and homologous recombinants. .

Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells (Sheep Methods such as positive selection, promoter selection, negative selection, poly A selection described in (Satoshisha) (1995) and the like can be used. For example, in the case of a target vector containing the hprt gene, after introduction into an ES cell lacking the hprt gene, the ES cell is cultured in a medium containing aminopterin, hypoxanthine and thymidine, and an aminopterin resistant strain is selected. Positive selection can be performed to select homologous recombinants containing the hprt gene. In the case of a target vector containing a neomycin resistance gene, positive selection that selects for homologous recombinants containing a neomycin resistance gene by culturing ES cells into which the vector has been introduced in a medium containing G418 and selecting G418 resistant strains. Can be performed. In the case of a target vector containing a DT gene, the ES cell into which the vector has been introduced is cultured, and the grown strain is selected (recombinants randomly inserted into the chromosome other than homologous recombination have the DT gene in the chromosome Since it is incorporated and expressed, it cannot grow due to the toxicity of DT), it is possible to perform negative selection for selecting homologous recombinants that do not contain the DT gene. Examples of a method for selecting a desired homologous recombinant from the selected cell lines include Southern hybridization to genomic DNA (Molecular Cloning 3rd edition), PCR, and the like.
(4) Incorporation of ES cells into fertilized eggs and production of non-human transgenic animals Uptake of ES cells into fertilized eggs and transplantation of fertilized eggs into non-human animals are as described in 1. above. (6) can be carried out by the method described in (ii).

From the obtained pups, the above-mentioned 1. According to the method described in (ii) of (6), a heterozygote having a gene replacement by homologous recombination in one of homologous chromosomes of cells of the whole body can be obtained. Furthermore, by mating males and females of the individual, homozygous transgenic non-human animals having gene replacement by homologous recombination in both homologous chromosomes can be obtained.
The homozygous transgenic non-human animal thus obtained is a transgenic non-human animal that expresses human CD16 encoded by the introduced gene and its human accessory molecule, and does not express the target gene it had It is. This homozygous transgenic non-human animal is the above-mentioned 1. Than the transgenic non-human animal obtained by the method of 4. And 5. As a non-human animal used for the pharmacological evaluation of the substance described later in (a) since the target gene originally expressed by the non-human animal is not expressed, the pharmacological effect via the target gene is used when used for pharmacological evaluation. There is no need to consider (b) the possibility of unpredictable secondary phenotypic changes such as the destruction of the gene present at the transgene introduction site or the activation of the gene around the introduction site by the transgene. Is preferable in that there is almost no.
3. Production of Non-Human Animal of the Present Invention Using a Method for Creating a Cloned Individual Using the Nucleus of the Targeted Genetically Modified Cell The non-human animal of the present invention is a clone sheep (I. Wilmut) described in the literature. Nature, 385 , 810, 1997), cloned cattle (JB Cibelli et al; Science, 280 , 1256, 1998, Akira Iriya; protein nucleic acid enzyme, 44 , 892, 1999), cloned goat (A. Baguisi et al; Nature Biotechnology , 17 , 456, 1999), cloned mice (T. Wakayama et al .; Nature, 394 , 369, 1998, T. Wakayama et al .; Nature Genetics, 22 , 127, 1999), for example, as follows. can do.

1 above. Or 2. Using the method described in 1), a gene containing human CD16 and its human accessory molecule expression unit is introduced onto the chromosome of any cell of a non-human animal. Initialize the nuclei of the obtained cells. Initializing a nucleus means performing an operation to return the nucleus to a state where it can be repeated again.
Development is initiated by injecting reprogrammed cell nuclei into enucleated non-fertilized unfertilized eggs.

  The non-human animal of the present invention that expresses the introduced gene can be obtained by artificially transplanting and implanting an embryo that has started development into a female non-human animal. In addition, the above-mentioned 2. A transgenic non-human animal obtained using the nucleus of a cell that has been subjected to gene replacement according to the method described in 1. is usually heterozygous in which a gene containing human CD16 and its human accessory molecule expression unit is introduced into one of the homologous chromosomes. Because it is a zygote, the resulting heterozygote is crossed to obtain a transgenic non-human animal that expresses human CD16 and its human accessory molecule without expressing the gene-replaced self target gene Can do. This homozygous transgenic non-human animal is also described in 2. As the non-human animal used for the pharmacological evaluation in the same manner as the homozygous transgenic non-human animal obtained by the method described in 1. above, It is preferable to the transgenic non-human animal obtained by the method described in 1. above.

  The method of reinitializing the cell nucleus differs depending on the type of non-human animal. When the non-human animal is a sheep, goat, cow, etc., the cells into which the foreign gene has been introduced are 5 to 30%, preferably 10% to 10% from the medium containing FCS (for example, M2 medium), preferably It is preferable to induce and initialize the cell cycle in a resting state (G0 phase or G1 phase) by culturing in an oligotrophic medium containing 0 to 1%, preferably 0.5% FCS for 5 days. When the non-human animal is a mouse or the like, the nucleus of a cell into which a foreign gene has been introduced is injected into an enucleated unfertilized egg of the same non-human animal and cultured for several hours, preferably about 1 to 6 hours. It is preferable to initialize.

The method for initiating development in an unfertilized egg that has been enucleated from an initialized nucleus varies depending on the type of non-human animal. If the non-human animal is a sheep, goat, cow, etc., the nucleus was induced by inducing the cell cycle to the resting state (G0 or G1) and then enucleated by the non-human animal of the same species by electrofusion. It is preferable to activate the ovum and initiate development by transplanting to an unfertilized egg. When the non-human animal is a mouse or the like, an unfertilized egg injected with a cell nucleus into which a foreign gene has been introduced is stimulated with an egg activator (eg, strontium) to inhibit cell division (eg, cytochalasin). It is preferable to start the generation by treating with B or the like to suppress the release of the second polar body.
4). Pharmacological evaluation method of substance using cells obtained from non-human animal of the present invention or progeny thereof Fc receptors such as natural killer cells, macrophages, monocytes and dendritic cells obtained from non-human animal of the present invention or progeny thereof Cells (effector cells) that express the phenotype can be excised and the effector activity such as antibody cytotoxicity can be evaluated. Methods for measuring the biological activity of antibodies include Monoclonal Antibodies: Principles and Practice, Third Edition, Acad. Press, 1993, or Antibody Engineering: A Practical Approach, IRL Press at Oxford University. Press, 1996) can be used.
As specific examples, the binding activity of a human antibody to an antigen or the binding activity of a human antibody to an antigen-positive cultured cell line can be determined by ELISA and fluorescent antibody methods [Cancer Immunol. Immunother. .), 36 , 373 (1993)]. Cytotoxic activity against an antigen-positive cultured cell line can be evaluated by measuring CDC activity, ADCC activity, etc. [Cancer Immunol. Immunother., 36 , 373 (1993)] .
Moreover, various types of cells can be obtained by inducing differentiation of ES cells obtained from the non-human animal of the present invention or its progeny. As a method of inducing differentiation, a method of inducing teratomas mixed with various tissues by transplanting ES cells subcutaneously in animals of the same type and lineage (Manipulating Mouse Embryo 2nd Edition) By in vitro culture under appropriate conditions, endoderm cells, ectoderm cells, mesoderm cells, blood cells, endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons, glial cells, epithelium A method of inducing differentiation into cells, melanocytes, and keratinocytes (Reprod. Fertil. Dev., 10 , 31, 1998) can be mentioned. This differentiated cell is brought into contact with the test substance, and compared to cells in the absence of the test substance, various cellular responses such as an increase in intracellular Ca ²⁺ concentration, changes in cell morphology, etc. By investigating the pharmacological action, the pharmacological evaluation of the test substance on the cells can be performed. By these methods, pharmacological evaluation can be performed on cells that are difficult to remove from a human body or cells that are present only in small numbers.
5. Pharmacological evaluation method of a substance using the non-human animal of the present invention or its progeny Compared with the animal to which a test substance, for example, an antibody is administered and the test substance is not administered Pharmacological evaluation of the test substance can be performed by measuring various physical parameters such as blood pressure, respiratory rate, body weight, appearance, behavioral observation, and histopathological examination of the animal. . In particular, the non-human animal of the present invention or its progeny are preferred for pharmacological evaluation based on effector activity such as ADCC activity.

Further, a pathological model animal in which various diseases are induced in the non-human animal of the present invention or its progeny is prepared, a test substance such as an antibody is administered to the pathological model animal, and the blood pressure and respiratory rate of the pathological model animal are prepared. The measurement of various physical parameters such as body weight, pathological condition, appearance, behavioral observation, histopathological examination, etc. are carried out in comparison with the pathological model animal not administered with the test substance. Pharmacological evaluation of efficacy and side effects for the disease can be performed. Moreover, based on this evaluation, a substance preferable as a therapeutic agent for the disease can be selected. In particular, there is a species difference in the binding of the Fc region of the antibody to the Fc receptor in the antibody, and it was difficult to evaluate the optimal effector activity and the like in the pharmacological evaluation using a normal disease model animal. By using the non-human animal of the present invention or its progeny, a more appropriate pharmacological evaluation of an antibody based on effector activity such as ADCC activity can be performed.
Examples of the disease induced in the non-human animal of the present invention or its progeny include heart diseases (for example, acute heart failure, chronic heart failure, myocarditis, etc.), respiratory diseases, joint diseases (for example, rheumatoid arthritis, osteoarthritis, etc.) ), Renal diseases (eg, renal failure, glomerulonephritis, IgA nephropathy, etc.), arteriosclerosis, psoriasis, hyperlipidemia, allergic diseases (eg, asthma, allergic rhinitis, atopic dermatitis, etc.), Bone disease (eg osteoporosis, rickets, osteomalacia, hypocalcemia), blood disease, cerebrovascular injury, traumatic brain injury, infection, dementia, cancer, diabetes, liver disease, skin disease, Examples include neurodegenerative diseases and chronic inflammatory diseases. The preparation of disease model animals is described in "Manual Disease Model Mice" (Molecular Medicine, 31 Special Issue, Nakayama Shoten (1994)), "Pathology Animal Models for Illustrative and Pharmacology" (Nishimura Shoten (1984)), ""Arthritis model animal" [Medical and dental publishing (1985)], "Neurological and muscle disease model animal" [Medical and dentistry publishing (1982)], "Reactive oxygen and pathology From disease model to bedside" [Academic Publishing Center (1992) )] Etc. can be used.

3. this pharmacological evaluation method; And 8. As test substances to be used in the pharmacological evaluation method to be described later, it is used for the prevention and treatment of various diseases including cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, or viral or bacterial infection And human antibodies.
Antibodies that recognize tumor-related antigens, antibodies that recognize antigens related to allergy or inflammation, antibodies that recognize antigens related to cardiovascular disease, antibodies that recognize antigens related to autoimmune diseases, or viruses or bacterial infections Specific examples of human antibodies that recognize relevant antigens are described below.

As antibodies that recognize tumor-associated antigens, anti-CA125 antibody (Immunology Today, 21 , 403-410, 2000), anti-17-1A antibody (Immunology Today, 21 , 403-410, 2000), anti-integrin αvβ3 antibody (Immunology) Today, 21 , 403-410, 2000), anti-CD33 antibody (Immunology Today, 21 , 403-410, 2000), anti-CD22 antibody (Immunology Today, 21 , 403-410, 2000), anti-HLA antibody (Immunology Today, 21 , 403-410, 2000), anti-HLA-DR antibody (Immunology Today, 21 , 403-410, 2000), anti-CD20 antibody (Immunology Today, 21 , 403-410, 2000), anti-CD19 antibody (Immunology Today, 21 , 403-410, 2000), anti-EGF receptor antibody (Immunology Today, 21 , 403-410, 2000), anti-CD10 antibody (American Journal of Clinical Pathology, 113 , 374-382, 2000; Proc. Natl. Acad Sci. USA, 79: 4386-4391, 1982), anti-GD2 antibody (Anticancer Res., 13 , 331-336, 1993), anti-GD3 antibody (Cancer Immunol. Immunother., 36 , 2) 60-266, 1993), anti-GM2 antibody (Cancer Res., 54 , 1511-1516, 1994), anti-HER2 antibody (Proc. Natl. Acad. Sci. USA, 89 , 4285-4289, 1992), anti-CD52 antibody (Nature, 332 , 323-327, 1988), anti-MAGE antibody (British J. Cancer, 83 , 493-497, 2000), anti-HM1.24 antibody (Molecular Immunol., 36 , 387-395, 1999), anti Parathyroid hormone-related protein (PTHrP) antibody (Cancer, 88 , 2909-2911, 2000), anti-FGF8 antibody (Proc. Natl. Acad. Sci. USA, 86 , 9911-9915, 1989) Anti-basic fibroblast proliferation Factor antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265 , 16455-16463, 1990), anti-basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody (J. Neurosci. Res. , 40 , 647-659, 1995), anti-insulin-like growth factor receptor antibody (J. Neurosci. Res., 40 , 647-659, 1995), anti-PMSA antibody (J. Urology, 160 , 2396-2401, 1998) ), Anti-vascular endothelial growth factor antibody (C ancer Res., 57 , 4593-4599, 1997) or an anti-vascular endothelial growth factor receptor antibody (Oncogene, 19 , 2138-2146, 2000).

Antibodies that recognize antigens related to allergy or inflammation include anti-IgE antibodies (Immunology Today, 21 , 403-410, 2000), anti-CD23 antibodies (Immunology Today, 21 , 403-410, 2000), anti-CD11a antibodies ( Immunology Today, 21 , 403-410, 2000), anti-CRTH2 antibody (J. Immunol., 162 , 1278-1286, 1999), anti-CCR8 antibody (WO99 / 25734), anti-CCR3 antibody (US6207155), anti-interleukin 6 Antibody (Immunol. Rev., 127 , 5-24, 1992), anti-interleukin 6 receptor antibody (Molecular Immunol., 31 , 371-381, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127 , 1992) 5-24, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3 , 562-567, 1991), anti-interleukin 4 receptor antibody (J. Immunol. Meth., 217 , 41) -50, 1998), anti-tumor necrosis factor antibody (Hybridoma, 13 , 183-190, 1994), anti-tumor necrosis factor receptor antibody (Molecula r Pharmacol., 58 , 237-245, 2000), anti-CCR4 antibody (Nature, 400 , 776-780, 1999), anti-chemokine antibody (J. Immunol. Meth., 174 , 249-257, 1994) or anti-chemokine And a receptor antibody (J. Exp. Med., 186 , 1373-1381, 1997).

Examples of antibodies that recognize antigens related to cardiovascular diseases include anti-GpIIb / IIIa antibodies (J. Immunol., 152 , 2968-2976, 1994), antiplatelet-derived growth factor antibodies (Science, 253 , 1129-1132, 1991). ), An antiplatelet-derived growth factor receptor antibody (J. Biol. Chem., 272 , 17400-17404, 1997) or an anticoagulant factor antibody (Circulation, 101 , 1158-1164, 2000).

Antibodies that recognize antigens associated with autoimmune diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis include anti-self DNA antibodies (Immunol. Letters, 72 , 61 -68, 2000), anti-CD11a antibody (Immunology Today, 21 , 403-410, 2000), anti-ICAM3 antibody (Immunology Today, 21 , 403-410, 2000), anti-CD80 antibody (Immunology Today, 21 , 403-410) , 2000), anti-CD2 antibody (Immunology Today, 21 , 403-410, 2000), anti-CD3 antibody (Immunology Today, 21 , 403-410, 2000), anti-CD4 antibody (Immunology Today, 21 , 403-410, 2000) ), Anti-integrin α4β7 antibody (Immunology Today, 21 , 403-410, 2000), anti-CD40L antibody (Immunology Today, 21 , 403-410, 2000), anti-IL-2 receptor antibody (Immunology Today, 21 , 403- 410, 2000).

Examples of antibodies that recognize antigens associated with viral or bacterial infection include anti-gp120 antibodies (Structure, 8 , 385-395, 2000), anti-CD4 antibodies (J. Rheumatology, 25 , 2065-2076, 1998), anti-CCR4 antibodies Or anti-verotoxin antibody (J. Clin. Microbiol., 37 , 396-399, 1999) etc. are mentioned.
The above antibodies can be obtained from public institutions such as ATCC (The American Type Culture Collection), RIKEN Cell Development Bank, Biotechnology Institute of Industrial Technology, Dainippon Pharmaceutical Co., Ltd., R & D SYSTEMS, PharMingen, Cosmo. It can be obtained from commercial reagent sales companies such as Bio Inc. and Funakoshi Co., Ltd.
6). Production of genetically modified animals using ES cells, eggs, sperm, and nuclei of non-human animals of the present invention or their progeny Using ES cells, eggs, sperm, or nuclei obtained from the non-human animals of the present invention or their progeny, 1 above. 2. And 3. The gene-modified non-human animal having a gene containing human CD16 and its human accessory molecule expression unit and having further modified the gene on the chromosome or its progeny can be obtained by the method described in 1. above. In particular, a gene known to cause a pathological condition that destroys the function of the gene is described in 2. above. Knockout animals that have been deleted using the homologous recombination technique described in 1) above, and dominant negative genes that inhibit the function of the gene are described above in 1. Or 3. The transgenic animal introduced and expressed by the method described in 1) is useful as a disease state model animal.
7). Production of genetically modified animals by crossing non-human animals of the present invention or their progeny with animals of the same species and other strains and use of the produced genetically modified animals Animals of the same species and other strains of the non-human animals of the present invention or their descendants ( For example, a gene having a gene containing human CD16 and its human accessory molecule expression unit on a chromosome and having a certain expression system (for example, a symptom similar to a human disease state) by mating with a human disease model animal) Modified mammals can be obtained. If the animal to be mated is a human disease model animal, a disease state model animal having a gene containing human CD16 and its human accessory molecule expression unit and exhibiting a symptom similar to a human disease state is obtained. Examples of mating methods include in vitro fertilization methods other than natural mating. As a disease model animal, any disease model animal can be used regardless of the disease, congenital or acquired. For example, acquired disease model animals include "Manual disease model mice" (Molecular Medicine, 31 extra edition, Nakayama Shoten (1994)), "Pathological animal models for illustration and pharmacology" (Nishimura Shoten (1984)). , "Arthritis model animal" [Medical and dental drug publication (1985)], "Neuro / muscular disease model animal" [Medical and dental drug publication (1982)], "Reactive oxygen and disease state From disease model to bedside" [Academic Publishing Center] (1992)] and the like.
8). 5. Method for pharmacological evaluation of substances using genetically modified animals And 7. A test substance, for example, an antibody or the like is administered to the genetically modified pathological model animal obtained by the method described in 1. to measure various physical parameters such as blood pressure, respiratory rate, and body weight, pathological condition, and appearance of the pathological model animal. The pharmacological evaluation of the efficacy and side effects of the test substance against the disease can be performed by performing behavioral observation, histopathological examination, and the like in comparison with the disease model animal not administered with the test substance. . Moreover, based on this evaluation, a substance preferable as a therapeutic agent for the disease state can be selected. In particular, with antibodies, there are species differences in the binding between the Fc region of antibodies and the Fc receptor, and it was difficult to evaluate optimal effector activity and the like in pharmacological evaluation using normal animal models of pathological conditions. By using the non-human animal of the invention or its progeny, more appropriate pharmacological evaluation based on effector activity such as ADCC activity can be performed.
9. Method for screening substance having pharmacological activity using non-human animal of the present invention or its progeny Compared with non-human animal of the present invention or its progeny by administering a test substance, for example, an antibody and not administering the test substance The pharmacological evaluation of the test substance is carried out by measuring various physical parameters such as blood pressure, respiratory rate, body weight, etc., observing pharmacological effects such as appearance and behavior, and histopathological examination, By selecting a test substance having a pharmacological activity from among the test substances, a test substance having a desired pharmacological activity can be screened. In particular, the non-human animal of the present invention or its progeny administers an antibody, evaluates the pharmacological activity based on the effector activity such as ADCC activity, and selects an antibody having the pharmacological activity based on the effector activity from the test antibody. Thus, an antibody having pharmacological activity in vivo can be screened.
Examples of the present invention are shown below.

Construction of human CD16 expression vector
1. Acquisition of cDNA encoding human CD16 Based on the cDNA sequence of human CD16 (also known as FcγRIIIa) (GenBank Accession No. NM_000569) (SEQ ID NO: 1), the restriction enzyme can be used to limit the sequence of the region including the translation initiation codon. A forward primer (SEQ ID NO: 10) to which a recognition sequence was added and a reverse primer (SEQ ID NO: 11) to which a restriction enzyme recognition sequence was added to the region containing the translation stop codon were designed.

  First, using DNA polymerase KOD (manufactured by Toyobo Co., Ltd.), 50 μL of a reaction solution containing 2 μL of library lysate of Human Leukocyte 5'-STRETCH PLUS cDNA Library (manufactured by Clontech) [2.5 units of DNA polymerase KOD, diluted 10-fold 10 × KOD Buffer # 1 (manufactured by Toyobo Co., Ltd.), 0.2 mmol / L dNTPs, 1 mmol / L MgCl2, 0.4 μmol / L The above primers (SEQ ID NO: 10 and SEQ ID NO: 11)] were prepared, and PCR was performed. PCR was performed for 30 cycles, with one cycle consisting of 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 74 ° C for 60 seconds. From the reaction solution after PCR, DNA was purified using QIAquick PCR Purification Kit (manufactured by QIAGEN) and dissolved in 20 μL of sterilized water. Subsequently, the purified DNA was digested with restriction enzymes HindIII (Takara Bio) and BamHI (Takara Bio), and then subjected to 1.5% agarose gel electrophoresis, and an approximately 0.8 kb PCR amplified fragment was subjected to QIAquick Gel Extraction Kit. Purification was performed using QIAGEN.

Next, 3 μg of plasmid pBluescript II SK (−) (Stratagene) was digested with restriction enzymes HindIII (Takara Bio) and BamHI (Takara Bio), and then subjected to 1.5% agarose gel electrophoresis to obtain about 2.9 kb. The DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
The PCR amplification fragment derived from Human Leukocyte 5'-STRETCH PLUS cDNA Library and the DNA fragment derived from plasmid pBluescript II SK (-) were mixed and ligated using DNA Ligation Kit Ver.2.0 (Takara Bio Inc.). Went. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, of the amino acid sequence of human CD16, the plasmid containing the cDNA (SEQ ID NO: 1) consisting of valine (hereinafter referred to as human CD16 (Val) or FcγRIIIa (V)) at position 176, and phenylalanine (position 176) Hereinafter, plasmids containing cDNA (SEQ ID NO: 2) consisting of human CD16 (Phe) or hFcγRIIIa (F)) were obtained and named pBshFcγRIIIa (V) and pBshFcγRIIIa (F), respectively.
2. Acquisition of cDNA encoding human CD3ζ Based on the human CD3ζ cDNA base sequence (GenBank Accession No. NM_000734) (SEQ ID NO: 5), a restriction enzyme recognition sequence was added to the sequence containing the translation initiation codon. A forward primer (SEQ ID NO: 12) and a reverse primer (SEQ ID NO: 13) in which a restriction enzyme recognition sequence was added to the sequence of the region containing the translation stop codon were designed.

  First, according to the method described in this section 1, PCR is performed using the above primers (SEQ ID NO: 12 and SEQ ID NO: 13), and the reaction solution after PCR is used with QIAquick PCR Purification Kit (manufactured by QIAGEN). DNA was purified and dissolved in 20 μL of sterile water. After digestion with restriction enzymes EcoRI (manufactured by Takara Bio Inc.) and KpnI (manufactured by Takara Bio Inc.), it was subjected to 1.5% agarose gel electrophoresis, and a PCR amplified fragment of about 0.5 kb was obtained using QIAquick Gel Extraction Kit (manufactured by QIAGEN) Purified.

Next, 3 μg of plasmid pBluescript II SK (−) (Stratagene) was digested with restriction enzymes EcoRI (Takara Bio) and KpnI (Takara Bio), and then subjected to 1.5% agarose gel electrophoresis to obtain about 2.9 kb. The DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
The PCR amplification fragment derived from Human Leukocyte 5'-STRETCH PLUS cDNA Library and the DNA fragment derived from plasmid pBluescript II SK (-) were mixed and ligated using DNA Ligation Kit Ver.2.0 (Takara Bio Inc.). Went. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid pBshCD3ζ containing cDNA of human CD3ζ (SEQ ID NO: 5) was obtained.
3. Acquisition of cDNA encoding human FcεRIγ Based on the cDNA sequence of human FcεRIγ cDNA (GenBank Accession No. NM_004106) (SEQ ID NO: 7), a restriction enzyme recognition sequence was added to the sequence containing the translation initiation codon. A forward primer (SEQ ID NO: 14) and a reverse primer (SEQ ID NO: 15) in which a restriction enzyme recognition sequence was added to the sequence of the region containing the translation stop codon were designed.

  First, according to the method described in this section 1, PCR is performed using the above primers (SEQ ID NO: 14 and SEQ ID NO: 15), and the reaction solution after PCR is used with a QIAquick PCR Purification Kit (manufactured by QIAGEN). DNA was purified and dissolved in 20 μL of sterile water. After digestion with restriction enzymes EcoRI (manufactured by Takara Bio Inc.) and KpnI (manufactured by Takara Bio Inc.), it was subjected to 1.5% agarose gel electrophoresis, and a PCR amplified fragment of about 0.3 kb was obtained using QIAquick Gel Extraction Kit (manufactured by QIAGEN) Purified.

The PCR amplification fragment derived from Human Leukocyte 5'-STRETCH PLUS cDNA Library obtained above and the DNA fragment of about 2.9 kbp described in this item 2 prepared from plasmid pBluescript II SK (-) were mixed, and DNA Ligation Kit Ver The ligation reaction was carried out according to .2.0 (manufactured by Takara Bio Inc.). Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid pBshFcεRIγ containing cDNA of human FcεRIγ (SEQ ID NO: 7) was obtained.
4.Construction of pKANTEX hFcγRIIIa (V) and pKANTEX hFcγRIIIa (F) hFcγRIIIa (V) and pKANTEX hFcγRIIIa (F) were constructed (FIG. 1).

The plasmids pBshFcγRIIIa (V) and pBshFcγRIIIa (F) obtained in this section 1 were each digested with restriction enzymes NotI (Takara Bio) and BamHI (Takara Bio) and subjected to 1.5% agarose gel electrophoresis. About 0.8 kb DNA fragments were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
Next, 10 μg of the plasmid pKANTEX93 described in WO97 / 10354 was digested with restriction enzymes NotI (Takara Bio) and BamHI (Takara Bio), and then subjected to 1.5% agarose gel electrophoresis to obtain a DNA fragment of about 11.7 kb. It refine | purified using QIAquick Gel Extraction Kit (made by QIAGEN).

A DNA fragment containing the cDNA of human CD16 (Val) (also known as FcγRIIIa (V)) or human CD16 (Phe) (also known as FcγRIIIa (F)) obtained above and a DNA fragment derived from plasmid pKANTEX93 are mixed, Ligation Kit Ver.2.0 (Takara Bio Inc.) was used for the ligation reaction. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, plasmids pKANTEX hFcγRIIIa (V) and pKANTEX hFcγRIIIa (F) containing cDNAs of the target human FcγRIIIa (V) and human FcγRIIIa (F) were obtained.
5.Construction of an expression vector that co-expresses human CD16 and its accessory molecules. Thus, an expression vector for co-expressing human CD16 (FcγRIIIa) and its accessory molecule was constructed according to the following procedure (FIG. 2).

  First, 5.0 μg of each of the plasmids pBshCD3ζ and pBshFcεRIγ obtained in Sections 2 and 3 were digested with restriction enzymes EcoRI (Takara Bio) and KpnI (Takara Bio), and then subjected to 1.5% agarose gel electrophoresis. A 0.5 kb DNA fragment derived from pBshCD3ζ and an approximately 0.3 kb DNA fragment derived from pBshFcεRIγ were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

  Next, after digesting each 2.0 μg of the plasmids pKANTEX hFcγRIIIa (V) and pKANTEX hFcγRIIIa (F) obtained in this section (4) with restriction enzymes EcoRI (Takara Bio) and NotI (Takara Bio), The samples were subjected to 1.5% agarose gel electrophoresis, and DNA fragments of about 10.0 kb and about 2.5 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Further, 1.5 μg of the about 2.5 kb DNA fragment derived from pKANTEX hFcγRIIIa (V) obtained was digested with restriction enzyme KpnI (manufactured by Takara Bio Inc.), and then subjected to 1.5% agarose gel electrophoresis, and the about 2.0 kb DNA fragment was obtained. It refine | purified using QIAquick Gel Extraction Kit (made by QIAGEN).

  About 0.5 kb DNA fragment derived from pBshCD3ζ obtained above, about 10.0 kb DNA fragment derived from pKANTEX hFcγRIIIa (V), and about 2.0 kb DNA fragment derived from pKANTEX hFcγRIIIa (V) were mixed, and DNA Ligation Kit Ver The ligation reaction was performed according to .2.0 (manufactured by Takara Bio Inc.). Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) The nucleotide sequence was analyzed. As a result, the human FcγRIIIa expression vector pKANTEX hFcγRIIIa (V) + hCD3ζ containing the cDNA of the target human FcγRIIIa (V) and human CD3ζ was obtained.

  Furthermore, using an about 0.3 kb DNA fragment derived from pBshFcεRIγ and an about 10.0 kb DNA fragment derived from pKANTEX hFcγRIIIa (F), hFcγRIIIa (V) and hFcεRIγ, hFcγRIIIa (F) and hCD3ζ, hFcγRIIIa An hFcγRIIIa expression vector containing each cDNA of (V) and hFcεRIγ was constructed. The obtained plasmids were named pKANTEX hFcγRIIIa (V) + hFcεRIγ, pKANTEX hFcγRIIIa (F) + hCD3ζ, and pKANTEX hFcγRIIIa (F) + hFcεRIγ, respectively.

Construction of plasmid SAT1-HR to insert human CD16 and human CD3ζ cDNA expression units controlled by human CD16 promoter into mouse HPRT gene region by homologous recombination method
1. Construction of plasmid SAT1-linker
Oligo DNA sense strand SAT1-link1 (SEQ ID NO: 16) consisting of recognition sequences of 12 types of restriction enzymes (KpnI, AscI, BstBI, XbaI, NsiI, PmeI, NruI, SpeI, ClaI, HpaI, SrfI, SacI) and antisense Chain SAT1-link2 (SEQ ID NO: 17) was synthesized by Fasmac. Prepare 100 μL of distilled water containing 10 pmol each of SAT1-link1 and SAT1-link2, heat at 90 ° C for 10 minutes, and slowly return to room temperature to anneal SAT1-link1 and SAT1-link2, Produced.

Subsequently, 5 μg of pBluescriptIISK (+) (manufactured by STRATAGENE) was digested with KpnI (manufactured by Takara Bio Inc.) and SacI, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered plasmid was dissolved in 50 μL of water. .
After preparing 10 μL of the reaction solution containing 0.2 pmol of the SAT1-linker obtained above and 50 ng of pBluescriptIISK (+) fragment (KpnI-SacI), add 5 μL of Ligation High (manufactured by Toyobo Co., Ltd.) and connect at 16 ° C. for 2 hours. Reaction was performed. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid SAT1-linker containing the designed SAT1-linker sequence was obtained (FIG. 3).
2. Construction of plasmid SAT1-IRES
5 μg of plasmid G68 (GenOway) was prepared with SmaI (New England Biolabs) and NsiI (New England Biolabs), and 5 μg of the plasmid SAT1-linker prepared in this section 1 was added to PmeI (New England Biolabs). And NsiI (manufactured by New England Biolabs), respectively, and then DNA fragments of plasmid G68 and plasmid SAT1-linker were subjected to 1.0% (W / V) agarose gel electrophoresis. A 3 kb DNA fragment was purified using QIAquick Gel Extraction Kit (QIAGEN).

Next, after preparing 10 μL of a reaction solution containing 20 ng of a plasmid G68-derived DNA fragment (NsiI-SmaI) and 80 ng of a plasmid SAT1-linker-derived DNA fragment (NsiI-PmeI), 5 μL of Ligation High (manufactured by Toyobo) was added, and 16 ° C. was added at 16 ° C. A time ligation reaction was performed. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, plasmid SAT1-IRES containing the IRES sequence derived from plasmid G68 was obtained (FIG. 4).
3. Construction of plasmid SAT1-IRES-polyA
5 μg of plasmid G147 (GenOway) was prepared with SmaI (New England Biolabs) and ClaI (New England Biolabs), and 5 μg of plasmid SAT1-IRES prepared in this section 2 was added to HpaI (New England Biolabs). And digested with ClaI (New England Biolabs), respectively, and then subjected to DNA fragment of plasmid G147 and plasmid SAT1-IRES on 1.0% (W / V) agarose gel electrophoresis, about 0.8 kb, about The 3.6 kb DNA fragment was purified using QIAquick Gel Extraction Kit (QIAGEN).

Next, after preparing 10 μL of a reaction solution containing 20 ng of the plasmid G147-derived DNA fragment (ClaI-SmaI) and 80 ng of the plasmid SAT1-IRES-derived DNA fragment (ClaI-HpaI), 5 μL of Ligation High (manufactured by Toyobo) was added, and 16 ° C. was added at 16 ° C. A time ligation reaction was performed. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, plasmid SAT1-IRES was obtained in which a human growth hormone-derived polyA sequence was ligated downstream of the IRES sequence (FIG. 5).
4. Preparation of plasmid CD3Zeta in pBS Based on the cDNA sequence of human CD3ζ (GenBank Accession No. NM_000734) (SEQ ID NO: 5), the restriction enzyme recognition sequence (EcoRI) and Kozak A forward primer (SEQ ID NO: 18) with a sequence added and a reverse primer (SEQ ID NO: 19) with a restriction enzyme recognition sequence (XbaI, SpeI) added to the sequence in the region containing the translation stop codon were prepared, and the following PCR was performed. . That is, 20 μL of reaction solution containing pKANTEX hFcγRIIIa (V) + hCD3ζ prepared in Example 1 as a template [Pyrobest DNA polymerase (manufactured by Takara Bio Inc.), 10 × PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L above Primers (SEQ ID NO: 18 and SEQ ID NO: 19)] were prepared, heated at 94 ° C. for 5 minutes, and then subjected to PCR in a 30-cycle reaction with 94 ° C. for 1 minute and 68 ° C. for 2 minutes as one cycle. The solution after the PCR reaction was subjected to 1.5% agarose gel electrophoresis, and an approximately 0.5 kb PCR amplified fragment was purified using a QIAquick Gel Extraction kit (QIAGEN).

  The PCR amplified fragment obtained above was ligated with the plasmid pCR4Blunt-TOPO using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Invitrogen). The product was transformed. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) The nucleotide sequence was analyzed. As a result, a plasmid CD3Zeta in pCR4Blunt containing the cDNA sequence of human CD3ζ was obtained (FIG. 6).

Subsequently, 5 μg of the plasmid CD3Zeta in pCR4Blunt prepared above was analyzed with EcoRI (Takara Bio) and SpeI (Takara Bio), and 5 μg of the plasmid pBluescriptIISK (+) (STRATAGENE) was EcoRI (Takara Bio). After digestion with SpeI and Takara Bio, respectively, the DNA fragments of plasmid CD3Zeta in pCR4Blunt and plasmid pBluescriptIISK (+) were subjected to 1.0% (W / V) agarose gel electrophoresis and about 0.5 kb each. And a DNA fragment of about 3.0 kb was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, after preparing 10 μL of a reaction solution containing 20 ng of DNA fragment (EcoRI-SpeI) derived from plasmid CD3Zeta in pCR4Blunt and 80 ng of DNA fragment (EcoRI-SpeI) derived from plasmid pBluescriptIISK (+), 5 μL of Ligation High (Toyobo Co., Ltd.) was added, The ligation reaction was carried out at 16 ° C. for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid CD3Zeta in pBS containing the cDNA sequence of human CD3ζ was obtained (FIG. 6).
5. Preparation of plasmid SAT1-IRES-CD3 Zeta 5 μg of plasmid SAT1-IRES-polyA prepared in this section 3 with NruI (New England Biolabs) and SpeI (Takara Bio) and this section 4 After digesting 5 μg of the plasmid CD3Zeta in pBS prepared in step 1 with EcoRV (Takara Bio) and SpeI (Takara Bio), the plasmid SAT1-IRES-polyA and the plasmid CD3Zeta in pBS DNA fragments were 1.0% ( W / V) Agarose gel electrophoresis was performed, and DNA fragments of about 4.4 kb and about 0.5 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

Next, after preparing 10 μL of a reaction solution containing plasmid SAT1-IRES-polyA-derived DNA fragment (NruI-SpeI) 80 ng, plasmid CD3Zeta in pBS-derived DNA fragment (EcoRV-SpeI) 20 ng, Ligation High (manufactured by Toyobo Co., Ltd.) was added 5 μL, The ligation reaction was performed at 16 ° C. for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid SAT1-IRES-CD3 Zeta in which the cDNA sequence of human CD3ζ was linked between the IRES sequence and the human growth hormone-derived polyA sequence was obtained (FIG. 7).
6. Preparation of plasmid CD16 in pBS Based on the nucleotide sequence of human CD16 cDNA (GenBank Accession No. NM_000569) (SEQ ID NO: 1), a forward primer consisting of a sequence containing a 5 ′ untranslated region and a translation initiation codon (sequence The reverse primer (sequence number 21) which added the restriction enzyme recognition sequence (XbaI, SpeI) to the arrangement | sequence of the area | region containing a number 20) and a translation stop codon was produced, and the following PCR was performed. That is, 20 μL of reaction solution containing pKANTEX hFcγRIIIa (V) + hCD3ζ prepared in Example 1 as a template [Pyrobest DNA polymerase (manufactured by Takara Bio Inc.), 10 × PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L above Primers (SEQ ID NO: 20 and SEQ ID NO: 21)] were prepared, heated at 94 ° C. for 5 minutes, and then subjected to PCR in a 30-cycle reaction with 94 ° C. for 1 minute and 68 ° C. for 2 minutes as one cycle. The solution after the PCR reaction was subjected to 1.5% agarose gel electrophoresis, and an approximately 0.8 kb PCR amplified fragment was purified using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

  The PCR amplified fragment obtained above was ligated with the plasmid pCR4Blunt-TOPO using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Invitrogen). The product was transformed. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) The nucleotide sequence was analyzed. As a result, a plasmid CD16 in pCR4Blunt containing the cDNA sequence of human CD3ζ was obtained (FIG. 8).

  Subsequently, 5 μg of the plasmid CD16 in pCR4Blunt prepared above was analyzed with EcoRI (Takara Bio) and SpeI (Takara Bio), and 5 μg of the plasmid pBluescriptIISK (+) (STRATAGENE) was EcoRI (Takara Bio). After digestion with SpeI (manufactured by Takara Bio Inc.) respectively, the DNA fragments of plasmid CD16 in pCR4Blunt and plasmid pBluescriptIISK (+) obtained above were subjected to 1.0% (W / V) agarose gel electrophoresis. The DNA fragments of about 0.8 kb and 3.0 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

Next, after preparing 10 μL of a reaction solution containing 20 ng of a plasmid CD16 in pCR4Blunt-derived DNA fragment (EcoRI-SpeI) and 80 ng of a plasmid pBluescriptIISK (+)-derived DNA fragment (EcoRI-SpeI), 5 μL of Ligation High (manufactured by Toyobo Co., Ltd.) was added, The ligation reaction was carried out at 16 ° C. for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid CD16 in pBS containing the cDNA sequence of human CD16 was obtained (FIG. 8).
7. Preparation of plasmid CD16Pmo Based on the promoter sequence of human CD16 (GenBank Accession No. AL_590385) (SEQ ID NO: 9), the forward primer (SEQ ID NO: 22) and reverse primer (SEQ ID NO: 23) were prepared, and the following PCR Was done. That is, 20 μL reaction solution containing Human Genomic DNA (Novagen) as a template [Pyrobest DNA polymerase (Takara Bio), 10 × PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L the above primers (SEQ ID NO: 22 and SEQ ID NO: 23)] were prepared, heated at 94 ° C. for 5 minutes, and then subjected to PCR in a 30-cycle reaction with 94 ° C. for 1 minute and 68 ° C. for 2 minutes as one cycle. The solution after the PCR reaction was subjected to 1.5% agarose gel electrophoresis, and an approximately 1.1 kb PCR amplified fragment was purified using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

  The PCR amplified fragment obtained above was ligated with the plasmid pCR4Blunt-TOPO using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Invitrogen). The product was transformed. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) The nucleotide sequence was analyzed. As a result, plasmid CD16Pmo-N containing the promoter sequence of human CD16 was obtained (FIG. 9).

  Next, based on the human CD16 promoter sequence (GenBank Accession No. AL_590385) (SEQ ID NO: 9), a forward primer (SEQ ID NO: 24) and a reverse primer (SEQ ID NO: 25) were prepared, and the following PCR was performed. . That is, 20 μL of reaction solution containing Human Genomic DNA (Novagen) as a template [Pyrobest DNA polymerase (Takara Bio), 10 × PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L above primers (SEQ ID NO: 24 and SEQ ID NO: 25)] were prepared, heated at 94 ° C. for 5 minutes, and then subjected to PCR in a 30-cycle reaction with 94 ° C. for 1 minute and 68 ° C. for 2 minutes as one cycle. The solution after the PCR reaction was subjected to 1.5% agarose gel electrophoresis, and an approximately 0.8 kb PCR amplified fragment was purified using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

  The PCR amplified fragment obtained above was ligated with the plasmid pCR4Blunt-TOPO using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Invitrogen). The product was transformed. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) The nucleotide sequence was analyzed. As a result, plasmid CD16Pmo-C containing the promoter sequence of human CD16 was obtained (FIG. 10).

Subsequently, 5 μg of the plasmid CD16Pmo-N prepared above is HindIII (Takara Bio) and ApaI (Takara Bio) and 5 μg of the plasmid CD16Pmo-C prepared above is HindIII (Takara Bio). ) And ApaI (manufactured by Takara Bio Inc.), respectively, and the DNA fragments of plasmid CD16Pmo-N and CD16Pmo-C obtained above were subjected to 1.0% (W / V) agarose gel electrophoresis, DNA fragments of about 1.1 kb and about 4.8 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, after preparing 10 μL of a reaction solution containing 20 ng of plasmid CD16Pmo-N-derived DNA fragment (HindIII-ApaI) and 80 ng of plasmid CD16Pmo-C-derived DNA fragment (HindIII-ApaI), 5 μL of Ligation High (manufactured by Toyobo Co., Ltd.) was added, The ligation reaction was carried out for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid CD16Pmo containing an about 1.8 kb human CD16 promoter sequence was obtained (FIG. 11).
8. Preparation of plasmid CD16Pmo + cDNA 5 μg of the plasmid CD16 in pBS prepared in this section 6 with BspEI (New England Biolabs) and HindIII (Takara Bio), and the plasmid CD16Pmo prepared in this section 7 After digesting with 5 μg each of BspEI (manufactured by New England Biolabs) and HindIII (manufactured by Takara Bio), the DNA fragments of plasmid CD16 in pBS and plasmid CD16Pmo obtained above were 1.0% (W / V) agarose gel. For electrophoresis, DNA fragments of about 3.8 kb and 1.8 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, after preparing 10 μL of a reaction solution containing 80 ng of a plasmid CD16 in pBS-derived DNA fragment (HindIII-BspEI) and 20 ng of a plasmid CD16Pmo-derived DNA fragment (HindIII-BspEI), 5 μL of Ligation High (manufactured by Toyobo) was added, and 16 ° C. was added at 16 ° C. A time ligation reaction was performed. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid CD16Pmo + cDNA in which the promoter sequence and cDNA sequence of human CD16 were linked was obtained (FIG. 12).
9. Preparation of plasmid SAT1-Tg 5 μg of plasmid SAT1-IRES-CD3 Zeta prepared in this section 5 was prepared in BstBI (New England Biolabs) and XbaI (Takara Bio) and in section 8. After digesting plasmid CD16Pmo + cDNA with ClaI (manufactured by New England Biolabs) and XbaI (manufactured by Takara Bio Inc.) in an amount of 5 μg, plasmid SAT1-IRES-CD3 Zeta and DNA fragments of plasmid CD16Pmo + cDNA obtained above Was subjected to 1.0% (W / V) agarose gel electrophoresis, and DNA fragments of about 4.9 kb and about 2.6 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, after preparing 10 μL of a reaction solution containing 80 ng of the plasmid SAT1-IRES-CD3 Zeta-derived DNA fragment (BstBI-XbaI) and 20 ng of the plasmid CD16Pmo + cDNA-derived DNA fragment (ClaI-XbaI), add 5 μL of Ligation High (Toyobo). The ligation reaction was performed at 16 ° C. for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid SAT1-Tg in which the human CD16 cDNA linked with the IRES sequence and the human CD3ζ cDNA sequence are arranged downstream of the promoter sequence of human CD16 and the human growth hormone-derived polyA sequence is further added downstream thereof. (Fig. 13).
10. Preparation of plasmid SAT1-HR A plasmid ready-to was prepared by recovering an approximately 4.6 kb DNA fragment generated by digesting plasmid SAT1-Tg prepared in this section 9 with restriction enzymes AscI and SrfI, and digesting with AscI and SwaI. -use Quick knock-in ^TM vector (GenOway) and ligase enzyme were used for ligation reaction. Using the obtained ligated plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, the mouse HPRT gene has a diphtheria toxin expression unit (DTA) as a negative selection marker, a neomycin resistance gene expression unit as a positive selection marker, and a 5 'arm for causing homologous recombination in the mouse HPRT gene region. The human CD16 promoter in the ready-to-use Quick knock-in ^TM vector containing the base sequence upstream of the first exon, and the third and second introns of the mouse HPRT gene as the 3 'arm. Plasmid SAT1-HR, in which a human CD16 cDNA sequence and a human CD3ζ cDNA sequence linked by an IRES sequence are arranged downstream, and a gene expression construct with a human growth hormone-derived polyA sequence added downstream. (Fig. 14).

Generation of genetically modified mice expressing human CD16 and human CD3ζ under the control of human CD16 promoter
1. Establishment of Genetically Modified ES Cell Line Using the following method, human CD16 cDNA and human CD3ζ expression unit controlled by human CD16 promoter were inserted upstream of exon 3 of HPRT gene of mouse ES cells. Gene transfer was carried out by the following procedure using a known electroporation method [Cytotechnology, 3, 133 (1990)].

After the plasmid SAT1-HR prepared in Example 2 was digested with PmeI (New England Biolabs), 40 μg of the linearized plasmid was 5 × 10 ⁶ cells of 129Ola mouse-derived E14 ES cells (GenOway) Gene transfer was performed under conditions of a pulse voltage of 260 V and an electric capacity of 500 μF. The 129Ola mouse is a mouse prepared by deleting exon 1 of the HPRT gene and its surrounding chromosomal region from the 129 strain mouse. Two days later, ES cells were selectively cultured in a medium containing 200 μg / ml of G418, and 400 clones of G418 resistant ES cell line were isolated. 400 clones were each cultured to confluence in 96-well plates, and then subcultured to two 96-well plates (for cell culture and for obtaining replica genomic DNA).

After obtaining genomic DNA from a 96-well plate for obtaining genomic DNA by a known method (WO03 / 085107 (Yamane's FUT8KO strain patent)), the target HPRT gene region is controlled by the human CD16 promoter by homologous recombination. Homologous recombination clones in which human CD16 cDNA and human CD3ζ expression units were inserted were detected using the genomic PCR method described below.
Confirmation of the 5 ′ side of the homologous recombination region was performed using PCR primer GW496 (SEQ ID NO: 26) and PCR primer GW497 (SEQ ID NO: 27) (FIG. 15). 50 μL reaction solution containing 100 ng of genomic DNA as a template [2.6 units Expand High Fidelity polymerase (Roche diagnostics), 10 × PCR buffer, 0.5 mmol / L dNTP mixture, 15 pmol each of primer GW496 (SEQ ID NO: 26 ) And GW497 (SEQ ID NO: 27)], heated at 94 ° C. for 2 minutes, and then subjected to PCR in 35 cycles of a reaction of 94 ° C. for 30 seconds, 62 ° C. for 1 minute, and 68 ° C. for 4 minutes. Was done. The PCR reaction completion solution was subjected to agarose gel electrophoresis, and an ES cell clone strain in which a 4529 bp PCR amplified fragment was detected was cloned (FIG. 16). As a result, a PCR amplified fragment of 4529 bp was observed in 37 clones out of 400 clones having G418 resistance. The following 3 ′ genomic PCR analysis was performed on 20 of the 37 clones.

  Confirmation of the 3 'side of the homologous recombination region was performed using PCR primer GX2922 (SEQ ID NO: 28) and PCR primer GX0025 (SEQ ID NO: 29) (Fig. 17). 50 μL reaction solution containing 100 ng of genomic DNA as a template [2.6 units Expand High Fidelity polymerase (Roche diagnostics), 10 × PCR buffer, 0.5 mmol / L dNTP mixture, 15 pmol each of primer GX2922 (SEQ ID NO: 28 ) And GX0025 (SEQ ID NO: 29)], and heated at 94 ° C for 2 minutes, followed by PCR in 35 cycles of 1 cycle of 94 ° C for 30 seconds, 62 ° C for 1 minute, and 68 ° C for 4 minutes Was done. The PCR reaction completed solution was subjected to agarose gel electrophoresis, and an ES cell clone strain in which a 5733 bp PCR amplified fragment was detected was identified (FIG. 17). As a result, a PCR amplified fragment of 5733 bp was observed in 17 out of 20 clones in which homologous recombination on the 5 'side was confirmed.

The following Southern blot analysis was performed on 17 clones in which specific bands were detected by 5 ′ and 3 ′ PCR analysis (FIG. 18). The genomic DNA of each clone was cleaved with PstI (New England Biolabs) for 16 hours, then subjected to agarose gel electrophoresis, and nylon using existing methods (J. Mol. Biol., 98 , 503, 1975) DNA was transferred to the filter. 65 ° C. of pre-hybridization solution after soaked filter prepared above in (4 × SSC, 1% SDS , 65 ℃ 0.5% skim milk, 20 mM EDTA, 100 μg / mL salmon sperm DNA), Ya further ³² P label A 3 ′ probe (SEQ ID NO: 30) labeled with a DIG label was added, and hybridization was performed at 65 ° C. for 18 hours. The 3 ′ probe was designed from the third exon sequence of mouse HPRT gene (GenBank Accession No. K01509). After hybridization, washing was performed twice for 15 minutes at 65 ° C using a solution containing 3xSSC and 1% SDS, and then at 65 ° C using a solution containing 0.5xSSC and 1% SDS. Two 15 minute washes were performed. The X-ray film was exposed to a filter after washing for 3 days and developed, and an ES cell clone strain (approx. 6.1 kb band was detected) in which the desired homologous recombination occurred was identified (FIG. 18). 19). As a result, a band of about 6.1 kb was observed in 4 clones of # 1C8, # 2A8, # 12A2, and # 15A9, and it was confirmed that homologous recombination was normally performed in these clones.

  The above ES clones (# 1C8, # 2A8, # 12A2, # 15A9) were injected into blastocysts derived from C57BL / 6 mice, then transplanted into the oviducts of pseudopregnant OF1 mice, and pups Got. As a result, from the blastocyst injected with ES cells of # 1C8, # 12A2, and # 15A9, it has a chimera rate of 95-100% (the ratio of hair color on the body surface is expressed as chimera) A total of 8 male chimeras were obtained. From these 8 mice, 2 chimeras derived from # 1C8, 1 chimera derived from # 12A2 and 1 chimera derived from # 15A9 were each mated with females of C57BL / 6 mice, and about F1 females born The following PCR analysis and Southern blot analysis were performed.

  Confirmation of the 5 ′ side of the homologous recombination region was performed using PCR primer GW496 (GenOway (SEQ ID NO: 26)) and PCR primer GW497 (GenOway (SEQ ID NO: 27)) (FIG. 15). . 50 μL of reaction solution containing 100 ng of genomic DNA obtained from the tail of female F1 body as a template (2.6 units Expand High Fidelity polymerase (Roche diagnostics), 10 × PCR buffer, 0.5 mmol / L dNTP mixture, 15 pmol primers GW496 (SEQ ID NO: 26) and GW497 (SEQ ID NO: 27)] were prepared and heated at 94 ° C. for 2 minutes, followed by 94 ° C. for 30 seconds, 62 ° C. for 1 minute, and 68 ° C. for 4 minutes. PCR was performed with a reaction of 35 cycles. The PCR reaction completed solution was subjected to agarose gel electrophoresis, and it was confirmed whether a 4529 bp PCR amplified fragment was detected (FIGS. 15 and 20). As a result, a PCR amplified fragment of 4529 bp was detected from the female F1 body produced from the chimeras derived from # 1C8, # 12A2 and # 15A9.

Subsequently, the 3 ′ side of the homologous recombination region was confirmed by Southern blot analysis. After the genomic DNA obtained from the tail of the F1 of females was cut 16 hours PstI (manufactured by New England Biolabs) and subjected to agarose gel electrophoresis, existing methods (J. Mol. Biol., 98 , 503, 1975) was used to transfer DNA to a nylon filter. 65 ° C. of pre-hybridization solution after soaked filter prepared above in (4 × SSC, 1% SDS , 65 ℃ 0.5% skim milk, 20 mM EDTA, 100 μg / mL salmon sperm DNA), Ya further ³² P label A 3 ′ probe (SEQ ID NO: 30) labeled with a DIG label was added, and hybridization was performed at 65 ° C. for 18 hours. The 3 ′ probe was designed from the third exon sequence of mouse HPRT gene (GenBank Accession No. K01509). After hybridization, washing was performed twice for 15 minutes at 65 ° C using a solution containing 3xSSC and 1% SDS, and then at 65 ° C using a solution containing 0.5xSSC and 1% SDS. Two 15 minute washes were performed. The X-ray film was exposed to a filter after washing for 3 days and developed, and the F1 body in which the desired homologous recombination occurred (a band of about 6.1 kb was detected) was identified (FIGS. 21 and 22). ). As a result, a C57Bl / 6 mouse chromosome-derived band (about 7.8 kb) and an about 6.1 kb homologous recombinant-derived band were detected in the F1 body in which a 4529 bp PCR amplified fragment was detected.

  As a result of the above, the germline of the male # 1C8, # 12A2 and # 15A9 derived chimera is derived from the ES cell line that had undergone the desired homologous recombination. As a result, it was confirmed that the FRT body in which the gene expression unit in which the cDNAs of human CD16 and human CD3ζ were linked by the IRES sequence downstream of the human CD16 promoter was produced in the HPRT gene region of one X chromosome.

Generation of genetically modified mice expressing human FcεRIγ and GFP under the control of human CD16 promoter
1. Construction of plasmid Epsilon in pCR4Blunt Based on the cDNA base sequence of human FcεRIγ (GenBank Accession No. NM_004106) (SEQ ID NO: 7), the restriction enzyme recognition sequence (BspEI) and human sequence are included in the sequence including the translation initiation codon. A forward primer (SEQ ID NO: 31) with a CD16 promoter sequence added and a reverse primer (SEQ ID NO: 32) with a restriction enzyme recognition sequence (SpeI) added to the sequence in the region containing the translation stop codon were prepared, and the following PCR was performed. . That is, 20 μL reaction solution containing pKANTEX hFcγRIIIa (V) + hFcεRIγ prepared in Example 1 as a template [Pyrobest DNA polymerase (manufactured by Takara Bio Inc.), 10 × PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L above Primers (SEQ ID NO: 31 and SEQ ID NO: 32)] were prepared, heated at 94 ° C. for 5 minutes, and then subjected to PCR in 30 cycles of a reaction of 94 ° C. for 1 minute and 68 ° C. for 2 minutes. The solution after the PCR reaction was subjected to 1.5% agarose gel electrophoresis, and an approximately 0.3 kb PCR amplified fragment was purified using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

The PCR-amplified fragment obtained above was ligated with the plasmid pCR4Blunt-TOPO using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen), and then E. coli DH5α strain (Toyobo Co., Ltd.) using this plasmid by the heat shock method. The product was transformed. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid Epsilon in pCR4Blunt containing the cDNA sequence of human FcεRIγ was obtained (FIG. 23).
2. Construction of plasmid GFP in pCR4Blunt Based on the known Green fluorescent protein (GFP) cDNA sequence (GenBank Accession No. M62653), a cDNA sequence of modified GFP in which stronger luminescence is observed is designed (SEQ ID NO: 33). ), And a synthetic oligo DNA was used to prepare a DNA strand containing this sequence.

  Subsequently, a restriction primer recognition sequence (SpeI) is added to the forward primer (SEQ ID NO: 34) in which a restriction enzyme recognition sequence (EcoRI) and a Kozak sequence are added to the sequence of the region including the translation start codon, and a sequence of the region including the translation stop codon. An added reverse primer (SEQ ID NO: 35) was prepared and subjected to the following PCR. That is, 20 μL of reaction solution containing the modified GFP cDNA sequence (SEQ ID NO: 33) as a template [Pyrobest DNA polymerase (Takara Bio Inc.), 10 × PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L above primers (SEQ ID NO: 34 and SEQ ID NO: 35) were prepared, heated at 94 ° C. for 5 minutes, and then subjected to PCR in a 30-cycle reaction with 94 ° C. for 1 minute and 68 ° C. for 2 minutes as one cycle. The solution after the PCR reaction was subjected to 1.5% agarose gel electrophoresis, and an approximately 0.8 kb PCR amplified fragment was purified using a QIAquick Gel Extraction kit (manufactured by QIAGEN).

The PCR-amplified fragment obtained above was ligated with the plasmid pCR4Blunt-TOPO using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen), and then E. coli DH5α strain (Toyobo Co., Ltd.) using this plasmid by the heat shock method. The product was transformed. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid GFP in pCR4Blunt containing the modified GFP cDNA sequence was obtained (FIG. 24).
3. Preparation of plasmid Pmo + epsilon 5 μg of plasmid CD16Pmo + cDNA prepared in Example 2 was prepared with BspEI (New England Biolabs) and SpeI (Takara Bio), and plasmid Epsilon prepared in this section 1. In pCR4Blunt was digested with 5 μg of BspEI (manufactured by New England Biolabs) and SpeI (manufactured by Takara Bio Inc.), and then the plasmid CD16Pmo + cDNA obtained above and the DNA fragment of plasmid Epsilon in pCR4Blunt were 1.0% (W / V) It was subjected to agarose gel electrophoresis, and DNA fragments of about 3.8 kb and about 0.3 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

Next, after preparing 10 μL of a reaction solution containing 50 ng of the plasmid CD16Pmo + cDNA-derived DNA fragment (BspEI-SpeI) and 50 ng of the plasmid Epsilon in pCR4Blunt-derived DNA fragment (BspEI-SpeI), 5 μL of Ligation High (Toyobo Co., Ltd.) was added, and 16 ° C. The ligation reaction was carried out for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, a plasmid Pmo + epsilon in which the cDNA sequence of human FcεRIγ was linked downstream of the promoter sequence of human CD16 was obtained (FIG. 25).
4. Preparation of plasmid SAT1-IRES-GFP 5 μg of plasmid SAT1-IRES-polyA prepared in Example 2 was added with NruI (New England Biolabs) and SpeI (Takara Bio) and in this section 2. After digesting the prepared plasmid GFP in pCR4Blunt with 5 μg of EcoRV (Takara Bio) and SpeI (Takara Bio), respectively, the DNA fragments of plasmid SAT1-IRES-polyA and plasmid GFP in pCR4Blunt obtained above Was subjected to 1.0% (W / V) agarose gel electrophoresis, and DNA fragments of about 4.4 kb and 0.8 kb were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, after preparing 10 μL of a reaction solution containing 50 ng of the plasmid SAT1-IRES-polyA-derived DNA fragment (NruI-SpeI) and plasmid GFP in pCR4Blunt-derived DNA fragment (EcoRV-SpeI), 5 μL of Ligation High (Toyobo) was added, The ligation reaction was performed at 16 ° C. for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, plasmid SAT1-IRES-GFP was obtained in which the cDNA sequence of modified GFP was linked between the IRES sequence and the human growth hormone-derived polyA sequence (FIG. 26).
5. Preparation of plasmid EpsGFP-Tg 5 μg of plasmid SAT1-IRES-GFP prepared in this section 4 in BstBI (manufactured by New England Biolabs) and XbaI (manufactured by Takara Bio Inc.) After digesting Pmo + epsilon with 5 μg of ClaI (New England Biolabs), XbaI (Takara Bio) and 10 μL of 10 × M buffer (Takara Bio), the plasmid SAT1- DNA fragments of IRES-GFP and plasmid Pmo + epsilon were subjected to 1.0% (W / V) agarose gel electrophoresis, and DNA fragments of about 5.0 kb and about 2.1 kb were respectively obtained using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Purified. Next, after preparing 10 μL of a reaction solution containing plasmid SAT1-IRES-GFP-derived DNA fragment (BstBI-XbaI) 80 ng and plasmid Pmo + epsilon-derived DNA fragment (ClaI-XbaI) 20 ng, 5 μL of Ligation High (manufactured by Toyobo) was added, The ligation reaction was performed at 16 ° C. for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. As a result, the plasmid EpsGFP-Tg, in which the human FcεRIγ cDNA and the modified GFP cDNA sequence linked to the IRES sequence downstream of the human CD16 promoter sequence and the human growth hormone-derived polyA sequence were added downstream of the cDNA sequence. Obtained (FIG. 27).
6. Preparation of genetically modified mice expressing human FcεRIγ and GFP under the control of the human CD16 promoter Plasmid for linearizing or inserting the plasmid EpsGFP-Tg prepared in this section 5 into a specific gene region of the mouse chromosome And then introduced into mouse fertilized eggs or ES cells to produce genetically modified mice expressing human FcεRIγ and GFP under the control of the human CD16 promoter. Further, by crossing this mouse with a genetically modified mouse expressing human CD16 and human CD3ζ under the control of the human CD16 promoter described in Example 3, human CD16, human CD3ζ, human FcεRIγ under the control of the human CD16 promoter. , And genetically modified mice expressing modified GFP.

  Further, the plasmid EpsGFP-Tg prepared in this section 5 was introduced into fertilized eggs or ES cells of genetically modified mice expressing human CD16 and human CD3ζ under the control of the human CD16 promoter described in Example 3, and human Genetically modified mice expressing human CD16, human CD3ζ, human FcεRIγ, and modified GFP under the control of the CD16 promoter were prepared.

A method for preparing plasmids pKANTEXhFcγRIIIa (V) and pKANTEXhFcγRIIIa (F) is shown. The methods for preparing plasmids pKANTEXhFcγRIIIa (V) + hCD3ζ, pKANTEXhFcγRIIIa (F) + hCD3ζ, pKANTEXhFcγRIIIa (V) + hFcεRIγ, and pKANTEXhFcγRIIIa (F) + hFcεRIγ are shown. The preparation method of plasmid SAT1-linker is shown. The preparation method of plasmid SAT1-IRES is shown. The preparation method of plasmid SAT1-IRES-polyA is shown. The preparation method of plasmid CD3Zeta in pBS is shown. The preparation method of plasmid SAT1-IRES-CD3Zeta is shown. The preparation method of plasmid CD16 in pBS is shown. The preparation method of plasmid CD16Pmo-N is shown. The preparation method of plasmid CD16Pmo-C is shown. The preparation method of plasmid CD16Pmo is shown. The preparation method of plasmid CD16Pmo + cDNA is shown. The preparation method of plasmid SAT1-Tg is shown. The preparation method of plasmid SAT1-HR is shown. The chromosomal structure of the expected homologous recombinant (HPRT gene region) and the positions of the 5 'and 3' PCR primers for detecting the homologous recombinant are schematically shown. An amplification band derived from genomic DNA of a homologous recombinant ES cell line detected by 5 'PCR is shown. Amplified bands derived from genomic DNA of homologous recombinant ES cell lines detected by 3 'PCR. The expected chromosomal structure (HPRT gene region) of the homologous recombinant and the position of the 3 'probe used for Southern blot analysis for detecting the homologous recombinant are schematically shown. The band derived from the genomic DNA of the host strain E14 ES cell line and homologous recombinant ES cell line detected by Southern blot analysis using the 3 'probe is shown. Amplified bands derived from genomic DNA of wild type and F1 body mice detected by 5 'PCR. The chromosomal structure (HPRT gene region) of the parent E14 ES cell line, the expected homologous recombinant, and the wild-type mouse, and the position of the 3 'probe used for Southern blot analysis are schematically shown. The band derived from the genomic DNA of the host strain E14 ES cell line, wild-type mouse, and F1 body, which is detected by Southern blot analysis using the 3 'probe, is shown. The preparation method of plasmid Epsilon in pCR4Blunt is shown. The preparation method of plasmid GFP in pCR4Blunt is shown. The preparation method of plasmid Pmo + epsilon is shown. The preparation method of plasmid SAT1-IRES-GFP is shown. The preparation method of plasmid EpsGFP-Tg is shown.

  INDUSTRIAL APPLICABILITY According to the present invention, there are provided a non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule, and a method for using the same. The non-human animals and their progeny of the present invention are useful for the biological activity and pharmacological evaluation of antibodies.

Claims (16)

A non-human animal or its progeny whose genome has been modified to co-express human CD16 and its human accessory molecule. The non-human animal or progeny thereof according to claim 1, wherein the human accessory molecule of human CD16 is a molecule selected from the group consisting of the following (a) to (c).
(a) the human CD3ζ chain;
(b) the human Fcε receptor Iγ chain;
(c) Human CD3ζ chain and human Fcε receptor Iγ chain. The non-human animal or progeny thereof according to claim 1 or 2, wherein the genome is modified such that expression of human CD16 or a human accessory molecule thereof is controlled by a human CD16 promoter region. The non-human animal or progeny thereof according to any one of claims 1 to 3, wherein human CD16 is a protein encoded by a DNA selected from the group consisting of the following (a) to (d).
(a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b) DNA consisting of the base sequence represented by SEQ ID NO: 2;
(c) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having an activity of binding to the Fc region of a human antibody;
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 2 under a stringent condition and encodes a protein having an activity of binding to the Fc region of a human antibody. The non-human animal or progeny thereof according to any one of claims 1 to 3, wherein the human CD16 is a protein selected from the group consisting of the following (a) to (f).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4;
(c) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 3 and having an activity of binding to the Fc region of a human antibody;
(d) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 4 and having an activity of binding to the Fc region of a human antibody;
(e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 and having an activity of binding to the Fc region of a human antibody;
(f) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having an activity of binding to the Fc region of a human antibody. The non-human animal or progeny thereof according to any one of claims 2 to 5, wherein the human CD3ζ chain is a protein encoded by the following DNA (a) or (b):
(a) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(b) A DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a protein that associates with human CD16 and has an intracellular signal transduction function. The non-human animal or progeny thereof according to any one of claims 2 to 5, wherein the human CD3ζ chain is a protein selected from the group consisting of the following (a) to (c).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 6;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6, and which is associated with human CD16 and has an intracellular signal transduction function ;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6, and being associated with human CD16 and having an intracellular information transmission function. The non-human animal or progeny thereof according to any one of claims 2 to 7, wherein the human Fcε receptor Iγ chain is a protein encoded by a DNA selected from the following (a) or (b).
(a) DNA consisting of the base sequence represented by SEQ ID NO: 7;
(b) A DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions and encodes a protein that associates with human CD16 and has an intracellular signal transduction function. The non-human animal or progeny thereof according to any one of claims 2 to 7, wherein the human Fcε receptor Iγ chain is a protein selected from the group consisting of the following (a) to (c).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 8, and is associated with human CD16 and has an intracellular signal transduction function ;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and being associated with human CD16 and having an intracellular information transmission function. The non-human animal or progeny thereof according to any one of claims 3 to 9, wherein the human CD16 promoter region is DNA selected from the following (a) or (b).
(a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b) hybridizes with DNA comprising the nucleotide sequence represented by SEQ ID NO: 9 under stringent conditions and expresses human CD16 or its human accessory molecule in natural killer cells, macrophages, monocytes or dendritic cells Active DNA; The non-human animal according to any one of claims 1 to 10, wherein the non-human animal is an animal selected from the group consisting of cow, sheep, goat, pig, horse, mouse, rat, chicken, monkey and rabbit. Or its descendants. A method for measuring antibody-dependent cytotoxic activity of an antibody, comprising the following steps (1) to (3).
(1) A step of isolating effector cells from the non-human animal or progeny thereof according to any one of claims 1 to 11;
(2) An isolated effector cell is mixed with an antibody and a cell expressing an antigen targeted by the antibody, and the effector cell, the antibody and the antigen targeted by the antibody are expressed in the reaction solution. Contacting with living cells;
(3) A step of quantifying the survival rate of cells expressing the target antigen in the reaction solution. A method for evaluating pharmacological activity based on antibody-dependent cytotoxic activity of an antibody, comprising the following step (1) and (2).
(1) A step of administering an antibody as a test substance to the non-human animal according to any one of claims 1 to 11 or a progeny thereof;
(2) A step of evaluating the pharmacological activity based on the antibody-dependent cytotoxic activity of the antibody as the test substance in the non-human animal or its offspring. An antibody screening method comprising the following steps (1) to (3):
(1) A step of administering an antibody as a test substance to the non-human animal according to any one of claims 1 to 11 or a progeny thereof;
(2) a step of evaluating pharmacological activity based on antibody-dependent cytotoxic activity of an antibody as a test substance in the non-human animal or its offspring;
(3) A step of selecting an antibody having the pharmacological activity. The method according to any one of claims 12 to 14, wherein the antibody is a protein selected from the group consisting of the following (a) to (e).
(a) a human antibody;
(b) a humanized antibody;
(c) Human chimeric antibody
(d) an antibody fragment comprising the Fc region of (a), (b) or (c);
(e) A fusion protein having the Fc region of (a), (b) or (c). 16. The method of claim 15, wherein the antibody class is IgG.

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