Anti-pcsk9 antibodies with ph-dependent binding characteristics

Anti-PCSK9 antibody with pH-dependent binding properties

ANTI-PCSK9 ANTIBODIES WITH pH-DEPENDENT BINDING CHARACTERISTICS

<b>Cross-reference of related applications</b>

This application is requesting U.S. Provisional Application No. 61/682,349 filed on August 13, 2012, and 61/703,342 filed on September 20, 2012; 61/734,398 filed on December 7, 2012; 61/761,383 filed on February 6, 2013; and 61/828,258 filed on May 29, 2013 under 35U.SC§ 119(e), the disclosures of which are incorporated by reference in their entirety In this article.

The present invention relates to antigen binding molecules that specifically interact with the proprotein convertase subtilisin/kexin type 9 (PCSK9), and the use of these molecules to treat hypercholesterolemia and other related diseases characterized by elevated cholesterol levels .

The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a proprotein convertase that belongs to the proteinase K subfamily of the secreted subtilase subtilase family. The encoded protein is synthesized into a soluble zymogen, which is processed by autocatalysis in the endoplasmic reticulum. Circulating PCSK9 binds to and destroys the low-density lipoproprotein receptor (LDLR) on the surface of liver cells. This process reduces the binding capacity of the liver and removes LDL cholesterol (LDL-C), thereby causing an increase in the amount of LDL-C. It specifically binds to PCSK9 and blocks its interaction with LDL receptors The acting antibodies have been shown to be therapeutically effective in reducing the amount of plasma LDL-C in human subjects. (See, for example, Stein et al.,<i>New Engl.J.Med.</i> 2012；366：1108-1118)。

The dosage and/or frequency of administration of antibodies required to produce therapeutic effects are generally determined by the number of antigens that can be neutralized by a single antibody molecule. For example, before the antibody becomes a degradation target in the host, if the antibody can only bind to and neutralize an antigen, a relatively large amount of the antibody must be administered to produce therapeutic effects and/or the antibody must be administered quite frequently. In other words, if a single antibody can repeatedly bind to multiple antigens before degradation, the number of antibodies that need to be administered is reduced, and it can be administered at a lower frequency, resulting in an effective therapeutic response.

In this technology, there is a need for new therapeutic molecules that can bind to PCSK9, produce an effective therapeutic response over a longer period of time, and/or lower the dose required by currently known and available PCSK9 antagonists.

The present invention provides antibodies and antigen-binding fragments thereof that have pH-dependent binding properties to the proprotein convertase subtilisin/kexin type 9 (PCSK9). For example, the present invention includes antibodies and antigen-binding fragments thereof that bind to PCSK9 with greater affinity at neutral pH than at acidic pH (that is, the binding affinity decreases at acidic pH). As shown in the examples described in the text, anti-PCSK9 antibodies with reduced binding affinity at acidic pH have various improved/enhanced biological properties compared to antibodies without reduced binding affinity at acidic pH. For example, the anti-PCSK9 antibody of the present invention with reduced binding affinity at acidic pH, when administered to an animal subject (including human patients), compared to the anti-PCSK9 antibody with no reduced binding affinity at acidic pH, it is circulating In has a long half-life. In other words, anti-PCSK9 antibodies of the present invention with reduced binding affinity at acidic pH are cleared from the circulation more slowly than anti-PCSK9 antibodies lacking pH-dependent binding. Slower antibody clearance (i.e. The longer circulating half-life is related to the longer cholesterol-lowering effect of the antibody of the present invention. Therefore, the antibody of the present invention can be administered to a subject at a lower frequency and/or at a lower dose, but will have the same (or better) efficacy than an anti-PCSK9 antibody that does not have a reduced binding affinity at acidic pH.

Without being bound by theory, it is believed that compared to neutral pH, anti-PCSK9 antibody with lower binding affinity at acidic pH is dissociated from the antigen in an acidic endosomal environment and recirculated to the plasma. Here it can perform another round of therapeutic antigen binding. This phenomenon is called "antibody recycling" or "capture and release" and can greatly improve the efficacy of antibodies in vivo, because a single antibody molecule can bind or neutralize multiple antigens. Conversely, compared to neutral pH, antibodies that bind to PCSK9 with equal or greater affinity at acidic pH will only bind to the antigen in the endosome through a single antibody-antigen binding. Follow a single path to the lysosomes for degradation.

The binding properties of anti-PCSK9 antibodies can be quantified in vitro by surface plasmon resonance, which provides a numerical value (e.g. ka, k_d, K_D, T½, etc.). These parameters can be used to determine whether the binding of antibodies to PCSK9 has pH-dependent binding properties. The present invention therefore includes, as measured by surface plasmon resonance, antibodies or antigen-binding fragments thereof that bind to PCSK9 with an affinity at least 5 times higher at neutral pH than at acidic pH (or conversely, such as surface As measured by plasmon resonance, the antibody binds to PCSK9 with an affinity at least 5 times lower at acidic pH than at neutral pH). The present invention also includes antibodies or antigen-binding fragments thereof. As measured by surface plasmon resonance, the t½ of binding to PCSK9 at acidic pH is at least 5 times shorter than the t½ of binding to PCSK9 at neutral pH (or vice versa , As measured by surface plasmon resonance, the t½ of antibody binding to PCSK9 at neutral pH is at least 5 times longer than the t½ of antibody binding to PCSK9 at acidic pH). According to a specific embodiment, it is provided that it binds to PCSK9 with at least 5 times higher affinity at neutral pH than at acidic pH, and the t½ of binding to PCSK9 at acidic pH is higher than that at neutral pH. The antibody binds to PCSK9 at neutral pH. The t½ is at least 5 times shorter than the anti-PCSK9 anti body.

According to a specific embodiment of the present invention, an anti-PCSK9 antibody is provided, which when administered to a subject at a dose of about 10 mg/kg, reduces serum LDL-C by at least 25% compared to baseline and maintains this reduced serum LDL- C at least 20 days.

The anti-PCSK9 antibody of the present invention can be pH-dependent by mutating the amino acid sequence of the maternal anti-PCSK9 antibody that does not have pH-dependent binding or only has intermediate pH-dependent binding Bound variant anti-PCSK9 antibody. For example, one or more amino acids in one or more complementarity determining regions (CDRs) of the maternal anti-PCSK9 antibody can be changed to histidine residues and the histidine variant antibody produced thereby can be adjusted to pH -Dependent binding is tested (for example, the affinity for PCSK9 decreases at acidic pH compared to neutral pH).

According to the present invention, an exemplary parental anti-PCSK9 antibody can be modified at the amino acid sequence level to produce a variant anti-PCSK9 antibody with enhanced pH-dependent binding properties, which is an antibody named 300N. In addition, any heavy chain and light chain variable regions (HCVR/LCVR) (that is, including SEQ ID NO: 218/226) comprising antibody 300N or heavy chain and light chain CDRs (HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3) (that is, including SEQ ID NOs: 220-222-224-228-230-232) anti-PCSK9 antibody, which can be used as a parent antibody, derived from it by mutagenicity of histidine substitution Anti-PCSK9 antibody with pH-dependent binding properties. In addition, any anti-PCSK9 antibody or antigen-binding fragment thereof that includes the amino acid sequence of HCVR/LCVR as described in Table 1 in the text, or the amino acid sequence of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, can be used as a parent Source antibody, from which anti-PCSK9 antibody with pH-dependent binding properties is derived through mutagenesis of histidine substitution.

The present invention includes methods of treating diseases and disorders, wherein the diseases and disorders are treatable and/or ameliorated by antagonizing PCSK9, for example, by blocking the interaction of PCSK9 with LDL receptor (LDLR). The method according to this aspect of the invention includes A pharmaceutical composition comprising an anti-PCSK9 antibody or an antigen-binding fragment thereof with pH-dependent binding properties is administered to a subject in need thereof. The method according to this aspect of the invention can be used to treat, for example, hypercholesterolemia and other related diseases or disorders as disclosed elsewhere in the text.

The present invention also includes therapeutic administration therapy, which includes administering multiple doses of anti-PCSK9 antibodies with pH-dependent binding properties to a subject in need thereof. According to specific embodiments in this aspect of the invention, individual doses of anti-PCSK9 antibodies with pH-dependent binding properties can be administered to a subject at a frequency of less than once a month (e.g., once every two months, every three months Once, once every four months, etc.).

Other embodiments will become apparent from the discussion of confirming the detailed description.

<b>Detailed description</b>

Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as these methods and conditions can be changed. It should also be understood that the terms used in the text are only for the purpose of describing specific embodiments and are not intended to be limited, because the scope of the present invention will only be limited by the scope of the attached patent application.

Unless otherwise specified, all technical and scientific terms used in the text have the same meanings as normally understood by those skilled in the art to which the present invention belongs. As used in the text, the term "approximately" when used in relation to a specific quoted value means that the value can vary by no more than 1% from the quoted value. For example, as used in the text, the term "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any of the methods and materials similar or equivalent to those described in the text can be used in the practice or testing of the present invention, the preferred methods and materials are currently described. All patents, applications and non-patent applications mentioned in this specification are incorporated herein by reference in their entirety.

<b>General definition</b>

The words "proprotein convertase subtilisin/kexin type 9", "PCSK9" or "PCSK9 fragment" and similar words, unless specified to be from a non-human species (e.g. "mouse PCSK9", "mouse PCSK9 fragment" , "Monkey PCSK9", "Monkey PCSK9 Fragment", etc.), otherwise it refers to the human PCSK9 protein as used in the text. Human PCSK9 (sometimes abbreviated as "hPCSK9" in the text) has the amino acid sequence set forth in SEQ ID NO:755.

The term "antibody" as used herein refers to any antigen binding molecule or molecular complex that includes at least one complementarity determining region (CDR) that specifically binds or interacts with a specific antigen (eg, PCSK9). The term "antibody" includes four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, and immunoglobulin molecules and their multimers (for example, IgM). Each heavy chain system includes a heavy chain variable region (abbreviated as HCVR or V in the text)_H) And a heavy chain constant region. Each heavy chain constant region includes three regions, C_H1. C_H2 and C_H3. Each light chain system includes a light chain variable region (abbreviated as LCVR or V in the text)_L) And a light chain constant region. The light chain constant region includes a region (C_L1). V_HAnd V_LRegions can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conservative regions, called framework regions (FR). Each V_HAnd V_LThe system is composed of three CDRs and four FRs, arranged from the amino end to the carboxyl end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FR of the antibody (or antigen-binding portion thereof) may be the same as the human germline sequence, or be modified naturally or artificially. The amino acid consensus sequence can be defined based on the side-by-side analysis of two or more CDRs.

The term "antibody", as used herein, also includes antigen-binding fragments of whole antibody molecules. The terms "antigen-binding portion" of antibody, "antigen-binding fragment" of antibody, etc., as used in the text, include any naturally-occurring, enzyme-prepared, synthetic or genetically engineered polypeptide or carbohydrate that specifically binds to the antigen to form a complex protein. Antigen-binding fragments of antibodies can use any suitable standard techniques, such as proteolytic digestion or DNA manipulation and expression involving the variable region and optionally constant region of the antibody. The current recombinant genetic engineering technology is derived from, for example, whole antibody molecules. This DNA is known and/or can be easily obtained from, for example, market sources, DNA databases (including, for example, phage-antibody databases), or can be synthesized. This DNA can be sequenced and manipulated chemically or by using molecular biotechnology, such as arranging one or more variable and/or constant regions into a suitable configuration, or introducing codons to generate cysteine residues, Modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; ( vi) dAb fragment; and (vii) the smallest recognition unit (such as isolated complementarity determining region (CDR), such as CDR3 peptide) or restricted FR3-CDR3- consisting of amino acid residues in the hypervariable region of an antibody mimicking FR4 peptide. Other engineered molecules, such as region-specific antibodies, single-domain antibodies, region-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-antibodies, tetra-antibodies, mini-antibodies, nano-antibodies (e.g. monovalent nano-antibodies, Bivalent nano-antibodies, etc.), SMIP and shark variable IgNAR regions are also included in the term "antigen-binding fragment" used in the text.

Antigen-binding fragments of antibodies typically include at least one variable region. The variable region can be a region of any size or amino acid composition and should generally include CDRs adjacent to or within the framework of one or more framework sequences. Having V_LZone and V_HIn the region-bound antigen-binding fragment, V_HAnd V_LThe zones can be located opposite each other in any suitable arrangement. For example, the variable region can be dimerized and contain V_H-V_H, V_H-V_LOr V_L-V_LDimer. In addition, the antigen-binding fragment of the antibody may contain monomer V_HOr V_LArea.

In a specific embodiment, the antigen-binding fragment of an antibody may contain at least one variable region and at least one constant region covalently linked. Non-limiting and exemplary configurations of variable and constant regions that can be found in the antigen-binding fragment of the antibody of the present invention include: (i) V_H-C_H1; (ii) V_H-C_H2; (iii) V_H-C_H3; (iv) V_H-C_H1-C_H2; (v)V_H-C_H1-C_H2-C_H3; (vi)V_H-C_H2-C_H3; (vii)V_H-C_L;(Viii)V_L-C_H1; (ix)V_L-C_H2; (x)V_L-C_H3; (xi)V_L-C_H1-C_H2; (xii)V_L-C_H1-C_H2-C_H3; (xiii) V_L-C_H2-C_H3; and (xiv)V_L-C_L. In any configuration of the variable region and the constant region, including any of the exemplary configurations listed above, the variable region and the constant region can be directly connected to each other or can be connected by a complete or partial hinge region or linker region . The hinge region can be composed of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, making the adjacent variable and/or constant regions of a single polypeptide molecule flexible And semi-flexible connection. Furthermore, the antigen-binding fragments of the antibody of the present invention may include non-covalently linked to each other and/or with one or more monomer V_HOr V_LA homodimer or heterodimer (or other multimer) of any of the above-listed variable and constant region configurations where the regions are connected (for example by disulfide bonds).

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or in vitro position-specific mutagenesis or somatic mutations in vivo), such as In CDR and especially CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been attached to human framework sequences.

The term "recombinant human antibody", as used herein, is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as the use of recombinant expression vectors to be transfected into host cells (further detailed below). Recombinant combinatorial human antibody library (further detailed below) isolated antibodies, antibodies isolated from animals (e.g., mice) transfected with human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295), or by any other method involving the splicing of human immunoglobulin gene sequences and DNA sequences to prepare, express, produce or isolate antibodies. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, these recombinant human antibodies are mutated in vitro (or when using human Ig sequence bases). Because when the animal is transformed, it is the somatic cell in the living body that induces mutation), and therefore the V of the recombinant antibody_HAnd V_LThe amino acid sequence of the region (when derived from the human germline V_HAnd V_LSequence or when it is related to it) it is a sequence that may not naturally exist in the germline repertoire of human antibodies in vivo.

Human antibodies can exist in two forms related to hinge heterogeneity. The first form includes a stable four-chain immunoglobulin molecule of about 150-160 kDa, in which the dimerization system is brought together by inter-chain heavy chain disulfide bonds. In the second form, the dimers are not connected via disulfide bonds and the 75-80kDa molecule is composed of covalently coupled light and heavy chains (half antibodies). These forms are extremely difficult to separate, even after affinity purification.

Among the various complete IgG isotypes, the frequency of the second form is due to (but not limited to) structural differences related to the isotype of the hinge region of the antibody. The single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the extent typically observed with the human IgG1 hinge . The present invention covers hinges, C_H2 or C_HAn antibody with one or more mutations in region 3, which may be desirable in manufacturing, for example, to improve the yield of the desired antibody form.

"Isolated antibody", as used herein, refers to an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody isolated or removed from at least one organism, or from a component of a tissue or cell, wherein for the purpose of the present invention, the antibody that exists in nature or is naturally produced is an "isolated antibody". Isolated antibodies also include in situ antibodies in recombinant cells. The isolated antibody is an antibody that has undergone at least one purification or separation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular materials and/or chemicals.

"Neutralizing" or "blocking" antibody, as used herein, hopefully refers to an antibody whose binding to PCSK9 is reduced or that can detectably inhibit the interaction between PCSK9 and LDL receptor (LDLR) or extracellular fragments of LDLR.

Compared with the corresponding germline sequence from which the antibody can be derived, the anti-PCSK9 antibody disclosed herein can include one or more amino acid substitutions in the framework and/or CDR regions of the heavy and light chain variable regions , Insert and/or delete. These mutations can be easily determined by comparing the amino acid sequence disclosed in the text with the germline sequence obtained from, for example, a published antibody sequence database. The specific amino acid changes that confer pH-dependent binding properties to the anti-PCSK9 antibodies of the present invention are discussed in detail elsewhere in the text. The present invention includes antibodies derived from any amino acid sequence disclosed in the text and antigen-binding fragments thereof, wherein one or more amino acids in one or more frameworks and/or CDR regions are mutated into the derivative antibody The corresponding residues of the germline sequence, or the corresponding residues of another human germline sequence, or the conservative amino acid substitutions of the corresponding germline residues (the changes in these sequences are collectively referred to as "germline mutations" in the text). Those of ordinary skill in this technology, starting from the heavy and light chain variable region sequences disclosed in the text, can easily produce many antibodies and antigen-binding fragments including one or more individual germline mutations or combinations thereof. In certain embodiments, V_<b>H</b>And/or V_<b>L</b>All framework and/or CDR residues in the region are mutated back to residues found in the primordial germline sequence from which the antibody was derived. In other embodiments, only specific residues are mutated back to the original germline sequence, for example, mutated residues are only found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only Mutated residues were found in CDR1, CDR2 or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated into corresponding residues of a different germline sequence (ie, a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibody of the present invention may contain any combination of two or more germline mutations in the framework and/or CDR region, for example, where specific individual residues are mutated to the corresponding residues of a specific germline sequence, and Other residues with different primordial germline sequences are maintained as they are or mutated into corresponding residues of different germline sequences. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, antagonistic or agonistic biological properties, or Enhance (depending on the situation), cause immunity Decrease etc. The antibodies and antigen-binding fragments obtained by this general method are encompassed in the present invention.

The present invention also includes anti-PCSK9 antibodies, which include any variant of the HCVR, LCVR and/or CDR amino acid sequence disclosed in the text with one or more conservative substitutions. For example, the present invention includes anti-PCSK9 antibodies, which, relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed in the text, have, for example, 10 or less, 8 or less, 6 or more amino acid sequences. HCVR, LCVR and/or CDR amino acid sequences with fewer, 4 or fewer conservative amino acid substitutions.

The term "epitope" refers to an epitope that interacts with a specific antigen binding site called a paratope in the variable region of an antibody molecule. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions of the antigen and can have different biological effects. Epitopes can be conformational or linear. The conformational epitope is generated by the spatially juxtaposed amino acids of different line segments of the linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in the polypeptide chain. In certain cases, the epitope may include a carbohydrate group, a phosphatidyl group or a sulfonyl group on the antigen.

The term "substantially identical" or "substantially identical" when referring to nucleic acids or fragments thereof means that when an appropriate nucleotide insertion or deletion is optimally aligned with another nucleic acid (or its complementary strand), Any well-known sequence identity algorithm, such as FASTA, BLAST or Gap as discussed below, has at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of nucleotide bases The base has nucleotide sequence identity. A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule, under certain circumstances, can encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term "substantially similar" or "substantially similar" refers to two peptide sequences that share at least 95% sequence identity when the GAP or BESTFIT program uses the built-in gap weight to optimize the alignment Sex, very preferably at least 98% or 99% sequence identity. Preferably, the positions of different residues are different in the conservative amine Base acid substitution. "Conservative amino acid substitution" refers to the substitution of an amino acid residue by another amino acid residue with a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally speaking, conservative amino acid substitutions will not substantially change the functional properties of the protein. In the case of different amino acid sequences with two or more conservative substitutions to each other, the percentage of sequence identity or similarity can be adjusted to modify the nature of the conservative substitution. The method of adjustment is well-known to those who are familiar with this technique. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups with side chains with similar chemical properties include (1) lipid side chains: glycine, alanine, valine, leucine and isoleucine; (2) lipid-hydroxyl Side chains: serine and threonine; (3) side chains containing amines: aspartic acid and glutamic acid; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; ( 5) Basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamine, and (7) sulfur-containing side chains with cysteine and methyl Thiamine. The preferred conservative amino acid substituent groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine Acid-aspartic acid and aspartic acid-glutamic acid. In addition, conservative substitutions are any changes that have positive values in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445 (which is incorporated herein by reference). "Moderate conservativeness" is replaced by any change that has a non-negative value in the PAM250 log-likelihood matrix.

The sequence similarity of polypeptides, which also refers to sequence identity, is typically measured using sequence analysis software. Protein analysis software uses similar metrics assigned to various substitutions, deletions, and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can use default parameters to determine the sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different biological species, or wild-type and its mutant proteins. sex. See, for example, GCG Version 6.1. Peptide sequence can also use FASTA, using default or suggested parameters, a program in GCG Version 6.1, for comparison. FASTA (e.g. FASTA2 And FASTA3) provide an alignment of the best overlap between the query sequence and the search sequence and the percent sequence identity (Pearson (2000) supra). When the sequence of the present invention is compared with a database containing a larger number of sequences from different organisms, another preferred algorithm is the computer program BLAST with default parameters, especially BLASTP or TBLASTN. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, each of which is incorporated herein by reference.

<b>Anti-PCSK9 antibody with pH-dependent binding properties</b>

The present invention provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding properties. As used in the text, the term "with pH-dependent binding properties" refers to antibodies or antigen-binding fragments that "compared to neutral pH, at acidic pH, binding to PCSK9 decreases" (for the purpose of this disclosure, The two words can be used interchangeably). For example, "with pH-dependent binding properties" include antibodies and antigen-binding fragments thereof that bind to PCSK9 with greater affinity at neutral pH than at acidic pH. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention are at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 at neutral pH than at acidic pH. , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more times the affinity binds to PCSK9. The term "antibody with pH-dependent binding properties" also includes antibodies with "intermediate pH-dependent binding properties" as defined elsewhere in the text.

For the purpose of this disclosure, the "affinity" of the antibody to the antigen (such as PCSK9) is based on the K of the antibody_DExpress. K of antibody_DRefers to the equilibrium dissociation constant of the antibody-antigen interaction. In terms of antibody binding to its antigen K_DThe larger the value, the weaker the binding affinity of the antibody for this specific antigen. Therefore, as used in the text, "has a higher affinity at neutral pH than at acidic pH" (or equivalent term "pH-dependent binding") refers to the K that the antibody binds to PCSK9 when it is acidic._DGreater than the K of the antibody binding to PCSK9 at neutral_D. For example, in the context of the present invention, if the K of the antibody binds to PCSK9 at acidic pH_DThe K of antibody binding to PCSK9 when the pH is greater than neutral_DAt least about 3 times, The anti-system is considered to bind to PCSK9 with higher affinity at neutral pH than at acidic pH. Therefore, the present invention includes the K in which the antibody and its antigen-binding fragment bind to PCSK9 at acidic pH_DK for binding of antibody to PCSK9 at neutral pH_DAt least about 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100 or more times (which means that the affinity of the antibody or antigen-binding fragment thereof to PCSK9 at neutral pH is greater than that at acidic pH by at least about 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or More times).

The binding properties of antibodies to specific antigens can also be determined by the k_dTo represent. K of antibody_dRefers to the dissociation rate constant of the antibody for a specific antigen and is expressed as the reciprocal of seconds (i.e., sec^-1). k_dAn increase in the value means that the antibody binds to its antigen weaker. The present invention therefore includes that compared to neutral pH, antibody binding to PCSK9 has a higher k at acidic pH._dvalue. The present invention includes the binding of antibodies and antigen-binding fragments thereof to PCSK9 at acidic pH_dThan when the antibody binds to PCSK9 at neutral pH_dAt least about 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100 or more times.

The binding properties of an antibody to a specific antigen can also be expressed by the t½ of the antibody. The t½ of an antibody refers to the half-life of the antibody-antigen interaction. Therefore, according to the present invention, antibodies with "pH-dependent binding properties" or equivalent words "compared to neutral pH, binding to PCSK9 at acidic pH is reduced", including antibodies that bind to PCSK9 at acidic pH ratios Shorter antibodies at neutral pH. For example, the present invention includes that the t½ of the antibody or its antigen-binding fragment that binds to PCSK9 at acidic pH is at least 5 times shorter than the t½ of the antibody binds to PCSK9 at neutral pH. For example, the present invention includes that the t½ of the antibody and its antigen-binding fragment bound to PCSK9 at acidic pH is at least about 5, 6, 7, 8, 9, 10, 15, and t½ shorter than the t½ at which the antibody binds to PCSK9 at neutral pH. 20. 25, 30, 35, 40, 45, 50, 55, 60 or more times. As shown in the example, if the anti-PCSK9 antibody has a t½ of 21 minutes at a neutral pH and a t½ of 3 minutes at an acidic pH, then for the purposes of this disclosure, the antibody binds to PCSK9 at an acidic pH of t½. It is 7 times shorter than the t½ of antibody binding to PCSK9 at neutral pH [ie 21 minutes divided by 3 minutes].

Under certain circumstances, "compared to neutral pH, binding to PCSK9 decreases at acidic pH" is the K of the antibody binding to PCSK9 at acidic pH_DValue and the K of antibody binding to PCSK9 at neutral pH_DValues are expressed as a ratio (or vice versa). For example, for the purpose of the present invention, if the antibody or antigen-binding fragment thereof has an acidic/neutral K of about 3.0 or greater_DRatio, the antibody or its antigen-binding fragment can be considered to have "compared to neutral pH, binding to PCSK9 is reduced at acidic pH". In a specific exemplary embodiment, the acidic/neutral K of the antibody or antigen-binding fragment thereof of the present invention_DThe ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

Under certain circumstances, "compared to neutral pH, binding to PCSK9 decreases at acidic pH" is the result of antibody binding to PCSK9 at acidic pH._dValue and k of antibody binding to PCSK9 at neutral pH_dValues are expressed as a ratio (or vice versa). For example, for the purpose of the present invention, if the antibody or antigen-binding fragment thereof has an acidic/neutral k of about 3.0 or greater_dRatio, the antibody or its antigen-binding fragment can be considered to have "compared to neutral pH, binding to PCSK9 is reduced at acidic pH". In a specific exemplary embodiment, the acid/neutral k of the antibody or antigen-binding fragment thereof of the present invention_dThe ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0.25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or higher.

Under certain circumstances, the "compared to neutral pH, the binding of PCSK9 to PCSK9 is reduced at acidic pH" to determine the t½ value of antibody binding to PCSK9 at acidic pH and the t½ value of antibody binding to PCSK9 at neutral pH. Values are expressed as a ratio (or vice versa). For example, for the purposes of the present invention, if the antibody or its antigen-binding fragment has an acidic/neutral t½ ratio of about 0.2 or less, the antibody or its antigen-binding fragment can be considered to have "compared to neutral pH, at acidic pH). Binding to PCSK9 decreases at pH". In specific exemplary embodiments, the acid/neutral t½ ratio of the antibody or antigen-binding fragment thereof of the present invention may be about 0.20, 0.15, 0.14.0.12, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 or lower.

Under certain circumstances, the antibody of the present invention can have a lower affinity (that is, a higher K_D) And the shorter t½ combined with PCSK9. For example, the present invention includes that it binds to PCSK9 with at least 5 times higher affinity at neutral pH than at acidic pH, and t½ at acidic pH is at least 5 times shorter than the t½ at which the antibody binds to PCSK9 at neutral pH. The antibody. However, in certain cases, it has a higher binding affinity to PCSK9 at neutral pH than at acidic pH (such as K_DValue indicated), it may not necessarily have a shorter t½ at acidic pH compared to neutral pH.

As used herein, the term "acidic pH" refers to a pH of 6.0 or lower (e.g., lower than about 6.0, lower than about 5.5, lower than about 5.0, etc.). The term "acidic pH" includes about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or Lower pH.

As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

K_DValue, k_dThe value and t½ time, as shown in the text, can be measured using a surface plasmon resonance-based biosensor to determine the antibody-antigen interaction Use the characteristics (see, for example, Example 3 in the text). K_DValue, k_dThe value and t½ time can be measured at 25°C or 37°C.

It has been found that, compared to neutral pH, antibodies and antigen-binding fragments that have decreased binding to PCSK9 at acidic pH are compared to antibodies and antigen-binding fragments that do not have decreased binding to PCSK9 at acidic pH compared to neutral pH. The combined fragments have improved pharmacokinetic properties. For example, as verified by the operation examples provided in the text, the specific antibody of the present invention, compared with neutral pH, has a decreased binding to PCSK9 at acidic pH, and when administered to an animal subject, compared to not having a pH-dependent Anti-PCSK9 antibody with sexual binding properties has a slower circulation clearance rate. According to this aspect of the present invention, there is provided an antibody with a decrease in binding to PCSK9 at acidic pH compared to neutral pH, and an antibody with no decrease in binding to PCSK9 at acidic pH compared to neutral pH, The line has at least 2 times slower circulation clearance rate. The clearance rate can be expressed by the half-life of the antibody, where a slower clearance rate is related to a longer half-life. The present invention also includes an anti-PCSK9 antibody with reduced binding to PCSK9 at acidic pH compared to neutral pH, wherein the antibody, when administered to an animal expressing human PCSK9 at a dose of about 1 mg/kg, is administered A concentration greater than about 1.0 μg/ml can be detected in the animal's serum at least 30 days after the administration.

It was also found that antibodies and their antigen-binding fragments that have decreased binding to PCSK9 at acidic pH compared to neutral pH, compared to antibodies that do not have decreased binding to PCSK9 at acidic pH compared to neutral pH, and Its antigen-binding fragment has improved and lasting cholesterol-lowering activity. For example, the present invention provides an anti-PCSK9 antibody that provides long-lasting LDL-C lowering ability compared to antibodies and antigen-binding fragments thereof that do not have reduced binding to PCSK9 at acidic pH. According to a specific embodiment of the present invention, an anti-PCSK9 antibody is provided, which when administered to a subject at a dose of about 10 mg/kg, reduces the amount of serum LDL-C by at least 25% compared to baseline and maintains this reduced serum LDL -C amount is at least 25 days. In specific cases, an anti-PCSK9 antibody is provided, which when administered to a subject at a dose of about 10 mg/kg, is comparable to baseline Compared with reducing the amount of serum LDL-C by at least 25% and maintaining this reduced amount of serum LDL-C, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more days. As used herein, the term "baseline" when it refers to LDL-C (or other related parameters) refers to the subject's serum before the anti-PCSK9 antibody (or other comparative therapeutic intervention) is administered to the subject The amount of LDL-C.

The inventors have found that, at least in certain therapeutic situations, a too high degree of pH sensitivity for binding to PCSK9 may be harmful to the antibody. That is, under certain circumstances, it may be desirable to bind the antibody with a lower affinity at acidic pH compared to neutral pH, but still retain a certain degree of binding affinity for PCSK9 at acidic pH. Therefore, according to a specific embodiment of the present invention, an anti-PCSK9 antibody with intermediate pH-dependent binding properties is provided.

As used in the text, the term "intermediate pH-dependent binding properties" means that the antibody or its antigen-binding fragment has an acidic/neutral K greater than 3.0 but less than 8.0_Dratio. In certain exemplary embodiments, the acidic/neutral K of the antibody with "intermediate pH-dependent binding properties"_DThe ratio is between 3.5 and 8.0; between 4.0 and 8.0; between 4.5 and 8.0; between 5.0 and 8.0; between 5.5 and 8.0; between 6.0 and 8.0; between 6.5 and 8.0; between 3.0 and 7.5; Between 3.0 and 7.0; between 3.0 and 6.5; between 3.0 and 8.0; between 3.5 and 7.5; between 4.0 and 7.0; between 4.5 and 7.0; between 5.0 and 7.0; or between 4.5 and 6.5. In certain exemplary embodiments, the acidic/neutral K of an antibody with "intermediate pH-dependent binding properties"_DThe ratio is about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. Anti-PCSK9 antibodies with "intermediate pH-dependent binding properties" may also have an acid/neutral t½ ratio of less than about 1.0 but greater than about 0.15. To determine whether an antibody has "intermediate pH-dependent binding properties" as defined in the text, acid/neutral K can be determined by surface plasmon resonance at 25°C_DRatio and/or acid/neutral t½ ratio.

As used herein, anti-PCSK9 antibodies with "intermediate pH-dependent binding properties" also include, as measured by surface plasmon resonance, at acidic pH (e.g. pH 6.0) and 25°C for less than about 35 minutes but Antibodies and antigen-binding fragments that bind to PCSK9 with t½ greater than 10.5 minutes. For example, the present invention includes anti-PCSK9 antibodies with "intermediate pH-dependent binding properties", which are less than about 20 minutes but greater than 10 minutes at acidic pH (e.g., pH 6.0) and 25°C; 20 minutes but greater than about 11 minutes; less than about 20 minutes but greater than about 12 minutes; less than about 20 minutes but greater than about 13 minutes; less than about 20 minutes but greater than about 14 minutes; less than about 20 minutes but greater than about 15 Minutes; less than about 30 minutes but greater than about 11 minutes; less than about 25 minutes but greater than about 12 minutes; less than about 18 minutes but greater than about 14 minutes; less than about 16 minutes but greater than about 13 minutes; or less than about T½ of 16 minutes but greater than about 14 minutes is combined with PCSK9.

Antibodies with "intermediate pH-dependent binding properties" also include those at acidic pH (eg pH 6.0) and 25°C for approximately 10.5 minutes, approximately 11.0 minutes, approximately 11.5 minutes, approximately 12.0 minutes, approximately 12.5 minutes, approximately 13.0 Minutes, about 13.5 minutes, about 14.0 minutes, about 14.5 minutes, about 15.0 minutes, about 15.5 minutes, about 16.0 minutes, about 16.5 minutes, about 17.0 minutes, about 17.5 minutes, about 18.0 minutes, about 18.5 minutes, about 19.0 minutes, About 19.5 minutes, about 20.0 minutes, about 20.5 minutes, about 21.0 minutes, about 22.0 minutes, about 23.0 minutes, about 24.0 minutes, about 25.0 minutes, about 26.0 minutes, about 27.0 minutes, about 28.0 minutes, about 29.0 minutes, about 30.0 Minutes, about 31.0 minutes, about 32.0 minutes, about 33.0 minutes, about 34.0 minutes or 35.0 minutes t½ of antibodies that bind to PCSK9.

<b>PH-dependent anti-PCSK9 antibody with histidine substitution</b>

The present invention provides anti-PCSK9 antibodies with pH-dependent binding properties, wherein these antibodies have one or more amino acid differences compared to the parental anti-PCSK9 antibodies. As used in the text, a "maternal" anti-PCSK9 antibody is an antibody that does not show pH-dependent binding properties or only shows intermediate pH-dependent binding properties. -PCSK9 antibody (for example, where the binding affinity of the parent antibody to PCSK9 at neutral pH is not greater than 3 times the binding affinity of the antibody to PCSK9 at acidic pH; or where the binding affinity of the parent antibody to PCSK9 at neutral pH is not Shorter than 3 times the t½ of the antibody binding to PCSK9 at acidic pH). In some cases, the "maternal" anti-PCSK9 antibody may be an anti-PCSK9 antibody that has increased binding to PCSK9 at acidic pH compared to neutral pH. In some embodiments, the "maternal" anti-PCSK9 antibody is produced by standard antibody production/isolation methods (e.g., mouse immunization, phage expression, etc.) without artificial Introduce any amino acid modification resulting from the antibody.

According to this aspect of the invention, an anti-PCSK9 antibody with pH-dependent binding properties may have one or more amino acid variations relative to the parental anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with pH-dependent binding properties may contain one or more histidine substitutions or insertions, such as in the CDRs of one or more maternal anti-PCSK9 antibodies. Therefore, according to a specific embodiment of the present invention, an anti-PCSK9 antibody is provided, which includes a CDR amino acid sequence (such as a heavy chain and a light chain CDR) that is the same as the CDR amino acid sequence of the parental anti-PCSK9 antibody, but One or more amino acids of one or more CDRs of the parent antibody are substituted with histidine residues. Anti-PCSK9 antibodies with pH-dependent binding, in a single CDR of the maternal antibody or the distribution of maternal anti-PCSK9 antibodies in the entire majority (for example, 2, 3, 4, 5, or 6) CDRs may have For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more histidine substitutions. For example, the present invention includes anti-PCSK9 antibodies with pH-dependent binding, which include one or more histidine substitutions in HCDR1 of the parental anti-PCSK9 antibody, and one or more histidine acids in HCDR2 Substitution, or one or more histidine in HCDR3, one or more histidine in LCDR1, one or more histidine in LCDR2, one or more histidine in LCDR3 .

A "maternal" anti-PCSK9 antibody package that can be modified, mutated, or otherwise engineered to have pH-dependent binding properties (or enhanced pH-dependent binding properties) Including: anti-PCSK9 antibody containing any complementarity determining region (CDR) or heavy chain and chain variable region (HCVR/LCVR) disclosed in U.S. Patent No. 8,062,640 (also summarized in Table 1 of Example 1 in the text ). A specific example of a maternal anti-PCSK9 antibody with only intermediate pH-dependent binding properties is the antibody referred to herein (and in US Patent No. 8,062,640) as "300N". 300N contains the HCVR/LCVR amino acid sequence with SEQ ID NO:218/226, and the heavy chain and light chain CDR sequences (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3). Therefore, any HCVR/LCVR amino acid sequence pair comprising SEQ ID NO: 218/226, or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 amine of SEQ ID NO: 220, 222, 224, 228, 230, 232 The anti-PCSK9 antibody or its antigen-binding fragment of the base acid sequence can be modified at the amino acid sequence level (for example, with one or more histidine substitutions and/or insertions in one or more CDRs) to produce Suitable maternal antibody for anti-PCSK9 antibody with pH-dependent binding properties.

Alternatively, any anti-PCSK9 antibody or antigen-binding fragment thereof, which includes the HCVR/LCVR amino acid sequence pair, or any of the examples described in US Patent No. 8,062,640 (to be summarized in Example 1 and Table 1 in the text) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 amino acid sequence of the anti-PCSK9 antibody can also be modified at the amino acid sequence level (for example, one or more histamines are included in one or more CDRs). Acid substitution and/or insertion) to produce a suitable maternal antibody of anti-PCSK9 antibody or antigen-binding fragment thereof with pH-dependent binding properties.

In a specific embodiment, the present invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof that exhibits pH-dependent binding properties and includes a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein The HCVR line includes any SEQ ID NO: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450, 454, 458, 474, 478, 482, 498, 502, 506,522,526,530,546,550,554,570,574,578,594,598,602,618,622,626,642,646,650,666,670,674,690,694,698, The amino acid sequences of 714, 718, 722, 738 and 742, or one or more of the amino acids in one or more of the heavy chain CDRs are of any of the foregoing amino acid sequences substituted with histidine residues Variants; and the LCVR line includes any SEQ ID NO: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260, 264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456, 466, 476, 480, 490, 500, 504, 514, 524, 528, 538, 548, 552, 562, 572, 576, 586, 596, 600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692, 696, 706, 716, 720, 730, 740 and 744 amino acid sequences, or one or more of them One or more amino acids in the light chain CDR are variants of any of the foregoing amino acid sequences substituted with histidine residues.

For example, the present invention provides a variant of an exemplary parental anti-PCSK9 antibody called "300N" (that is, a variant of an antibody comprising the HCVR/LCVR amino acid sequence pair of SEQ ID NO:218/226). In particular, the present invention provides anti-PCSK9 antibodies or antigen-binding fragments thereof that exhibit pH-dependent binding properties and include a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR system includes SEQ ID NO: 218 or a variant of SEQ ID NO: 218 comprising one or more amino acid substitutions selected from the group consisting of N52H, Q53H, I100H, V101H, V104H, D106H, M107H, D108H, and Y112H ; And the LCVR system includes SEQ ID NO: 226 or a variant of SEQ ID NO: 226 substituted with one or more amino acids selected from the group consisting of: L29H, L30H, N33H, G34H, Y37H, L97H, T99H and P100H.

According to an exemplary embodiment, the present invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof that exhibits pH-dependent binding properties and includes a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein The HCVR system includes a variant of SEQ ID NO: 218 that includes a D106H amino acid substitution, and the LCVR system includes SEQ ID NO: 226. The D106H amino acid substitution is located in the heavy chain CDR3 (HCDR3). The variant HCDR3 including the D106H amino acid substitution is represented by the amino acid sequence of SEQ ID NO: 788 shown in Table 3 in the text.

According to another exemplary embodiment, the present invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof having pH-dependent binding properties and including a heavy chain variable region (HCVR) and a light chain variable region (LCVR), The HCVR system includes SEQ ID NO: 218, and the LCVR system includes a variant of SEQ ID NO: 226 including an L30H amino acid substitution. The L30H amino acid substitution is located in the light chain CDR1 (LCDR1). The variant LCDR1 including the L30H amino acid substitution is represented by the amino acid sequence of SEQ ID NO: 802 shown in Table 3 in the text.

According to another exemplary embodiment, the present invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof that exhibits pH-dependent binding properties and includes a heavy chain variable region (HCVR) and a light chain variable region (LCVR), The HCVR system includes a variant of SEQ ID NO: 218 including a D106H amino acid substitution, and the LCVR includes a variant of SEQ ID NO: 226 including a L30H amino acid substitution.

In a specific embodiment, the present invention provides a solution that exhibits pH-dependent binding characteristics and includes 3 heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and 3 light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). The anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the HCDR1 line comprises SEQ ID NO: 220 (maternal); wherein the HCDR2 line comprises SEQ ID NO: 222 (maternal), 772 (N52H) and 773 (Q53H) The amino acid sequence selected from the group; wherein HCDR3 contains Selected from the group consisting of SEQ ID NO: 224 (maternal), 782 (I100H), 783 (V101H), 786 (V104H), 788 (D106H), 789 (M107H), 790 (D108H) and 794 (Y112H) The amino acid sequence; wherein LCDR1 is selected from the group consisting of SEQ ID NO: 228 (maternal), 801 (L29H), 802 (L30H), 804 (N33H), 805 (G34H) and 808 (Y37H) The amino acid sequence; where LCDR2 contains SEQ ID NO: 230 (maternal source); and where LCDR3 contains SEQ ID NO: 232 (maternal source), 815 (L97H), 817 (T99H) and 818 (P100H) The amino acid sequence selected from the group consisting of.

According to specific embodiments, the present invention provides antibodies that exhibit pH-dependent binding characteristics and include 3 heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and 3 light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). -PCSK9 antibody or antigen-binding fragment thereof, wherein HCDR1 line comprises SEQ ID NO: 220 (maternal origin); wherein HCDR2 line comprises SEQ ID NO: 222 (maternal origin); wherein HCDR3 line comprises SEQ ID NO: 224 (maternal origin) Or 788 (D106H); wherein LCDR1 contains SEQ ID NO: 228 (maternal source) or 802 (L30H); wherein LCDR2 contains SEQ ID NO: 230 (parent source); and wherein LCDR3 contains SEQ ID NO: 232 ( Mother source).

According to specific embodiments, the present invention provides antibodies with pH-dependent binding properties and comprising 3 heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and 3 light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3). -PCSK9 antibody or antigen-binding fragment thereof, wherein the HCDR1 line comprises SEQ ID NO: 220 (maternal origin); wherein the HCDR2 line comprises SEQ ID NO: 222 (maternal origin); wherein the HCDR3 line comprises SEQ ID NO: 788 (D106H); Wherein LCDR1 contains SEQ ID NO: 228 (maternal source); wherein LCDR2 contains SEQ ID NO: 230 (maternal source); and LCDR3 contains SEQ ID NO: 232 (maternal source).

According to specific embodiments, the present invention provides antibodies with pH-dependent binding properties and comprising 3 heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and 3 light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3). -PCSK9 The antibody or antigen-binding fragment thereof, wherein the HCDR1 line comprises SEQ ID NO: 220 (maternal origin); wherein the HCDR2 line comprises SEQ ID NO: 222 (maternal origin); wherein the HCDR3 line comprises SEQ ID NO: 224 (maternal origin); wherein LCDR1 includes SEQ ID NO: 802 (L30H); wherein LCDR2 includes SEQ ID NO: 230 (maternal source); and LCDR3 includes SEQ ID NO: 232 (maternal source).

According to specific embodiments, the present invention provides antibodies with pH-dependent binding properties and comprising 3 heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and 3 light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3). -PCSK9 antibody or antigen-binding fragment thereof, wherein HCDR1 line comprises SEQ ID NO: 220 (maternal origin); wherein HCDR2 line comprises SEQ ID NO: 222 (maternal origin); wherein HCDR3 line comprises SEQ ID NO: 788 (D106H); Wherein, LCDR1 includes SEQ ID NO: 802 (L30H); where LCDR2 includes SEQ ID NO: 230 (maternal source); and where LCDR3 includes SEQ ID NO: 232 (maternal source).

<b>Anti-PCSK9 antibodies including Fc variants</b>

According to a specific embodiment of the present invention, there is provided an anti-PCSK9 antibody comprising an Fc region, wherein the Fc region comprises, for example, one or more of enhancing or reducing the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. Mutations. For example, the present invention includes C in the Fc region_H2 or C_HRegion 3 contains a mutant anti-PCSK9 antibody, where this mutation increases the affinity of the Fc region to FcRn in an acidic environment (for example, in endosomes where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of these Fc modifications include, for example, positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., , S or T) and 256 (for example, S/R/Q/E/D or T) modification; or position 428 and/or 433 (for example, H/L/R/S/P/Q or K) and / Or modification of 434 (for example, H/F or Y); or modification of position 250 and/or 428; or modification of position 307 or 308 (for example, 308F, V308F) and 434. In one embodiment, this modification includes 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (E.g., H433K) and 434 (e.g., 434Y) modifications 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and/or 308 modifications (e.g., , 308F or 308P).

The present invention includes anti-PCSK9 antibodies comprising two of the following: (1) A variant CDR sequence comprising one or more histidine substitutions, which reduces the binding of the antibody to PCSK9 at acidic pH compared to neutral pH Affinity; and (2) a variant Fc region sequence containing one or more mutations, which increases the affinity of the Fc region for FcRn at acidic pH compared to neutral pH. According to this aspect of the present invention, a heavy chain or light chain variable region (HCVR/LCVR) or CDR (see, for example, Table 3) containing any histidine substitution as described herein can be constructed, and an Fc containing any of the above mutations can be constructed Region of anti-PCSK9 antibody, where these mutations make the Fc region bind to FcRn with greater affinity at acidic pH. For example, the present invention includes anti-PCSK9 antibodies containing CDR amino acid sequences, for example, the histidine variant anti-PCSK9 antibody called "VH-D106H" in the text or the histamine called "VK-L30H" in the text An acid variant anti-PCSK9 antibody, and an Fc region including one or more mutations selected from the group consisting of: T250Q/M248L; M252Y/S254T/T256E; M428L/N434S; and H433K/N434F. All possible CDR histidine substitution mutations and Fc region mutations described in the text are included in the scope of the present invention.

<b>Biological properties of antibodies</b>

In addition to having pH-dependent binding properties, the anti-PCSK9 antibody of the present invention may also have one or more additional beneficial biological properties. For example, the present invention includes anti-PCSK9 antibodies that effectively block the interaction between PCSK9 and low-density lipoproprotein receptor (LDLR). In a specific embodiment, the antibody of the present invention is less than about 1 nM at neutral pH, for example, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, IC of less than about 400pM, less than about 300pM, less than about 200pM, or less than about 100pM₅₀Block the interaction between PCSK9 and LDLR, for example, using the one described in Example 4 in the text Measured by blocking ELISA or a substantially similar analysis mode.

In a specific embodiment, the antibody of the present invention can block the PCSK9/LDLR interaction more effectively at neutral pH than at acidic pH (for example, at acidic pH, it reflects that the binding of the antibody to PCSK9 is reduced). The ability of anti-PCSK9 antibodies to block the PCSK9/LDLR interaction can be determined by IC₅₀Value to quantify, for example, at neutral and acidic pH. (See, for example, Example 4 in the text). Compared to acidic pH, the degree to which the antibody blocks the PCSK9/LDLR interaction at neutral pH can be determined by the antibody IC measured at acidic pH.₅₀Value and antibody IC measured at neutral pH₅₀The ratio of the value is expressed. Higher acid/neutral IC in this type of analysis mode₅₀The ratio reflects the decreased ability to block the PCSK9/LDLR interaction at acidic pH compared to neutral pH. Therefore, the present invention includes anti-PCSK9 antibodies, in which, as measured using the analysis mode of Example 4 or a substantially similar analysis, the antibody system is greater than about 1, greater than about 5, greater than about 10, greater than about 20, and greater than about 30. , Greater than about 32, greater than about 34, greater than about 36, greater than about 38, greater than about 40, greater than about 50, greater than about 60, greater than about 70, greater than about 80, greater than about 90, greater than about 100, greater than about 110, greater than About 120, greater than about 130, greater than about 140, greater than about 150, greater than about 160, greater than about 170, greater than about 180, greater than about 190, greater than about 200, greater than about 210, greater than about 220, greater than about 230 or greater The acid/neutral IC50 ratio blocks the PCSK9/LDLR interaction.

The present invention also includes anti-PCSK9 antibodies with pH-dependent binding properties, wherein the antibody system blocks the inhibition of PCSK9-mediated LDL absorption. The cell-based LDL uptake analysis, as shown in Example 5, can be used to determine whether the anti-PCSK9 antibody can block the inhibition of PCSK9-mediated LDL uptake and/or its extent. According to a specific embodiment, an anti-PCSK9 antibody with pH-dependent binding properties is provided, wherein the antibody, for example, as measured using the in vitro LDL absorption analysis described in Example 5 herein or a substantially similar analysis, Can be less than about 40nM, less than about 35nM, less than about 30nM, less than about 25nM, less than about 20nM, low IC of about 15nM or less than about 10nM₅₀Blocks the inhibitory effect of PCSK9-mediated LDL absorption.

The antibody of the present invention may have one or more of the aforementioned biological properties, or any combination thereof. The biological properties of the antibodies of the invention listed above are not exhaustive. Those who are familiar with this technology will better understand other biological characteristics of the antibody of the present invention by reviewing this disclosure (including the operation examples in the text).

<b>Method for producing antibodies with pH-dependent binding properties</b>

The present invention also provides methods for producing antibodies with pH-dependent binding properties. The method according to this aspect of the invention includes screening for antibodies with at least intermediate pH-dependent binding properties and then subjecting the antibody to further mutagenesis to increase the pH-dependence of the antibody to its antigen. The screening step can include any method or process, whereby antibodies with intermediate pH-dependent binding properties can be identified among the antibody populations specific for a particular antigen. In a specific embodiment, the initial antibody population is obtained by immunizing animals or by screening a phage expression database for antibodies that specifically bind to a specific relevant antigen. These antibodies, in specific embodiments, may be fully human antibodies. In a specific embodiment, the screening step includes measuring one or more binding parameters of individual antibodies in the initial antibody group at acidic pH and neutral pH (e.g., K_DOr t½). The binding parameters of antibodies can be measured using, for example, surface plasmon resonance or any other analytical method that can quantitatively and qualitatively evaluate the binding properties of antibodies to specific antigens. According to a specific embodiment of this aspect of the present invention, the screening step includes identifying an acid/neutral K greater than about 3.0 but less than about 8.0_DThe ratio of the antibody bound to the antigen. Alternatively, this screening step may include identifying antibodies that bind to the antigen with an acid/neutral t½ ratio of less than about 1.0 but greater than about 0.15. In still other embodiments, this screening step may include identifying antibodies that have a t½ of less than 40 minutes but greater than 20 minutes (e.g., at 25°C) at an acidic pH (e.g., pH 6.0).

Once an antibody with intermediate pH-dependent binding properties is identified, the identified antibody is subjected to mutagenesis to increase the antibodys pH-dependence on the antigen Sexual combination. "Enhanced pH-dependent binding" means that the mutant version of the antibody has a larger acidic/neutral K than the original maternal (i.e., intermediate pH-dependent) version of the antibody before mutagenesis._DRatio, or the smaller acid/neutral t½ ratio. In a specific embodiment, "enhanced pH-dependent binding" means that the t½ of the antibody binding to its antigen at acidic pH (for example, pH 6.0) is less than the t½ of the antibody before mutagenesis. In a specific embodiment, "enhanced pH-dependent binding" means that the t½ of the binding of an antibody to its antigen at acidic pH (for example, pH 6.0) is less than about 16 minutes, less than about 10 minutes, and less than about 5 minutes. Minutes, less than about 2 minutes or less than about 1.5 minutes (for example at 25°C).

According to this aspect of the present invention, the mutagenesis step may include deleting, substituting or adding an amino acid in the heavy chain and/or light chain of the antibody. According to specific embodiments, the mutagenesis is performed in one or more variable regions of the antibody, for example, in one or more CDRs. For example, mutagenesis can include substituting an amino acid in one or more of the CDRs of the antibody with another amino acid. In a specific embodiment, the mutagenicity includes substituting histidine for one or more amino acids in at least one CDR of the antibody.

In the operation example described in the text, the anti-PCSK9 antibody with pH-dependent binding properties (for example, the fully human anti-PCSK9 antibody) was produced using the screening/mutagenic method described above; however, according to this The method of this aspect of the invention can be used to produce antibodies with pH-dependent binding properties, which binding to any antigen should be useful or desirable in terms of pH-dependent properties. The method according to this aspect of the invention can be used to produce antibodies that have an extended serum half-life when administered to a subject or patient.

<b>"Bihistidine" (His-His) is mutagenic to produce pH-dependent antibodies</b>

Based on the specific experiments described in the text, it was unexpectedly discovered that in the CDR residues located adjacent to (for example, immediately above or below) the naturally-occurring histidine residues, the histidine substitution was introduced into the CDR of the antibody By this, a His-His amino acid sequence is generated, which can transform an antibody with intermediate pH-dependent binding characteristics into an antibody with more obvious pH-dependent binding characteristics. As used in the text, "more pronounced pH-dependent binding properties" means that the antibody, after the introduction of histidine substitution, is better than the introduction of histidine substitution. The front shows one or more of the following: (a) Greater acid/neutral K_DRatio; (b) greater acid/neutral k_dRatio; and/or (c) a smaller acid/neutral t½ ratio. For example, the antibody referred to herein as 300N has intermediate pH-dependent binding properties and contains a single naturally-occurring histidine at the fifth amino acid position of LCDR1 (see SEQ ID NO: 228). By introducing histidine into the fifth amino acid position of LCDR1 (produced the "VK-L30H" antibody containing LCDR1 with SEQ ID NO: 802), it was found that the generated antibody has a more pronounced pH than 300N. Dependent binding characteristics are shown in Examples 3A and 3B in the text. This "double-His" mutation strategy is an applicable method generally used to generate antibodies with obvious pH-dependent binding characteristics. Therefore, the present invention includes a method for improving the pH-dependent properties of antibodies, which includes selecting an antibody with intermediate pH-dependent binding properties and placing it at the amino acid position adjacent to the existing histidine residues. , The introduction of a set of amino acid substitutions into one or more of the CDRs of the antibody, thereby producing antibodies with more pronounced pH-dependent binding characteristics (for example, having larger Acid/Neutral K_Dratio). This method can also be applied to antibodies that normally lack any histamine residues in the CDRs, for example, introducing two or more histidine substitutions at adjacent amino acid positions in one or more CDRs.

<b>Epitope mapping and related technologies</b>

The present invention includes anti-PCSK9 antibodies that interact with one or more amino acids (amino acids 1 to 152 of SEQ ID NO: 755) found in the prodomain of PCSK9. The present invention also includes anti-PCSK9 antibodies that interact with one or more amino acids (153 to 425 amino acids of SEQ ID NO: 755) found in the catalytic region of PCSK9. The present invention also includes anti-PCSK9 antibodies that interact with one or more amino acids (426 to 692 amino acids of SEQ ID NO: 755) found in the C-terminal region of PCSK9. Under certain circumstances, the anti-PCSK9 antibody system of the present invention interacts with amino acids located in two adjacent regions of PCSK9. The epitope that binds to the antibody can be 3 or more (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acid Consisting of a single continuous sequence. In addition, the epitope can be composed of a plurality of non-contiguous amino acids (or amino acid sequences) located in one or more PCSK9 regions.

Various techniques known to those skilled in the art can be used to determine whether the antigen-binding region of an antibody interacts with "one or more amino acids" in a polypeptide or protein. Example techniques include, for example, the conventional cross-blocking analysis, such as described in<b>Antibodies</b>, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide ink spot analysis (Reineke (2004), Methods Mol Biol 248:443-463) and peptide cleavage analysis . In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids in polypeptides that interact with the antigen-binding region of antibodies is to detect hydrogen/deuterium exchange by mass spectrometry. Generally speaking, the hydrogen/deuterium exchange method involves deuterium-labeled related proteins, and then the antibody is bound to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange for all residues except for the residues protected by the antibody (which are still deuterium calibrated). After dissociating the antibody, the target protein is cleaved with protease and analyzed by mass spectrometry to find the deuterium-labeled residue, which is equivalent to the specific amino acid that interacts with the antibody. See, for example, Ehring (1999)<i>Analytical Biochemistry</i>267(2): 252-259; Engen and Smith (2001)<i>Anal.Chem.73</i>: 256A-265A.

The invention further includes anti-PCSK9 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein. For example, the present invention includes anti-PCSK9 antibodies (e.g., VH-G26H, VH-F27H, VH-T28H, VH-F29H, VH -S30H, VH-S31H, VH-W33H, VH-I51H, VH-N52H, VH-Q53H, VH-D54H, VH-G55H, VH-S56H, VH-E57H, VH-K58H, VH-A97, VH-R98H , VH-D99H, VH-I100H, VH-V101H, VH-L102H, VH-M103H, VH-V104H, VH-Y105H, VH-D106H, VH-M107H, VH-D108H, VH-Y109H, VH-Y110H, VH-Y111H, VH-Y112H, VH-G113H, VH-M114H, VH-D115H, VH-V116H, VK-Q27H, VK-S28H, VK-L29H, VK-L30H, VK- S32H, VK-N33H, VK-G34H, VK-N35H, VK-N36H, VK-Y37H, VK-L55H, VK-G56H, VK-S57H, VK-M94H, VK-Q95H, VK-T96H, VK-L97H, VK-Q98H, VK-T99H, VK-P100H, VK-L101H, VK-T102H). Similarly, the present invention also includes anti-PCSK9 antibodies that compete with any of the histidine substitution variant antibodies listed in Table 3 in the text to bind to PCSK9 (e.g., VH-G26H, VH-F27H, VH-T28H, VH-F29H, VH-S30H, VH-S31H, VH-W33H, VH-I51H, VH-N52H, VH-Q53H, VH-D54H, VH-G55H, VH-S56H, VH-E57H, VH-K58H, VH-A97, VH- R98H, VH-D99H, VH-I100H, VH-V101H, VH-L102H, VH-M103H, VH-V104H, VH-Y105H, VH-D106H, VH-M107H, VH-D108H, VH-Y109H, VH-Y110H, VH-Y111H, VH-Y112H, VH-G113H, VH-M114H, VH-D115H, VH-V116H, VK-Q27H, VK-S28H, VK-L29H, VK-L30H, VK-S32H, VK-N33H, VK- G34H, VK-N35H, VK-N36H, VK-Y37H, VK-L55H, VK-G56H, VK-S57H, VK-M94H, VK-Q95H, VK-T96H, VK-L97H, VK-Q98H, VK-T99H, VK-P100H, VK-L101H, VK-T102H).

By using conventional methods known in this technology, it is easy to determine whether an antibody binds to the same epitope as the reference anti-PCSK9 antibody or competes for binding with it. For example, to determine whether the test antibody binds to the same epitope as the reference anti-PCSK9 antibody of the present invention, the reference antibody binds to the PCSK9 protein. Next, the ability of the test antibody to bind to the PCSK9 molecule was evaluated. If the test antibody can bind to PCSK9 after saturation binding of the reference anti-PCSK9 antibody, it can be concluded that the test antibody system binds to a different epitope of the reference anti-PCSK9 antibody. On the other hand, if the test antibody cannot bind to the PCSK9 molecule after the reference anti-PCSK9 antibody is fully bound, the test antibody may bind to the reference anti-PCSK9 antibody of the present invention. Binding of the same epitope. Additional routine experiments (such as peptide mutation and binding analysis) can then be performed to determine whether the observed absence of test antibody binding is in fact due to binding to the same epitope as the reference anti-PCSK9 antibody, or whether it is Stereoblocking (or another phenomenon) caused no binding to be observed. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or other other quantitative or qualitative antibody-binding assays available in this technology. According to a specific embodiment of the present invention, as measured in a competitive binding analysis, if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of antibody inhibits the binding of another antibody by at least 50%, but more Preferably 75%, 90% or even 99%, the two antibody systems bind to the same (or overlapping) epitope (see, for example, Junghans et al., Cancer Res. 199050:1495-1502). In addition, if substantially reducing or eliminating all amino acid mutations in the antigen bound by one antibody is reducing or eliminating the binding of the other antibody, the two antibodies are considered to bind to the same epitope. If only one subgroup of amino acid mutations reduces or eliminates the binding of one antibody or eliminates the binding of another antibody, then the two antibodies are considered to have "overlapping epitopes."

In order to determine whether an antibody competes with the reference anti-PCSK9 antibody for binding, the above-mentioned binding method is performed in two orientations: the first orientation: let the reference antibody bind to PCSK9 under saturated conditions, and then evaluate the binding of the test antibody to the PCSK9 molecule . Second orientation: Let the test antibody bind to the PCSK9 molecule under saturated conditions, and then evaluate the binding of the reference antibody to the PCSK9 molecule. If in the two orientations, only the first (saturated) antibody can bind to the PCSK9 molecule, it is concluded that the test antibody and the reference antibody system compete for binding to PCSK9. Those of ordinary skill in this technology should understand that antibodies that compete with the reference antibody for binding may not necessarily bind to the same epitope as the reference antibody, but may block the reference stereoscopically by binding to overlapping or adjacent epitopes. The binding of antibodies.

<b>Preparation of human antibodies</b>

Methods of producing monoclonal antibodies (including full-length human monoclonal antibodies) are known in the art. Any of these known methods can be used in the context of the present invention, Used to produce human antibodies that specifically bind to human PCSK9. These antibodies can then be used as parent antibodies from which histidine substituted variant antibodies (e.g., histidine substituted variant antibodies with pH-dependent binding properties) can be derived.

Use VELOCIMMUNE^TMTechnology or any other known method of producing monoclonal antibodies, initially isolated chimeric antibodies with human variable regions and mouse constant regions with high affinity to PCSK9. The antibody is qualitative and selected based on the desired properties, including affinity, selectivity, epitope, etc. Replace the mouse constant region with the desired human constant region, such as wild-type or modified IgG1 or IgG4, to produce the fully human antibody of the present invention. When the constant region selected according to the specific application can be different, the high-affinity antigen binding and target specificity characteristics remain in the variable region.

<b>Bioequivalence</b>

In addition to the histidine substitutions specified in the text as examples, the present invention also encompasses antibodies with amino acid sequences that are different from the antibodies described in the text but retain the ability to bind to human PCSK9 with pH-dependent binding properties. These variant antibodies and antibody fragments, when compared with the parental sequence, include the addition, deletion or substitution of one or more amino acids, but have a biological activity substantially equivalent to that of the antibody. Similarly, the anti-PCSK9 antibody encoding DNA sequence of the present invention, when compared with the disclosed sequence, includes the addition, deletion or substitution of one or more nucleotides, but the anti-PCSK9 antibody encoded by it is or The antibody fragment is substantially bioequivalent to the anti-PCSK9 antibody or antibody fragment of the present invention.

Two kinds of antigen-binding proteins or antibodies, for example, if they are pharmaceutical equivalents or pharmaceutical substitutes, when they are administered in a single dose or multiple doses of the same molar dose under similar experimental conditions, their absorption rate and extent are not If it shows a significant difference, it is considered to be bioequivalent. Certain antibodies with similar absorption levels but different absorption rates should be regarded as equivalents or pharmaceutical substitutes, and can still be regarded as bioequivalent, because these differences in absorption rates are deliberate and reflected on the label. For example, the effective body drug concentration for long-term use is not necessary, and the research on specific drug products The above is regarded as not medically significant.

In one example, if the two antigen binding proteins do not have clinically meaningful differences in their safety, purity, and efficacy, they are bioequivalent.

In one embodiment, if compared to continuous treatment without such changes, the patient can make one or more changes between the reference product and the biological product without the risk of unexpected harmful effects (including clinically immunogenic Significant change or reduced utility), then the two antigen binding proteins are bioequivalent.

In one example, if two antigen-binding proteins are used for symptoms or symptoms by a common mechanism or mechanism of action, to the extent that these mechanisms are known, then the two are bioequivalent.

Bioequivalence can be verified by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals, which measure the concentration of antibodies or their metabolites in blood, plasma, serum or other biological fluids over time; (b) with humans In vivo bioavailability data are correlated and can reasonably predict the data in vitro; (c) In vivo tests in humans or other mammals, in which the appropriate acuteness of the antibody (or its target) that changes over time is measured Pharmacological effects; and (d) Well-controlled clinical trials have been established for the safety, efficacy, or bioavailability or bioequivalence of antibodies.

Bioequivalent variants of the anti-PCSK9 antibody of the present invention can be constructed by, for example, making substitutions of various residues or sequences, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues not required for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges due to denaturation. Among other things, bioequivalent antibodies may include anti-PCSK9 antibody variants that include amino acid changes that modify the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

<b>Species selectivity and species cross-reactivity</b>

According to a specific embodiment of the present invention, the anti-PCSK9 anti-system is compatible with human PCSK9 binds but does not bind to PCSK9 from other species. The invention also includes anti-PCSK9 antibodies that bind to human PCSK9 and PCSK9 from one or more non-human species. For example, the anti-PCSK9 antibody of the present invention can bind to human PCSK9, and can be combined with or without (as the case may be) one or more kinds of mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, Goat, sheep, cattle, horse, camel, macaque, marmoset, rhesus or chimpanzee PCSK9 combined.

<b>Multispecific antibodies</b>

The antibodies of the present invention can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a target polypeptide or may contain antigen binding regions specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The anti-PCSK9 antibody of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked to one or more other molecular entities, such as another antibody or antibody fragment (for example, by chemical coupling, gene fusion, non-covalent linkage, or other), resulting in a second binding Specific bispecific or multispecific antibodies. For example, the present invention includes bispecific antibodies, wherein one arm of the immunoglobulin is specific to human PCSK9 or a fragment thereof, and the other arm of the immunoglobulin is specific to the second therapeutic target, or has a curative effect Component bonding.

Exemplary bispecific antibody models that can be used in the context of the present invention include the use of the first immunoglobulin (Ig) C_HZone 3 and second Ig C_HZone 3, where the first and second Ig C_HIn the 3 regions, at least one amino acid is different from each other, and compared with the bispecific antibody without the amino acid difference, at least one amino acid difference reduces the binding of the bispecific antibody to protein A. In one embodiment, the first Ig C_HRegion 3 binds protein A and second Ig C_HRegion 3 contains mutations that reduce or eliminate protein A binding, such as H95R modification (IMGT exon numbering; EU numbering is H435R). Second C_H3 may further include Y96F modification (IMGT; EU is Y436F). In the second C_HAdditional modifications can be found in 3, For IgG1 antibodies, the lines include: D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; EU is D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, the lines include: N44S, K52N, and V82I (IMGT; EU is N384S, K392N and V422I); and for IgG4 antibodies, it includes: Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (IMGT; EU is Q355R, N384S, K392N, V397M, R409K, E419Q and V422I). Variations in the above-mentioned bispecific antibody model are encompassed within the scope of the present invention.

<b>Therapeutic formulations and administration</b>

The present invention provides a pharmaceutical composition comprising the anti-PCSK9 antibody of the present invention or an antigen-binding fragment thereof. The pharmaceutical composition of the present invention is formulated with suitable carriers, excipients and other agents that provide improved transfer, transportation, tolerance and similar properties. Many suitable formulations can be found in the formulae known to all medicinal chemists: Remington's Pharmaceutical Sciences, Easton Mark Publishing Company, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN^TM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels and carbowax ) The semi-solid mixture. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311

The dose of antibody administered to the patient may vary according to the age and size of the patient, the underlying disease, symptoms, route of administration, and similar factors. The preferred dosage is typically calculated based on body weight or body surface area. The effective dose and time course of anti-PCSK9 antibody administration can be determined based on experience; for example, regular evaluation can be used to monitor the patient's progress, and the dose can be adjusted accordingly. Furthermore, the measurement of doses between species can be carried out using methods well known in this technology (for example, Mordenti et al., 1991,<i>Pharmaceut.Res.8</i> ：1351)。

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulated liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see , For example Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, such as infusion or bolus injection, to be absorbed through epithelial or mucosal layers (such as oral mucosa, rectum and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Administration can be systemic or local.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle or syringe. In addition, in terms of subcutaneous delivery, a pen-type delivery device can be easily used to deliver the pharmaceutical composition of the present invention. The pen-type delivery device can be a reusable type or a disposable type. Reusable pen-type delivery devices generally utilize replaceable refill cartridges containing medical components. Once all the medical components in the cassette are injected and the replenishment cassette becomes empty, the empty cassette can be easily discarded and replaced with a new replenishment cassette containing the medical composition. Then the pen-type delivery device can be used again. There is no replaceable refill box in a disposable pen-type delivery device. Instead, the disposable pen-type delivery device is pre-filled with medical composition and stored in a storage tank in the device. Once the storage tank for the pharmaceutical composition becomes empty, the entire device can be discarded.

Many reusable pen-type and auto-injector delivery devices have been used to deliver the pharmaceutical composition of the present invention subcutaneously. Examples include (but are not limited to) AUTOPEN^TM(Owen Mumford, Inc., Woodstock, UK), DISETRONIC^TMPen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25^TMPen, HUMALOG^TMPen, HUMALIN 70/30^TMPen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN^TMI, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR^TM(Novo Nordisk, Copenhagen, Denmark), BD^TMPen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN^TM, OPTIPEN PRO^TM, OPTIPEN STARLET^TMAnd OPTICLIK^TM(sanofi-aventis, Frankfurt, Germany), only to mention some. Disposable pen-type delivery devices for subcutaneous delivery of the pharmaceutical composition of the present invention include (but are not limited to) SOLOSTAR^TMPen (sanofi-aventis), FLEXPEN^TM(Novo Nordisk) and KWIKPEN^TM(Eli Lilly), SURECLICK^TMAuto-injector (Amgen, Thousand Oaks, CA), PENLET^TM(Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA^TMPens (Abbott Labs, Abbott Park IL), only a few are proposed.

Under certain circumstances, the pharmaceutical composition can be delivered by a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric substances can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed close to the target of the composition, so only a part of the systemic dose is required (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp .115-138). Other controlled release systems are discussed in Langer, 1990, Science 249: 1527-1533 in the review.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, drip infusion, and the like. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily vehicle conventionally used for injection. Aqueous vehicles for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be combined with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol) , Non-ionic surfactants [for example, polysorbate 80, HCO-50 (polyethylene oxide (50 mol) adduct of hydrogenated castor oil)], etc. are used in combination. Oily vehicle, sesame oil, soybean oil, etc. can be used, which can be used to solubilize Agents such as benzyl benzoate, benzyl alcohol and the like are used in combination. Therefore, the prepared injection is preferably filled in an appropriate ampoule.

Advantageously, the aforementioned pharmaceutical composition for oral or parenteral use is prepared into a unit dosage form suitable for the dosage of the active ingredient. These unit dosage forms include, for example, lozenges, tablets, capsules, injections (ampoules), suppositories, and the like. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per unit dosage form; especially in the form of injection, the aforementioned antibody system preferably contains about 5 to about 100 mg and other dosage forms are about 10 to about 250 mg. .

<b>Therapeutic use of antibodies</b>

The present invention includes a method of administering a therapeutic composition comprising an anti-PCSK9 antibody (for example, an anti-PCSK9 antibody with pH-dependent binding properties) to a subject in need thereof. This therapeutic composition may include any anti-PCSK9 antibody or fragment thereof as described herein. As used in the text, the term "objects in need" refers to humans or non-humans who have one or more symptoms or indications of hypercholesterolemia, or those who have been diagnosed with hypercholesterolemia, or other beneficial Those who have decreased total serum cholesterol, LDL, triglycerides, or VLDL, or those who benefit from increased HDL. The present invention also includes a method for reducing the amount of lipoprotein (a) [Lp(a)] by administering an anti-PCSK9 antibody (for example, an anti-PCSK9 antibody with pH-dependent binding properties) of the present invention.

In some cases, the patients treated with the therapeutic formulations of the present invention are healthy but have elevated amounts of lipid cholesterol, lipids, triglycerides, or lipoproteins. For example, the patient may not have any other risk factors for cardiovascular, thrombosis, or other diseases or disorders during the treatment period. However, in other cases, the patient is selected on the basis of the diagnosis or risk of a disease or condition caused by, related to, or associated with elevated serum cholesterol, lipids, triglycerides, or lipoproteins. . For example, at or before the administration of the pharmaceutical composition of the present invention, the patient may be diagnosed or identified as having a cardiovascular disease or condition, such as coronary artery disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, Stroke, peripheral arterial obstruction disease, etc. risk. This cardiovascular disease or condition, in some cases, is hypercholesterolemia. For example, if a patient is diagnosed or identified as having symptoms of hypercholesterolemia, such as heterozygote familial hypercholesterolemia (heFH), homozygote familial hypercholesterolemia (hoFH), and the occurrence of different family hypercholesterolemia For the risk of hypercholesterolemia (nonFH) of sexual hypercholesterolemia, this patient can choose to be treated with the pharmaceutical composition of the present invention.

In other cases, the patient may be diagnosed or identified as being at risk of developing thromboembolic diseases or conditions, such as pulmonary embolism, central retinal vein embolism, etc., at or before the administration of the pharmaceutical composition of the present invention. In a specific embodiment, the patient is selected based on the diagnosis or risk of developing a combination of two or more of the above-mentioned diseases or disorders. For example, the patient may be diagnosed or identified as being at risk of developing coronary artery disease and pulmonary embolism at the time of or before the administration of the pharmaceutical composition of the present invention. Other diagnostic combinations (for example, atherosclerosis and central retinal vein thrombosis, heFH and stroke, etc.) are also included in the definition of the patient group that can be treated with the pharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention can also be used to treat hypercholesterolemia or dyslipidemia caused by or related to underlying diseases or conditions selected from the following group: metabolic syndrome, diabetes, hypothyroidism, renal syndrome, renal failure , Cushings syndrome, biliary cirrhosis, glycogen storage disease, liver tumor, cholestasis, growth hormone deficiency. The pharmaceutical composition of the present invention can also be used to treat hypercholesterolemia or dyslipidemia caused by or related to previous treatments, such as estrogen therapy, progesterone therapy, beta blockers or diuretics.

In other cases, the patients treated with the pharmaceutical composition of the present invention are selected based on one or more factors selected from the following group: age (for example, greater than 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old), race, gender (male or female), exercise habits (e.g., regular exercisers, non-exercisers), other pre-existing medical symptoms (e.g., type II diabetes, high Blood pressure, etc.) and current medication status (for example, currently taking statins (for example, cerivastatin) (cerivastatin), atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin (pravastatin), etc.], beta blockers, nicotinic acid, etc.). Potential patients can be selected/screened based on one or more of these factors before treatment with the method of the present invention (for example, by questionnaire, diagnostic evaluation, etc.).

The present invention also includes a method for increasing intestinal cholesterol secretion (TICE) in a subject by administering a PCSK9 inhibitor to the subject. For example, the present invention provides a method for adding TICE to a subject by administering an anti-PCSK9 antibody with pH-dependent binding properties to the subject. According to specific embodiments, the present invention includes a method of identifying a subject whose improvement of TICE should be beneficial, or identifying a subject with a TICE disorder, and administering a PCSK9 inhibitor to the subject.

Statins are known to increase the amount of PCSK9 in patients (see, for example, Dubuc et al., Aug. 2004, Arterioscler. Thromb. Vasc. Biol.<i>24</i>: 1454-1459). Patients receiving the conventional anti-PCSK9 therapeutic agent, statin-treated patients, have a faster anti-PCSK9 clearance rate than non-statin-treated patients. Without being bound by theory, it is proposed that the elevated amount of PCSK9 in patients taking statins may be eliminated by the target-mediator, leading to faster elimination of anti-PCSK9 antibodies. Therefore, patients taking statins may require larger doses and/or more frequent administration of conventional anti-PCSK9 therapeutic agents (for example, anti-PCSK9 antibodies) to achieve the best cholesterol lowering. As used in the text, the term "conventional anti-PCSK9 therapeutic agent" refers to any PCSK9-binding molecule that does not have pH-dependent binding properties, that is, compared to neutral pH, there is no decrease in binding to PCSK9 at acidic pH Of the molecule. The inventors have conceived that this statin-induced target-mediated clearance can be avoided or circumvented by using anti-PCSK9 antibodies that can be effectively recycled in patients treated with statins. Therefore, the present invention includes administering a therapeutically effective amount of an anti-PCSK9 antibody with pH-dependent binding properties to a subject undergoing statin therapy therapy to overcome/avoid the statin-induced anti-PCSK9 binding agent The purpose of Mark-Medium removal method. The present invention also includes methods for reducing the amount of anti-PCSK9 agents that must be administered to statin-taking patients in order to achieve an appropriate cholesterol-lowering effect, and/or methods for reducing the frequency of administration of anti-PCSK9 agents to statin-taking patients, These methods include replacing the conventional anti-PCSK9 agent initially administered to the patient with an anti-PCSK9 antibody with pH-dependent binding properties to modify the patient's therapeutic dosing therapy. Any of the pH-dependent anti-PCSK9 antibodies described in the text can be used in the aforementioned methods.

<b>Combination therapy</b>

The present invention also provides treatment methods that include the administration of a pharmaceutical composition comprising any of the exemplary anti-PCSK9 antibodies described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that can be administered in combination with the anti-PCSK9 antibody of the present invention include, for example, statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, Pravastatin, rosuvastatin, simvastatin, etc.), nicotinic acid, fibric acid, cholic acid chelator (for example, cholestyramine), colesevelam, colesevelam (colestipol), ezetimibe, anti-hypertensive agent, anti-diabetic agent, angiopoietin-like protein 3 (ANGPTL3) or angiopoietin-like protein-4 (ANGPTL4) antagonist (e.g., anti -ANGPTL3 antibody [e.g., anti-ANGPTL3 antibody as described in WO2008/073300 or US 7,935,796] or anti-ANGPTL4 antibody [e.g., anti-ANGPTL4 as described in WO2006/0074228 or WO2007/109307 or WO 2011/0792575 Antibody]) and any combination of the aforementioned additional therapeutic agents.

The other therapeutically active agent can be administered before, at the same time or shortly after the anti-PCSK9 antibody of the present invention is administered (for the purposes of this disclosure, these administration therapies are regarded as the anti-PCSK9 antibody of the present invention and the other The therapeutically active agent "combination" administration). The present invention includes a pharmaceutical composition in which the anti-PCSK9 antibody system of the present invention is co-formulated with one or more additional therapeutically active ingredients as described elsewhere in the text.

The present invention also provides a pharmaceutical composition comprising any of the exemplary anti-PCSK9 antibodies described in the text, and administering it at the time of administration of the pharmaceutical composition of the present invention Or patients who are undergoing treatment for hypercholesterolemia or related symptoms before. For example, a patient who has been previously diagnosed with hypercholesterolemia may have been prescribed a stable treatment therapy with another agent before and/or at the same time administering the pharmaceutical composition comprising the anti-PCSK9 antibody of the present invention. Prior or simultaneous treatments may include, for example, (1) agents that induce cell elimination of cholesterol synthesis by inhibiting 3-hydroxy-3-methyl-glutaryl (HMG)-coenzyme A (CoA) reductase, such as statins Class (for example, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin and pravastatin, etc.); (2) inhibition of cholesterol absorption and/or bile Acid reabsorption agents; (3) agents that increase lipoprotein catabolism (such as nicotinic acid); and/or (4) activators of LXR transcription involved in cholesterol elimination, such as 22-hydroxycholesterol. In a specific embodiment, the patient is taking a fixed composition of therapeutic agent such as ezetimibe + simvastatin before and/or at the same time as the anti-PCSK9 antibody is administered; Leenamine, colestipol, colesevelam); nicotinic acid + statins (such as nicotinic acid and lovastatin); or other hypolipidemic drugs such as omega-3-fatty acid ethyl esters (such as omacor).

<b>dose</b>

According to the method and administration therapy of the present invention, the amount of anti-PCSK9 antibody administered to a subject is generally a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an anti-PCSK9 antibody that produces a detectable dose of anti-PCSK9 antibody that reduces serum LDL-C, or inhibits, prevents, reduces or delays hypercholesterolemia and/or related symptoms. PCSK9 antibody dosage. In the case of anti-PCSK9 antibodies with pH-dependent binding properties, the therapeutically effective amount may range from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0mg, about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 75mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg , About 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, About 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg , About 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570 mg, about 580 mg, about 590 mg or about 600 mg of anti-PCSK9 antibody.

The amount of anti-PCSK9 antibody contained in an individual dose can be expressed in milligrams of antibody per kilogram of the patient's body weight (ie, mg/kg). For example, anti-PCSK9 can be administered to the patient at a dose of about 0.0001 to about 10 mg/kg of the patient's body weight.

<b>Administration therapy</b>

According to specific embodiments of the present invention, multiple doses of anti-PCSK9 antibodies (for example, pharmaceutical compositions including anti-PCSK9 antibodies with pH-dependent binding properties) can be administered to a subject at a defined time course. The method according to this aspect of the invention comprises successively administering multiple doses of the anti-PCSK9 antibody of the invention to a subject. As used herein, "sequential administration" means that each dose of the anti-PCSK9 antibody system is administered to the subject at different time points, such as different days (eg, hours, days, weeks, or months) separated by expected intervals. The present invention includes the steps of sequentially administering a single initial dose of anti-PCSK9 antibody to the patient, followed by one or more second doses of anti-PCSK9 antibody, and optionally followed by one or more third doses of anti-PCSK9 antibody. PCSK9 antibody method.

The terms "initial dose", "second dose" and "third dose" refer to the temporary sequence of administration of anti-PCSK9 antibodies. Therefore, the "initial dose" is the dose administered at the beginning of the treatment (also referred to as the "baseline" dose); the "second dose" is the dose administered after the initial dose; and the "third dose" It is the dose administered after the second dose. The initial, second, and third doses can all contain the same amount of anti-PCSK9 antibody, but in terms of frequency of administration, they can generally be different from each other. In a specific embodiment, however, the anti-PCSK9 antibody contained in the initial, second and/or third doses The amount can be different during the treatment period (for example, adjusted up or down if appropriate). In a specific embodiment, two or more (for example, 2, 3, 4, or 5) doses are administered at the beginning of the treatment therapy as the "bearer dose", and then the subsequent doses are administered on a lower frequency basis ( For example, "maintenance dose"). The carrier dose can be administered, for example, once a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, and the like.

In an exemplary embodiment of the present invention, each second and/or third dose is 1 to 60 weeks immediately after the previous dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60 or Longer) administration. The term "immediately preceding dose", as used in the text, refers to the dose of anti-PCSK9 antibody in a multiple dosing sequence, in the sequence without intervening dose before each adjacent dose is administered Administration.

The method according to this aspect of the invention can include administering any number of second and/or third doses of anti-PCSK9 antibody to a patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Likewise, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient. In some cases, the second and/or third dose may be administered at a specific frequency for many years or for the life of a subject.

In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose can be administered to the patient 1 to 60 weeks immediately after the previous dose. Likewise, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose can be administered to the patient 1 to 60 weeks immediately after the previous dose. In addition, the frequency of the second and/or third dose administered to the patient may be different. During the treatment period, the frequency of administration can also be adjusted by the physician after the clinical examination according to the needs of individual patients.

<b>figure 1</b>The line shows the concentration of anti-PCSK9 antibody measured at different time points after subcutaneous administration of 1 mg/kg of anti-PCSK9 antibody in mice that only express mouse PCSK9 (that is, do not express human PCSK9).

<b>Figures 2A, 2B and 2C</b>The line shows the concentration of anti-PCSK9 antibody measured at different time points after subcutaneous administration of 1 mg/kg of anti-PCSK9 antibody in mice that only express human PCSK9 (replacing mouse PCSK9). The description of the antibodies used in the experiments depicted in Figures 2A, 2B and 2C is shown in Table 5 herein.

<b>Figure 3A-3G</b>It shows the induction map from the surface plasmon resonance binding experiment, in which the anti-PCSK9 antibody is combined with the human PCSK9 antigen at neutral pH (pH 7.4), and then changed to various pH (7.4, 7.2, 6.0 and 5.75) The buffer goes through the dissociation phase. The antibodies tested in these experiments are: 316P (v1) and 300N (v2) (<b>Figure 3A</b>); VH-D106H and VK-L30H(<b>Figure 3B</b>); VH-D106H/VK-L30H and comparator 1<b>(Figure 3C</b>); Comparator 2 and Comparator 3(<b>Figure 3D</b>); Comparator 4 and Comparator 5(<b>Figure 3E</b>); Comparator 6 and Comparator 7(<b>Figure 3F</b>); and comparator 8 and comparator 9(<b>Figure 3G</b>). The individual lines in each graph represent the binding reactions of individual antibodies at different concentrations. The description of the antibodies used in these experiments is shown in Table 5 in the text. All experiments were performed at 37°C. The dissociation half-life value (t½) is marked above the respective induction graph.

<b>Instance</b>

The following examples are presented to provide a complete disclosure and description of how those skilled in the art make and use the method and composition of the present invention, but do not intend to limit the scope of the invention as the inventor thinks. Although every effort has been made to ensure the correctness of the relevant figures (such as quantity, temperature, etc.) used, some experimental errors and deviations should still be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is expressed in degrees Celsius, and pressure is at or near atmospheric pressure.

<b>Example 1. Production of human antibodies against human PCSK9</b>

The human anti-PCSK9 antibody system was produced as described in US Patent No. 8,062,640. Table 1 lists the heavy chain and light chain variable region amino acid sequence pairs of the selected anti-PCSK9 antibodies of the present invention and the identifiers of the CDR amino acid sequences and their corresponding antibody names.

<tables><img alt="" file="twi596115b_d0001.tif" he="1531" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2097" /></tables><tables><img alt="" file="twi596115b_d0002.tif" he="2962" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2088" /></tables><tables><img alt="" file="twi596115b_d0003.tif" he="2952" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2093" /></tables><tables><img alt="" file="twi596115b_d0004.tif" he="2516" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2075" /></tables>

Any of the anti-PCSK9 antibodies listed in Table 1, related to the amino acid sequence of the individual heavy chain and chain variable region and/or CDR, can be used as a parent antibody, as shown in the following non-limiting operating examples, PH-dependent histidine substitution variants can be derived from it body.

<b>Example 2. Construction of human anti-PCSK9 antibody with histidine substitution mutation</b>

The anti-PCSK9 antibody named 300N is known to have intermediate pH-dependent binding properties with reduced binding affinity for PCSK9 at acidic pH, and enhanced pharmacokinetics (see US Patent No. 8,062,640). Want to produce more pH-dependent binding properties (that is, compared to neutral pH, binding decreases at low pH) and improved efficacy in vivo (for example, longer antibody serum half-life, intestinal cholesterol lowering 300N variants of activity, etc., are constructed to construct a series of variant antibodies. Specifically, a mutant version of 300N was constructed, in which each amino acid in the complementarity determining region (CDR) of 300N was individually mutated to histidine. As shown in Table 1, the heavy chain variable region (HCVR) of the maternal 300N antibody includes the amino acid sequence of SEQ ID NO: 218, and the light chain variable region (LCVR) of the maternal 300N antibody includes SEQ ID NO: 226 amino acid sequence. The CDR sequences of the maternal 300N antibody are shown in Table 2. The His-substitution mutation and the corresponding antibody name (for example, VH-G26H, VH-F27H, etc.) of the variant antibody derived from the histidine substitution of 300N are shown in Table 3.

<tables><img alt="" file="twi596115b_d0005.tif" he="755" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1703" /></tables>

<tables><img alt="" file="twi596115b_d0006.tif" he="2944" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1803" /></tables><tables><img alt="" file="twi596115b_d0007.tif" he="2404" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1813" /></tables>

For each variant antibody listed in Table 3, except for the mutant CDR sequences indicated in the table, all CDR sequences are the same as those of the maternal 300N antibody (including SEQ ID NO: 220, 222, 224, 228, 230, The CDR sequence of 232). E.g, The histidine-substituted variant resistance system called "VH-D106H" includes SEQ ID NO: 220, 222,<u style="single">788</u>, 228, 230, 232 amino acid sequences of the heavy chain and light chain CDR sequences (wherein the HCDR3 sequence of SEQ ID NO: 224 is replaced by the variant HCDR3 sequence of SEQ ID NO: 788). Similarly, the histidine-substituted variant resistance system called "VK-L30H" includes SEQ ID NO: 220, 222, 224,<u style="single">802</u>The heavy chain and light chain CDR sequences of, 230, and 232 amino acid sequences (the LCDR1 sequence of SEQ ID NO: 228 is replaced by the variant LCDR1 sequence of SEQ ID NO: 802).

<b>Example 3A. Binding properties of variant anti-PCSK9 antibodies at neutral and acidic pH</b>

The histidine-substituted variant antibody of Example 2 was analyzed with an instant plasmon resonance biosensor (Biacore T200) at 25°C, and the pH-dependent binding to human PCSK9 was performed at pH 5.75 and pH 7.2 test. The purpose of this experiment is to identify variant antibodies that have histidine substitutions with reduced binding to human PCSK9 at acidic pH relative to neutral pH.

The Biacore CM4 sensor chip is derived from a single strain of mouse anti-human Fc antibody to capture human antibodies. After being temporarily expressed in Chinese hamster ovary (CHO) cells, the histidine-substituted variant anti-PCSK9 antibody was captured from the culture medium to the surface of the anti-human Fc sensor. Human PCSK9 (SEQ ID NO: 755) with a C-terminal myc-myc-hexahistidine tag (hPCSK9-mmH) ranging from 3.125nM to 500nM was injected at a flow rate of 50μl/min in the capture On the surface of the anti-PCSK9 monoclonal antibody. The antibody-antigen binding is monitored for 4 or 5 minutes and then the dissociation of the antigen from the captured monoclonal antibody is monitored for 5 or 8 minutes. By using the Scrubber 2.0 curve fitting software to process the data and fit it with a 1:1 combination model to determine the dynamic binding (<i>k</i>_a) And dissociation (<i>k</i>_d) Rate constant. Binding and dissociation equilibrium constant (<i>K</i>_D) And dissociated half Lifetime (t_1/2) Is calculated by the power rate constant:<i>K</i>_D(M)=<i>k</i>_d /<i>k</i>_a; And t_1/2(min)=(ln2/(60*<i>k</i>_d )。

K at pH 7.2 (neutral) and pH 5.75 (acidic) when each histidine substituted variant anti-PCSK9 antibody binds to human PCSK9_DValue and t_1/2The values, and the pH 5.75/pH 7.2 ratio of these individual values, are shown in Table 4. The value of the maternal 300N antibody is shown in the bottom row of this table. K_DThe value is expressed in volume molar concentration (M) and the t½ value is expressed in minutes (min)

<tables><img alt="" file="twi596115b_d0008.tif" he="1978" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1962" /></tables><tables><img alt="" file="twi596115b_d0009.tif" he="2946" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1998" /></tables><tables><img alt="" file="twi596115b_d0010.tif" he="1640" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1981" /></tables>

As shown in Table 4 (all measurements are at 25°C), the maternal (300N) antibody showed moderate binding affinity (K) at pH 7.2 and pH 5.75._D~0.9-1.0nM), and compared to pH 7.2, t at pH 5.75_1/2The system drops more than 3-fold (that is, it dissociates faster at pH 5.75; the ratio of pH 5.75/pH 7.2 = 0.26).

Several single histidine substitutions caused a substantial decrease in binding at pH 7.2 and pH 5.75, and compared to the original sequence, other substitutions had minimal effect on pH-dependent binding. However, importantly, relative to the parent antibody, several single histidine substitutions resulted in a substantially faster dissociation rate of the antibody at pH 5.75 compared to pH 7.2 (at pH 5.75)._1/2T at pH 7.2_1/2At least 5-fold lower). These antibodies with pH-dependent binding properties include antibodies with heavy chain CDR substitutions: VH-W33H, VH-Q53H, VH-I100H, VH-V104H, VH-D106H, VH-M107H and VH-Y112H; and antibodies with light chain CDR substitutions: VK-L29H, VK-L30H, VK-N33H, VK-L97H, VK-T99H and VK-P100H. The histidine substituted variant antibodies VH-D106H and VK-L30H have particularly obvious pH-dependent binding and were selected for further research.

In order to further study the pH-dependent binding properties of the histidine-substituted variant anti-PCSK9 antibodies VH-D106H and VK-L30H and the bis-histidine-substituted variant (VH-D106H/VK-L30H), the antibody Purified and tested for binding to human PCSK9 at neutral pH (pH 7.4) (Table 6) and acidic pH (pH 6.0) (Table 7) using conditions similar to the above. The ratio of the binding properties of acidic pH to neutral pH is shown in Table 8. Several control/comparative antibodies are also included in these experiments. A summary of the antibodies tested in this analysis is shown in Table 5. All measurements are performed at 25°C.

<tables><img alt="" file="twi596115b_d0011.tif" he="1350" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2000" /></tables><tables><img alt="" file="twi596115b_d0012.tif" he="1546" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1987" /></tables>

<tables><img alt="" file="twi596115b_d0013.tif" he="1366" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1997" /></tables><tables><img alt="" file="twi596115b_d0014.tif" he="408" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1965" /></tables>

<tables><img alt="" file="twi596115b_d0015.tif" he="1888" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2007" /></tables>

<tables><img alt="" file="twi596115b_d0016.tif" he="498" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1988" /></tables><tables><img alt="" file="twi596115b_d0017.tif" he="1584" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2018" /></tables>

High value (for example, greater than about 12) of k_dAnd K_DThe acid/neutral ratio of t½ and the acid/neutral ratio of t½ at low values (for example, less than about 0.20) indicate pH-dependent binding. According to these standards, the histidine-substituted variant antibody VK-L30H and the double mutant VH-D106H/VK-L30H have the largest pH-dependent binding properties of all the tested antibodies. In particular, these antibodies each have a k greater than about 28_dAcid/neutral ratio, greater than approximately 14 K_DAcid/neutral ratio, and t½ acid/neutral ratio less than about 0.05.

<b>Example 3B. Binding characteristics of histidine variant anti-PCSK9 antibody to human PCSK9:</b><b>Combines at neutral pH and dissociates at neutral and acidic pH ranges</b>

To further evaluate the pH-dependence of the anti-PCSK9 antibody of the present invention For the binding properties, the binding experiment was performed, in which the antibody/antigen binding phase was observed at a neutral pH of 37°C and the antibody/antigen dissociation phase was observed at a neutral or acidic pH range.

The Biacore CM4 sensor chip is derived from multiple strains of Fab'2 anti-human Fc antibodies to capture human antibodies. Variant anti-PCSK9 antibodies (VH-D106H, VK-L30H and VH-D016H/VK-L30H) and maternal antibodies (316P and 300N) and comparison antibodies (comparators 1-7) substituted with the selected purified histidine , See Table 5) Capture to the surface of the anti-human Fc sensor. Human PCSK9 with a C-terminal myc-myc-hexahistidine tag (hPCSK9-mmH) ranging from 3.125nM to 50nM was injected into the anti-PCSK9 monoclonal antibody capture surface at a flow rate of 30μl/min . Observe the antibody-antigen binding at pH 7.4 for 6 minutes and then observe the dissociation of the antigen from the captured monoclonal antibody at pH 7.4, 7.2, 6.0 or 5.75 for 5 minutes. By using the Scrubber 2.0 curve fitting software to process and fit the data, determine the dissociation (<i>k</i>_d) Rate constant. Dissociation half-life (t_1/2) Is calculated from the dissociation rate constant: t_1/2(min)=(ln2/<i>k</i>_d)/60. The sensor graphs depicting the binding/dissociation characteristics of antibodies under various pH conditions are shown in the graphs in Figures 3A to 3G.

These experimental results confirmed that, compared to the parent antibody, the histidine-substituted variant anti-PCSK9 antibodies VH-D106H, VK-L30H, and VH-D016H/VK-L30H exhibited more rapid dissociation from the antigen at low pH ( As shown in Figures 3A-3G, the response level at the 360 second point dropped rapidly in the pH 6.0 and 5.75 experiments).

<b>Example 4. Receptor blocking activity of variant anti-PCSK9 antibodies</b>

First, an ELISA-based immunoassay was used to test the ability of the selected histidine substitution variant anti-PCSK9 antibody to block the binding of recombinant human PCSK9 to human LDLR (hLDLR) at neutral pH.

In short, the human LDLR-like epidermal growth factor A region (313-355 amino acid of SEQ ID NO:758) expressed by the C-terminal human Fc tag ("hLDLR EGFA-hFc") is 2μg/ml The PBS solution was coated on a 69-well microtiter plate at 4°C to overnight, and then blocked with 0.5% (weight/volume) BSA in PBS. Use this plate to measure free PCSK9 (maternal or histidine substituted variant) in hPCSK9-mmH solution pre-equilibrated with different concentrations of anti-hPCSK9 antibody at neutral pH (pH 7.2), As shown in Table 9A.

As an initial experiment to measure the blocking properties of antibodies at neutral pH (pH 7.2), hPCSK9-mmH (see Example 3) at a fixed final concentration of 500 pM was premixed with antibody serial dilutions ranging from 0 to about 100 nM, and then Incubate at room temperature for 1 hour to allow the binding to reach equilibrium. The equilibrated sample solution was then transferred to the LDLR EGFA-hFc coated dish prepared as described above. After 1 hour incubation, wash the receptor-coated microtiter plate and use the HRP-conjugated anti-myc secondary antibody (Novus, #NB600-341) to detect the plate-bound hPCSK9-mmH, and use the TMB HRP matrix (BD Biosciences, # 555214) expand the colorimetric signal. The absorbance at 450 nm was recorded to reflect the free hPCSK9-mmh concentration that can bind to the disc-coated LDLR receptor in the pre-equilibrated PCSK9-antibody solution. IC₅₀The value, defined as the antibody concentration that produces 50% reduction in the PCSK9-mmH binding signal of the sample compared with no antibody, was measured by the data using Prism software (GraphPad) and is shown in Table 9A. (Perform two separate experiments; not every antibody is tested in each experiment, as shown by the dotted line [--] in Table 9A).

<tables><img alt="" file="twi596115b_d0018.tif" he="385" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1524" /></tables><tables><img alt="" file="twi596115b_d0019.tif" he="746" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1508" /></tables>

The maternal antibody 300N shows an IC of about 0.20 nM₅₀value. Compared to 300N, histidine-substituted variant antibodies generally have a slight decrease in potency, but they all maintain IC₅₀Value &lt;1.0nM. The VK-L30H variant maintains a blocking potency close to that of the parent antibody (IC₅₀Values are 0.17 and 0.20 nM).

Then the histidine-substituted variant anti-PCSK9 antibody subgroups and comparison antibodies of the present invention were used to block the ability of recombinant human PCSK9 to bind to human LDLR (hLDLR) under neutral and low pH conditions, using a similar ELISA- Tests based on immunoassays.

In short, hLDLR EGFA-hFc was coated on a 96-well microtiter plate with a 2 μg/mL PBS solution at 4° C. to overnight, and then blocked with a 0.5% (weight/volume) BSA solution in PBS. This disc is used to measure the free hPCSK9-mmH in the hPCSK9-mmH solution pre-equilibrated with different concentrations of anti-hPCSK9 antibody at neutral pH (pH 7.2) or low (pH 5.75) pH.

For the blocking test, hPCSK9-mmH (see Example 3) at a fixed final concentration of 500 pM was premixed with antibody serial dilutions ranging from 0 to about 200 nM, and then incubated at room temperature for 1 hour to allow the binding to reach equilibrium. One group of this mixture is pre-bound in pH 7.2 buffer and the second group is pre-bound in pH 5.75 buffer. Then transfer the balanced sample solution to the coated LDLR EGFA-hFc In the plate. After 1 hour incubation, wash the receptor-coated microtiter plate, and use HRP-conjugated anti-myc secondary antibody (Novus, #NB600-341) to detect hPCSK9-mmH bound to the plate, and use TMB HRP matrix ( BD Biosciences, # 555214) to expand the colorimetric signal. Record the absorbance at 450 nm to reflect the free hPCSK9-mmh concentration and plot the antibody concentration. IC₅₀The value is defined as the antibody concentration that produces a 50% reduction in the free PCSK9-mmH signal in the absence of antibody, measured by the data using Prism software (GraphPad). Two analyzed ICs₅₀The values and the calculated ratio reflecting the pH dependence of the binding capacity are shown in Table 9B.

<tables><img alt="" file="twi596115b_d0020.tif" he="1952" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2006" /></tables><tables><img alt="" file="twi596115b_d0021.tif" he="135" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2010" /></tables>IC recorded as &lt;1.25E-10₅₀The data has a calculated value below the theoretical bottom line of the analysis assuming that an antibody can bind to two ligand binding sites. Use the value actually obtained in parentheses to generate the ratio. Unable to judge = due to the irregular bell-shaped antibody dose-response curve, there is no IC that can be calculated₅₀ 。

As shown in Table 9B, most of the comparative antibodies have no or minimally reduced blocking activity at acidic pH compared to neutral pH (see, for example, comparators 2, 5, 6, 7, 8 and 9 At neutral pH and acidic pH, the blocking activity is less than a 3-fold decrease). Comparator 3 and 4 show a moderate reduction in blocking ability, with acidic/neutral IC₅₀The ratios are 35.9 and 19.2, respectively. In contrast, the two exemplary histidine substituted variant anti-PCSK9 antibodies of the present invention, VK-L30H and VH-D106H/VK-L30H, have a dramatic decrease in PCSK9/LDLR blocking activity at acidic pH, where the acidic /Neutral IC₅₀The ratio is greater than about 200.

The results of this experiment confirm that the pH-dependent binding properties of the histidine variant anti-PCSK9 antibodies of the present invention reflect the extent to which these antibodies can block the interaction between PCSK9 and LDLR at neutral and acidic pH.

Example 5. Ability of in vitro variant anti-PCSK9 antibodies to block the inhibition of PCSK9-mediated LDL absorption

The ability of the selected histidine substitution variant anti-PCSK9 antibody to increase LDL uptake in vitro was measured using a human hepatocellular carcinoma cell line (HepG2, ATCC # HB-8065). HepG2 cells were implanted into DMEM containing 5% lipoprotein-deficient serum (LPDS, Millipore, # LP4) in a 96-well plate at 2 x 104 cells/well, and cultured at 37°C and 5% CO2 until overnight to form HepG2 Single layer. 2nM recombinant human PCSK9 (SEQ ID NO: 755, with C-terminal myc-myc hexahistidine tag and D374Y mutation; "hPCSK9-D374Y-mmH") or 50nM recombinant macaque PCSK9 (with C-terminal myc -myc hexahistidine label performance; MfPCSK9-mmH; SEQ ID NO: 761) was added to LPDS medium with various antibody concentrations (from 50 nM to 0.098 nM serial dilutions). After culturing overnight, LPDS medium of BODIPY-LPL (Invitrogen, L3483) was added to the cells to a final concentration of 0.01 mg/mL. The absorption of BODIPY-LPL was incubated at 37°C for 6 hours and then detected by a fluorescent disc reader (Molecular Devices Flexstation III) using excitation/emission filters set at 390nm/520nm. The IC50 values of the anti-PCSK9 antibodies in each test are shown in Table 10 (IC50=antibody concentration at which LDL absorption increases by 50%).

<tables><img alt="" file="twi596115b_d0022.tif" he="1427" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1646" /></tables>

As shown in Table 10, all the histidine substitution variant anti-PCSK9 antibodies tested in this analysis have an IC of less than 5 nM₅₀The value blocks the inhibitory effect of hPCSK9-D374Y-mmH-mediated LDL absorption (that is, increases LDL absorption), and with an IC lower than 22nM₅₀The value blocks the inhibitory effect of MfPCSK9-mmH-mediated LDL absorption (that is, enhances LDL absorption).

The ability of the anti-PCSK9 antibody subgroups of the present invention and the comparative antibodies (see Table 5) to increase LDL uptake in vitro also uses the same human hepatocellular carcinoma cell line analysis method described above, but is based on recombinant wild-type human PCSK9 (SEQ ID NO: 755, represented by the C-terminal myc-myc hexahistidine tag; "hPCSK9-mmh"). Add 100 nM constant hPCSK9-mmH and different concentrations of antibody (serial dilutions from 2000 nM to 0.034 nM) in LPDS medium. IC of anti-PCSK9 antibody in each test₅₀The values are shown in Table 11A (IC₅₀= The antibody concentration when LDL absorption increases by 50%).

<tables><img alt="" file="twi596115b_d0023.tif" he="1723" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1485" /></tables>

As shown in Table 11A, the 10 anti-PCSK9 antibodies tested have an IC ranging from 26 nM to 37 nM₅₀Value suppresses hPCSK9-mmh, comparator 1 is only 18nM IC₅₀The value partially suppresses hPCSK9-mmh.

The ability of the anti-PCSK9 antibody of the present invention and the comparative antibody subgroup (see Table 5) to increase LDL uptake in vitro also uses the same analysis method for human hepatocellular carcinoma cell lines as described above, using recombinant wild-type human PCSK9 (SEQ ID NO : 755, expressed by the C-terminal myc-myc hexahistidine tag; "hPCSK9-mmh"), human PCSK9 (expressed by the C-terminal myc-myc hexahistidine tag and the D374Y mutation; "hPCSK9-D374Y-mmH ") or recombinant macaque PCSK9 (expressed as a C-terminal myc-myc hexahistidine tag; MfPCSK9-mmH; SEQ ID NO: 761). Add 50nM constant hPCSK9-mmH, 2nM constant hPCSK9-D374Y-mmH or 50nM constant MfPCSK9-mmH and different concentrations of antibody (for hPCSK9-mmH or MfPCSK9-mmH blocking, the antibody concentration starts from 500nM in a continuous 1:2 Dilution; for hPCSK9-D374Y-mmH blocking, the antibody concentration starts from 50 nM and serially diluted 1:2) in LPDS medium. IC of anti-PCSK9 antibody in each test₅₀The values are shown in Table 11B (IC₅₀= The antibody concentration when LDL absorption increases by 50%).

<tables><img alt="" file="twi596115b_d0024.tif" he="954" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2038" /></tables><tables><img alt="" file="twi596115b_d0025.tif" he="234" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2027" /></tables>

As shown in Table 11B, VK-L30H is a 9nM IC₅₀The value blocks hPCSK9-mmH, while 316(v1) and 300N(v2) are ICs of 7.8nM and 10.3nM, respectively₅₀Value blocking hPCSK9-mmH. The comparative resistance system tested in this analysis has an IC ranging from 8.9nM to 39.3nMnM₅₀Value blocking hPCSK9-mmH. VK-L30H is a 3.3nM IC₅₀Value blocking hPCSK9-D374Y-mmH, and 316(v1) and 300N(v2) are both with 2nM IC₅₀Value blocking hPCSK9-mmH. Comparator 7 with 0.97nM IC₅₀Value blocking hPCSK9-D374Y-mmH, while comparator 9 has an IC of 10.6nM₅₀The value partially blocked hPCSK9-D374Y-mmH and Comparator 8 did not show any measurable hPCSK9-D374Y-mmH blocking properties. VK-L30H is 26.4nM IC₅₀Value blocks MfPCSK9-mmH, while 316(v1) and 300N(v2) are ICs of 10.3nM and 15.7nM, respectively₅₀Value blocking MfPCSK9-mmH. Comparator 7 and 9 are respectively 10.4nM and 33.9nM IC₅₀Value blocking MfPCSK9-mmH, while comparator 8 uses 77.2nM IC₅₀Value blocking MfPCSK9-mmH.

<b>Example 6. Pharmacokinetic analysis of variant anti-PCSK9 antibodies in wild-type and PCSK9 humanized mice</b>

In wild-type (WT) mice and mice with homozygous expression of human PCSK9 instead of mouse PCSK9 (humanized PCSK9 mice), all mouse lines have the same strain background (75% C57BL6 and 25 % 129Sv), comparison of the pharmacokinetic clearance rate of three variant anti-PCSK9 antibodies (VH-D106H, VK-L30H and VH-D106H/VK-L30H) with histidine substitution and its parent antibody molecule. Each antibody system was tested in 5 WT and 5 humanized PCSK9 mice. All anti-systems were administered subcutaneously at a dose of 1 mg/kg. Except for collection on the day before antibody injection In addition to blood (taken before), blood was collected at 6h, 1, 2, 3, 4, 7, 10, 14, 21, 30, 39, 50, 60, and 74 days after injection. Separate the blood serum part and use ELISA immunoassay for total serum antibody analysis. In short, 1 μg/mL of goat anti-human IgG multi-strain antibody (Jackson ImmunoResearch, #109-005-098) was coated on a 96-well plate by incubating at 4°C until overnight. The next day, the assay plate was blocked with BSA and then washed. Then six-dose serial dilutions of serum samples and 12-dose serial dilutions of individual antibody reference standards were added to the assay plate, and incubated at room temperature for 1 hour. After washing and removing the unbound antibody, the human antibody captured by the dish was detected with goat anti-human IgG multi-strain antibody (Jackson ImmunoResearch, # 109-005-098). Clean the measurement disc and then use the colorimetric tetramethylbenzidine (TMB) matrix according to the manufacturer (BD Pharmingen's suggestion shows off. Measure the absorbance at 450nm and use the reference standard curve generated in the sample pan to calculate the concentration of human IgG in the serum sample. The results are shown in Figures 1 and 2A, which show the time course of the concentration changes of the four anti-PCSK9 antibodies tested in WT and humanized mice, respectively. The average serum antibody concentration (μg/ml±SEM) of each group during the experimental period is shown in Table 12 (days 14, 21 and 30), Table 13 (days 39, 50 and 60) and Table 14 (day 74) Shown.

<tables><img alt="" file="twi596115b_d0026.tif" he="845" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2148" /></tables>

<tables><img alt="" file="twi596115b_d0027.tif" he="823" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2132" /></tables>

<tables><img alt="" file="twi596115b_d0028.tif" he="829" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1241" /></tables>

As shown in Figure 1, the antibodies of the four tests reached a similar C at about the first 1-2 days._max, And showed similar clearance rates in WT mice with overlapping pharmacokinetic profiles. In humanized PCSK9 mice (Figure 2A), the maternal antibody 300N showed a faster clearance rate than the tested histidine substitution variant anti-PCSK9 antibody. On the 14th day, the antibody concentration of 300N in humanized PCSK9 mice was below the detection limit (&lt;0.02μg/ml), compared to about 8μg/ml in WT mice, which shows the rapidity of the maternal antibody Human PCSK9-media clearance rate. The histidine-substituted variant antibody VH-D106H showed slower clearance than the parent antibody in humanized PCSK9 mice The average antibody concentration on day 14 in humanized PCSK9 mice was about 0.7 μg/ml. In humanized PCSK9 mice, the antibody concentration of VH-D106H fell below the detection limit at about the 50th day. In humanized PCSK9 mice, compared to VH-D106H or the parent antibody, the histidine-substituted variant antibodies VK-L30H and VH-D106H/VK-L30H showed a slower clearance rate. The daily average antibody concentration is approximately 10 μg/ml and 5 μg/ml, respectively. The amount of antibodies in the serum of VK-L30H and VH-D106H/VK-L30H remained within the detectable range (&gt;0.02μg/ml) until at least the 74th day. In particular, the serum concentration of VK-L30H in humanized PCSK9 mice remained above 0.25 μg/ml until day 74.

Then, compare the pharmacokinetic clearance rates of VH-D106H and VK-L30H with their maternal antibody (300N) and six comparator anti-PCSK9 antibodies (as defined in Table 5, comparator 1, 2, 3, 4, 5 and 6). This set of experiments was performed in humanized PCSK9 mice with 75% C57BL6 and 25% 129Sv strain background. Each anti-system was tested with a group of 5 mice, and all anti-systems were administered subcutaneously at a dose of 1 mg/kg. In addition to collecting blood before antibody injection (pre-blotting), blood was collected at 6h, 1, 2, 3, 4, 7, 10, 14, 21, 30, 45, and 74 days after injection. The analysis of total human antibodies in individual samples is performed by ELISA to detect human IgG Fc. The result is a graph of the time course of total human antibody amount in Figure 2B. The average serum antibody concentration of each group over time is shown in Table 15 (days 14, 21 and 30) and Table 16A (days 45 and 74).

<tables><img alt="" file="twi596115b_d0029.tif" he="466" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1846" /></tables><tables><img alt="" file="twi596115b_d0030.tif" he="752" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1871" /></tables>

<tables><img alt="" file="twi596115b_d0031.tif" he="1305" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1588" /></tables>

As shown in Figure 2B, all tested antibodies reached their maximum serum concentration (C_max), of which six antibodies (300N, VH-D106H, VK-L30H, Comparator 1, Comparator 3, and Comparator 4) have similar C_max; The other three antibodies (Comparator 2, Comparator 5 and Comparator 6) have approximately 2-3 times lower C_max. Compared with other test antibodies, antibody 300N, comparator 2, comparator 5 and comparator 6 Has a faster clearance rate. As shown in Table 15, the antibody concentration of the four antibodies was below the detection limit (&lt;0.02ug/ml) on the 14th day. In contrast, VH-D106H antibody, Comparator 1 and Comparator 4 had serum concentrations ranging from 0.5 μg/mL to 2 μg/mL on day 14; while VK-L30H antibody and Comparator 3 had approximately 7 μg on day 14 /mL serum concentration. On the 30th day, VH-D106H antibody, comparator 1 antibody, VK-L30H and comparator 3 were still detectable, and the average drug serum concentrations of each group were 0.07, 0.07, 1.04, and 1.85 μg/mL, respectively. The serum amount of VK-L30H and comparator 3 antibody remained in the detectable range (&gt;0.02 μg/mL) until at least day 74 (Table 16A).

Then conduct an additional study to compare anti-PCSK9 antibodies, including 316P(v1), 316P(v2), 300N(v1), 300N(v2), VK-L30H and three comparator anti-PCSK9 antibodies (Comparator 7, 8 and 9, as defined in Table 5) pharmacokinetic clearance rate. This set of experiments was performed in humanized PCSK9 mice with 75% C57BL6 and 25% 129Sv strain background. Each anti-system was tested with a group of 5 mice, and all anti-systems were administered subcutaneously at a dose of 1 mg/kg. In addition to collecting blood before antibody injection (pre-blood collection), blood was collected at 6h, 1, 2, 3, 4, 8, 10, 14, 21, and 30 days after injection. The analysis of total human antibodies in individual samples is performed by ELISA to detect human IgG Fc. The results are plotted in Figure 2C as the total human antibody amount over time. The average serum antibody concentration of each group over time is shown in Table 16B (days 14, 21 and 30).

<tables><img alt="" file="twi596115b_d0032.tif" he="652" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1862" /></tables><tables><img alt="" file="twi596115b_d0033.tif" he="479" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1865" /></tables>

As shown in Figure 2C, all test antibodies reached their maximum serum concentration (C_max), of which seven antibodies [316P(v1), 316(v2), 300N(v1), 300N(v2), Comparator 7, Comparator 8, and Comparator 9] have similar C_max; Compared with other test antibodies, VK-L30H has about 1.5 to 2 times higher C_max. 316P (v1) antibody, 316P (v2), 300N (v2) and Comparator 7 have faster clearance rates. As shown in Table 16B, the antibody concentration of the four antibodies was below the detection limit (&lt;0.02ug/ml) on the 14th day. In contrast, 300N (v1) antibody, comparator 8 and comparator 9 had serum concentrations ranging from 0.4 μg/mL to 0.7 μg/mL on day 14; while VK-L30H antibody had about 7 μg/mL on day 14 The serum concentration. On the 30th day, the VK-L30H antibody and comparator 8 were still detectable, and the average drug serum concentrations of the two groups were 0.09 and 3.34 μg/mL, respectively.

This example shows that compared to anti-PCSK9 antibodies that do not have pH-dependent binding properties or only have intermediate pH-dependent binding properties (for example, 300N and comparators 2, 5, and 6), they are pH-dependent Anti-PCSK9 antibodies with binding properties (e.g., VH-106H, VK-L30H, and VH-D106H/VK-L30H) have enhanced pharmacokinetic properties (e.g., long-term higher serum antibody levels).

<b>Example 7. Cholesterol-lowering activity of in vitro variant anti-PCSK9 antibodies</b>

The effect of in vitro anti-human PCSK9 antibody on serum LDL-C levels is to use homozygote to express human PCSK9 instead of mouse PCSK9 and heterozygote expressing mice (<i>Pcsk9</i>^hum/hum<i>Ldlr</i>^+/-) In mice. Place the mice before the experiment 5 Blood was taken in advance on the same day and divided into treatment groups based on the amount of LDL-C, so that the average amount of LDL-C in each group was equal. Then on day 0 of the study, mice were injected subcutaneously with anti-PCSK9 antibody or an isotype control antibody of irrelevant specificity at a dose of 10 mg/kg. For this study, two non-modified maternal anti-PCSK9 antibodies (316P and 300N) and two histidine substituted variant anti-PCSK9 antibodies (VK-L30H and VH-D106H) were used. This experiment system uses two 316P versions, 316P (v1) and 316P (v2). 316P (v1) has human IgG1 Fc, and 316P (v2) has human IgG4 Fc. All other test antibodies have human IgG4 Fc. (The "300N" antibody system used in this example is the same as the "300N(v2)" antibody used in Example 3 in the text). Five mice were used in each treatment group.

The mice were bled on the 4th, 7, 14, 20, 26, 33, 42, 46 and 52 days after the injection. The amount of LDL-C in the serum was determined using ADVIA® 1800 Chemistry System (Siemens). Then, the average LDL-C in the serum was calculated for each treatment group at each time point, and the results were expressed as (mean±SEM), as shown in Table 17. The value is expressed as the average amount of LDL-C (mg/dL) (±SEM). Table 18 shows the percentage decrease in the amount of LDL-C compared to the baseline.

<tables><img alt="" file="twi596115b_d0034.tif" he="1111" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2188" /></tables><tables><img alt="" file="twi596115b_d0035.tif" he="308" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2132" /></tables>

<tables><img alt="" file="twi596115b_d0036.tif" he="1238" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2103" /></tables>

As shown in Tables 17 and 18, a single 10 mg/kg dose of histidine substituted variant antibodies (VK-L30H and VH-D106H) was administered<i>Pcsk9</i>^hum/hum<i>Ldlr</i>^+/-In mice, each caused greater than about 45% on day 7 compared with baseline, and greater than about 44% of LDL-C reduction on day 14 compared with baseline. Furthermore, mice treated with a single dose of VK-L30H had at least 33% sustained LDL-C reduction compared to baseline until day 33. After a single dose of 10 mg/kg, the amount of LDL-C in mice treated with VK-L30H was still lower than baseline by almost 20% on day 46. The mice given VH-D106H had a similar initial decline as VK-L30H, but the amount of LDL-C only decreased by about 13% compared to baseline on day 33. A single dose of 316P (v1), compared with baseline, also caused approximately the same initial LDL-C reduction percentage as VK-L30H (in antibody administration On the 7th day afterwards, it decreased by approximately 49% compared to baseline), but the LDL-C lowering effect was not as prolonged as the histidine-substituted mutant. 316P(v2) and 300N showed the best short-term LDL-C lowering effect (each antibody decreased by about 58% compared with baseline on day 7), but compared with histidine-substituted mutants, they have a shorter duration effect.

The amount of circulating human antibodies in the mice of each treatment group was also determined using standard ELISA analysis. The assay plate was coated with a goat anti-human Fc antibody (Jackson ImmunoResearch, #109-005-098) with a 1 μg/ml PBS solution at 4°C for 18 hours. The assay plate was then blocked at room temperature (RT) for 3 hours. A standard curve is generated, and each antibody is added to the dish at a 2-fold dilution. From the 4th, 7th, 14th, 20th, 26th, 33rd, 42nd and 46th days after the antibody injection, the mouse serum is 1:100, 1:200, 1:500, 1:2000, 1:4000 and 1:8000 The dilution was added to the dish and then incubated at RT for 2 hours. Use goat anti-human IgG HRP conjugation antibody (Jackson ImmunoResearch, #109-035-098) to detect the captured antibody and use 3,3',5,5'-tetramethylbenzidine (TMB) (MP Biomedicals, #109-035-098) 152346) The matrix develops the colorimetric signal. At 2.0MH₂SO₄Stop the reaction and record the absorbance at 450nm to measure the total amount of human antibodies in the mouse serum. The average antibody amount at each time point in the treatment group of the calculation test and the results are shown in Table 19. The value is expressed as the average total serum antibody amount (μg/mL) (±SEM).

<tables><img alt="" file="twi596115b_d0037.tif" he="655" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2141" /></tables><tables><img alt="" file="twi596115b_d0038.tif" he="579" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2139" /></tables>

The maternal antibodies 316P (v1), 316P (v2), and 300N were cleared from the circulation on days 14, 20, and 26, respectively. They were not found in the serum samples of mice treated with these antibodies at the indicated time points. Human antibodies are detected. In contrast, human antibodies were detected in serum samples of mice treated with histidine-substituted mutant antibodies VK-L30H and VH-D106H until at least day 55. The amount of human antibodies is roughly related to the degree of cholesterol reduction observed at each time point. Therefore, the histidine substituted variant antibody of the present invention stays in the circulation of the treated animal for a longer time than the maternal antibody, and lowers serum LDL-C for a longer time than the maternal antibody.

Finally, the total amount of human PCSK9 in the serum of mice in each treatment group was measured at each time point. The results are expressed in ng/mL of human PCSK9, as shown in Table 20.

<tables><img alt="" file="twi596115b_d0039.tif" he="933" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2033" /></tables><tables><img alt="" file="twi596115b_d0040.tif" he="1263" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2051" /></tables>

At least 42 days after antibody administration, the total PCSK9 in mice treated with the histidine-substituted variants VK-L30H and VH-D106H remained above 1500ng/mL. In contrast, in all other treatment groups, the total PCSK9 amount fell below 1000 ng/mL on day 20 or earlier.

Next, use<i>Pcsk9</i>^hum/hum<i>Ldlr</i>^+/-The mouse evaluates the histidine-substituted variant anti-PCSK9 antibody VK-L30H relative to various comparator anti-PCSK9 antibodies (comparators 1, 2, 3, and 4 as defined in Table 5) in terms of serum LDL-C The effect. The mice were pre-collected 8 days before the experiment and divided into treatment groups based on their LDL-C amount, so that the average LDL-C amount of each group was equal. Then on day 0 of the study, mice (n=5/per treatment group) were injected subcutaneously with anti-PCSK9 antibody or an isotype (hIgG4) control antibody of irrelevant specificity at a dose of 10 mg/kg. Blood was taken from mice on days 4, 7, 14, 21, 28, 42, 49, 63, and 77 after the injection, and the amount of LDL-C in the serum was determined by ADVIA® 1800 Chemistry System (Siemens). Then calculate for each treatment group at each time point The average LDL-C in the serum and the results are expressed as the average amount of LDL-C (mg/dL) (±SEM), as shown in Table 21. Table 22 shows the percentage decrease in the amount of LDL-C compared to the baseline (ie, day 8).

<tables><img alt="" file="twi596115b_d0041.tif" he="2341" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2084" /></tables>

<b>Table 22: Compared with baseline [Day 8] Percentage change of LDL-C amount</b><tables><img alt="" file="twi596115b_d0042.tif" he="1420" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1996" /></tables>

As shown in Tables 21 and 22, a single 10 mg/kg dose of histidine substituted variant antibody VK-L30H was administered<i>Pcsk9</i>^hum/hum<i>Ldlr</i>^+/-Mice, causing a decrease in LDL-C greater than 30% compared to baseline in all 77-day measurements, with approximately 52% of the largest decrease in LDL-C compared to baseline on day 14. In contrast, mice dosed with Comparator 2 had a maximum LDL-C decrease of about 40% on day 7 after antibody administration, but at any time point on or after 14 days after antibody administration, The degree of this reduction is not obvious. A single dose of Comparator 1 showed a prolonged LDL-C decrease (wherein the largest LDL-C decrease of about 37% compared with the risk on day 14), but from the 42nd day to the 77th day of the end of the experiment, this The degree of LDL-C decrease compared with baseline is only about 9% to 24%. Although the presence of antibodies in the circulation was confirmed by ELISA, Comparators 3 and 4 did not show measurable effects in reducing LDL-C.

The amount of human antibodies circulating in the mice of each treatment group was using ELISA Method to detect total human IgG Fc. Calculate the average amount of antibody in mouse serum on 4th, 7th, 14, 21, 28, 42, 49, 63, and 77 days in the treatment group of the experiment, and calculate the average total serum antibody amount (μg/mL) ( ±SEM), as shown in Table 23.

<tables><img alt="" file="twi596115b_d0043.tif" he="2023" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2123" /></tables>

Comparator 2 and Comparator 4 were cleared from the circulation on days 28 and 77, respectively. After the indicated time point, mice treated with these antibodies were taken from serum samples No human antibodies have been detected in. In contrast, human antibodies were detected in the serum samples of mice treated with the histidine-substituted variant antibody VK-L30H and comparators 1 and 3, and human antibodies were still detected on day 77. On day 77, compared to all other treatment groups, the VK-L30H treatment group had the highest measurable human antibody.

Finally, the total amount of human PCSK9 in the serum of mice in each treatment group was measured at each time point. The results are expressed as the average amount of human PCSK9 (ng/mL) (±SEM), as shown in Table 24.

<tables><img alt="" file="twi596115b_d0044.tif" he="1955" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2125" /></tables><tables><img alt="" file="twi596115b_d0045.tif" he="404" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="2111" /></tables>

In this experiment, after antibody administration, the total human PCSK9 amount in mice treated with the histidine-substituted variant antibody VK-L30H and comparator 3 was maintained above 2500ng/mL for at least 77 days. In contrast, the total PCSK9 amount in the treatment groups of Comparator 2 and Comparator 4 fell below 1000 ng/mL on the 21st and 42nd days. The total amount of PCSK9 in the Comparator 1 treatment group has never been higher than 1000 ng/mL.

The present invention is not limited to the scope of the specific embodiments described in the text. In fact, in addition to those described in the text, from the foregoing description and accompanying drawings, various modifications of the present invention will be obvious to those who are familiar with the art. These modifications are hoped to fall within the scope of the attached patent application.

&lt;110&gt; George D.Yancopoulos Nicholas J.Papapopoulos Andrew J.Murphy Neil Stahl

&lt;120&gt; 具有pH-依賴性結合特性之抗-PCSK9抗體

&lt;130&gt; 8450A

&lt;140&gt; 分派至

&lt;141&gt; 隨附提交

&lt;150&gt; 61/682,349

&lt;151&gt; 2012-08-13

&lt;150&gt; 61/703,342

&lt;151&gt; 2012-09-20

&lt;150&gt; 61/734,398

&lt;151&gt; 2012-12-07

&lt;150&gt; 61/761,383

&lt;151&gt; 2013-02-06

&lt;150&gt; 61/828,258

&lt;151&gt; 2013-05-29

&lt;160&gt; 820

&lt;170&gt; FastSEQ for Windows Version 4.0

&lt;210&gt; 1

&lt;211&gt; 351

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 1<img alt="" file="TWI596115B_D0046.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1655" />

&lt;210&gt; 2

&lt;211&gt; 117

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 2<img alt="" file="TWI596115B_D0047.tif" he="781" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1578" />

&lt;210&gt; 3

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 3<img alt="" file="TWI596115B_D0048.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1620" />

&lt;210&gt; 4

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 4<img alt="" file="TWI596115B_D0049.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="810" />

&lt;210&gt; 5

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 5<img alt="" file="TWI596115B_D0050.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1617" />

&lt;210&gt; 6

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 6<img alt="" file="TWI596115B_D0051.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="694" />

&lt;210&gt; 7

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 7<img alt="" file="TWI596115B_D0052.tif" he="77" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1638" />

&lt;210&gt; 8

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 8<img alt="" file="TWI596115B_D0053.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1089" />

&lt;210&gt; 9

&lt;211&gt; 327

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 9<img alt="" file="TWI596115B_D0054.tif" he="308" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" />

&lt;210&gt; 10

&lt;211&gt; 109

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 10<img alt="" file="TWI596115B_D0055.tif" he="343" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1548" /><img alt="" file="TWI596115B_D0056.tif" he="356" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1536" />

&lt;210&gt; 11

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 11<img alt="" file="TWI596115B_D0057.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1645" />

&lt;210&gt; 12

&lt;211&gt; 6

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 12<img alt="" file="TWI596115B_D0058.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="633" />

&lt;210&gt; 13

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 13<img alt="" file="TWI596115B_D0059.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1609" />

&lt;210&gt; 14

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 14<img alt="" file="TWI596115B_D0060.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="334" />

&lt;210&gt; 15

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 15<img alt="" file="TWI596115B_D0061.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1663" />

&lt;210&gt; 16

&lt;211&gt; 10

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 16<img alt="" file="TWI596115B_D0062.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="955" />

&lt;210&gt; 17

&lt;211&gt; 351

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 17<img alt="" file="TWI596115B_D0063.tif" he="299" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1676" />

&lt;210&gt; 18

&lt;211&gt; 117

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 18<img alt="" file="TWI596115B_D0064.tif" he="772" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1576" />

&lt;210&gt; 19

&lt;211&gt; 324

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 19<img alt="" file="TWI596115B_D0065.tif" he="311" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1687" />

&lt;210&gt; 20

&lt;211&gt; 108

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 20<img alt="" file="TWI596115B_D0066.tif" he="659" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1575" />

&lt;210&gt; 21

&lt;211&gt; 351

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 21<img alt="" file="TWI596115B_D0067.tif" he="308" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1642" />

&lt;210&gt; 22

&lt;211&gt; 117

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 22<img alt="" file="TWI596115B_D0068.tif" he="761" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1536" />

&lt;210&gt; 23

&lt;211&gt; 324

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 23<img alt="" file="TWI596115B_D0069.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1658" />

&lt;210&gt; 24

&lt;211&gt; 108

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 24<img alt="" file="TWI596115B_D0070.tif" he="540" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1594" /><img alt="" file="TWI596115B_D0071.tif" he="170" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1462" />

&lt;210&gt; 25

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 25<img alt="" file="TWI596115B_D0072.tif" he="311" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1639" />

&lt;210&gt; 26

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 26<img alt="" file="TWI596115B_D0073.tif" he="726" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1517" />

&lt;210&gt; 27

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 27<img alt="" file="TWI596115B_D0074.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1645" />

&lt;210&gt; 28

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 28<img alt="" file="TWI596115B_D0075.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="781" />

&lt;210&gt; 29

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 29<img alt="" file="TWI596115B_D0076.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1613" />

&lt;210&gt; 30

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 30<img alt="" file="TWI596115B_D0077.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="800" />

&lt;210&gt; 31

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 31<img alt="" file="TWI596115B_D0078.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1594" />

&lt;210&gt; 32

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 32<img alt="" file="TWI596115B_D0079.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="726" />

&lt;210&gt; 33

&lt;211&gt; 321

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 33<img alt="" file="TWI596115B_D0080.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1678" />

&lt;210&gt; 34

&lt;211&gt; 107

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 34<img alt="" file="TWI596115B_D0081.tif" he="668" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1568" />

&lt;210&gt; 35

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 35<img alt="" file="TWI596115B_D0082.tif" he="54" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1626" />

&lt;210&gt; 36

&lt;211&gt; 6

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 36<img alt="" file="TWI596115B_D0083.tif" he="118" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="633" />

&lt;210&gt; 37

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 37<img alt="" file="TWI596115B_D0084.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1611" />

&lt;210&gt; 38

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 38<img alt="" file="TWI596115B_D0085.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="337" />

&lt;210&gt; 39

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 39<img alt="" file="TWI596115B_D0086.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1620" />

&lt;210&gt; 40

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 40<img alt="" file="TWI596115B_D0087.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="829" />

&lt;210&gt; 41

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 41<img alt="" file="TWI596115B_D0088.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1714" /><img alt="" file="TWI596115B_D0089.tif" he="205" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1649" />

&lt;210&gt; 42

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 42<img alt="" file="TWI596115B_D0090.tif" he="713" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1565" />

&lt;210&gt; 43

&lt;211&gt; 318

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 43<img alt="" file="TWI596115B_D0091.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1684" />

&lt;210&gt; 44

&lt;211&gt; 106

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 44<img alt="" file="TWI596115B_D0092.tif" he="202" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1559" /><img alt="" file="TWI596115B_D0093.tif" he="475" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1533" />

&lt;210&gt; 45

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 45<img alt="" file="TWI596115B_D0094.tif" he="292" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1694" />

&lt;210&gt; 46

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 46<img alt="" file="TWI596115B_D0095.tif" he="732" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1552" />

&lt;210&gt; 47

&lt;211&gt; 319

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 47<img alt="" file="TWI596115B_D0096.tif" he="308" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1661" />

&lt;210&gt; 48

&lt;211&gt; 106

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 48<img alt="" file="TWI596115B_D0097.tif" he="661" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1548" />

&lt;210&gt; 49

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 49<img alt="" file="TWI596115B_D0098.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1687" />

&lt;210&gt; 50

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 50<img alt="" file="TWI596115B_D0099.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1520" /><img alt="" file="TWI596115B_D0100.tif" he="675" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1576" />

&lt;210&gt; 51

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 51<img alt="" file="TWI596115B_D0101.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1611" />

&lt;210&gt; 52

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 52<img alt="" file="TWI596115B_D0102.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="855" />

&lt;210&gt; 53

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 53<img alt="" file="TWI596115B_D0103.tif" he="74" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1618" />

&lt;210&gt; 54

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 54<img alt="" file="TWI596115B_D0104.tif" he="135" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="769" />

&lt;210&gt; 55

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 55<img alt="" file="TWI596115B_D0105.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1635" />

&lt;210&gt; 56

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 56<img alt="" file="TWI596115B_D0106.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="720" />

&lt;210&gt; 57

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 57<img alt="" file="TWI596115B_D0107.tif" he="305" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1709" />

&lt;210&gt; 58

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 58<img alt="" file="TWI596115B_D0108.tif" he="392" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1583" /><img alt="" file="TWI596115B_D0109.tif" he="343" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1538" />

&lt;210&gt; 59

&lt;211&gt; 36

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 59<img alt="" file="TWI596115B_D0110.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1618" />

&lt;210&gt; 60

&lt;211&gt; 12

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 60<img alt="" file="TWI596115B_D0111.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1149" />

&lt;210&gt; 61

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 61<img alt="" file="TWI596115B_D0112.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1622" />

&lt;210&gt; 62

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 62<img alt="" file="TWI596115B_D0113.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="318" />

&lt;210&gt; 63

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 63<img alt="" file="TWI596115B_D0114.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1613" />

&lt;210&gt; 64

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 64<img alt="" file="TWI596115B_D0115.tif" he="102" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="909" />

&lt;210&gt; 65

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 65<img alt="" file="TWI596115B_D0116.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1654" />

&lt;210&gt; 66

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 66<img alt="" file="TWI596115B_D0117.tif" he="719" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1545" />

&lt;210&gt; 67

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 67<img alt="" file="TWI596115B_D0118.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1674" />

&lt;210&gt; 68

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 68<img alt="" file="TWI596115B_D0119.tif" he="707" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1559" />

&lt;210&gt; 69

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 69<img alt="" file="TWI596115B_D0120.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1694" />

&lt;210&gt; 70

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 70<img alt="" file="TWI596115B_D0121.tif" he="723" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1582" />

&lt;210&gt; 71

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 71<img alt="" file="TWI596115B_D0122.tif" he="308" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1669" />

&lt;210&gt; 72

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 72<img alt="" file="TWI596115B_D0123.tif" he="369" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1582" /><img alt="" file="TWI596115B_D0124.tif" he="324" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1562" />

&lt;210&gt; 73

&lt;211&gt; 354

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 73<img alt="" file="TWI596115B_D0125.tif" he="311" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1703" />

&lt;210&gt; 74

&lt;211&gt; 118

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 74<img alt="" file="TWI596115B_D0126.tif" he="768" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1536" />

&lt;210&gt; 75

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 75<img alt="" file="TWI596115B_D0127.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1633" />

&lt;210&gt; 76

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 76<img alt="" file="TWI596115B_D0128.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="813" />

&lt;210&gt; 77

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 77<img alt="" file="TWI596115B_D0129.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1617" />

&lt;210&gt; 78

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 78<img alt="" file="TWI596115B_D0130.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="826" />

&lt;210&gt; 79

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 79<img alt="" file="TWI596115B_D0131.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1624" />

&lt;210&gt; 80

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 80<img alt="" file="TWI596115B_D0132.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1099" /><img alt="" file="TWI596115B_D0133.tif" he="77" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="896" />

&lt;210&gt; 81

&lt;211&gt; 342

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 81<img alt="" file="TWI596115B_D0134.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1699" />

&lt;210&gt; 82

&lt;211&gt; 114

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 82<img alt="" file="TWI596115B_D0135.tif" he="722" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1551" />

&lt;210&gt; 83

&lt;211&gt; 36

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 83<img alt="" file="TWI596115B_D0136.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1663" />

&lt;210&gt; 84

&lt;211&gt; 12

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 84<img alt="" file="TWI596115B_D0137.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1186" />

&lt;210&gt; 85

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 85<img alt="" file="TWI596115B_D0138.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1623" />

&lt;210&gt; 86

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 86<img alt="" file="TWI596115B_D0139.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="356" />

&lt;210&gt; 87

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 87<img alt="" file="TWI596115B_D0140.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1620" />

&lt;210&gt; 88

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 88<img alt="" file="TWI596115B_D0141.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="929" />

&lt;210&gt; 89

&lt;211&gt; 354

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 89<img alt="" file="TWI596115B_D0142.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1687" />

&lt;210&gt; 90

&lt;211&gt; 118

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 90<img alt="" file="TWI596115B_D0143.tif" he="755" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1603" />

&lt;210&gt; 91

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 91<img alt="" file="TWI596115B_D0144.tif" he="305" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1696" />

&lt;210&gt; 92

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 92<img alt="" file="TWI596115B_D0145.tif" he="720" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1527" />

&lt;210&gt; 93

&lt;211&gt; 354

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 93<img alt="" file="TWI596115B_D0146.tif" he="305" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1701" />

&lt;210&gt; 94

&lt;211&gt; 118

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 94<img alt="" file="TWI596115B_D0147.tif" he="717" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1540" /><img alt="" file="TWI596115B_D0148.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="343" />

&lt;210&gt; 95

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 95<img alt="" file="TWI596115B_D0149.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1664" />

&lt;210&gt; 96

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 96<img alt="" file="TWI596115B_D0150.tif" he="729" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1568" />

&lt;210&gt; 97

&lt;211&gt; 351

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 97<img alt="" file="TWI596115B_D0151.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1674" />

&lt;210&gt; 98

&lt;211&gt; 117

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 98<img alt="" file="TWI596115B_D0152.tif" he="774" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1574" />

&lt;210&gt; 99

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 99<img alt="" file="TWI596115B_D0153.tif" he="54" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1603" />

&lt;210&gt; 100

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 100<img alt="" file="TWI596115B_D0154.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="796" />

&lt;210&gt; 101

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 101<img alt="" file="TWI596115B_D0155.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1632" />

&lt;210&gt; 102

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 102<img alt="" file="TWI596115B_D0156.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="694" />

&lt;210&gt; 103

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 103<img alt="" file="TWI596115B_D0157.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1616" />

&lt;210&gt; 104

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 104<img alt="" file="TWI596115B_D0158.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1079" />

&lt;210&gt; 105

&lt;211&gt; 324

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 105<img alt="" file="TWI596115B_D0159.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1635" />

&lt;210&gt; 106

&lt;211&gt; 108

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 106<img alt="" file="TWI596115B_D0160.tif" he="675" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1555" />

&lt;210&gt; 107

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 107<img alt="" file="TWI596115B_D0161.tif" he="77" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1604" />

&lt;210&gt; 108

&lt;211&gt; 6

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 108<img alt="" file="TWI596115B_D0162.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="639" />

&lt;210&gt; 109

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 109<img alt="" file="TWI596115B_D0163.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1613" />

&lt;210&gt; 110

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 110<img alt="" file="TWI596115B_D0164.tif" he="89" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="327" />

&lt;210&gt; 111

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 111<img alt="" file="TWI596115B_D0165.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1616" />

&lt;210&gt; 112

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 112<img alt="" file="TWI596115B_D0166.tif" he="118" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="880" />

&lt;210&gt; 113

&lt;211&gt; 351

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 113<img alt="" file="TWI596115B_D0167.tif" he="292" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1719" />

&lt;210&gt; 114

&lt;211&gt; 117

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 114<img alt="" file="TWI596115B_D0168.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1542" /><img alt="" file="TWI596115B_D0169.tif" he="482" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1553" />

&lt;210&gt; 115

&lt;211&gt; 321

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 115<img alt="" file="TWI596115B_D0170.tif" he="308" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1714" />

&lt;210&gt; 116

&lt;211&gt; 107

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 116<img alt="" file="TWI596115B_D0171.tif" he="675" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1598" />

&lt;210&gt; 117

&lt;211&gt; 351

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 117<img alt="" file="TWI596115B_D0172.tif" he="289" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1645" />

&lt;210&gt; 118

&lt;211&gt; 117

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 118<img alt="" file="TWI596115B_D0173.tif" he="755" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1584" />

&lt;210&gt; 119

&lt;211&gt; 321

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 119<img alt="" file="TWI596115B_D0174.tif" he="286" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1674" />

&lt;210&gt; 120

&lt;211&gt; 107

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 120<img alt="" file="TWI596115B_D0175.tif" he="77" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1532" /><img alt="" file="TWI596115B_D0176.tif" he="642" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1561" />

&lt;210&gt; 121

&lt;211&gt; 384

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 121<img alt="" file="TWI596115B_D0177.tif" he="343" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1706" />

&lt;210&gt; 122

&lt;211&gt; 128

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 122<img alt="" file="TWI596115B_D0178.tif" he="768" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1584" />

&lt;210&gt; 123

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 123<img alt="" file="TWI596115B_D0179.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1649" />

&lt;210&gt; 124

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 124<img alt="" file="TWI596115B_D0180.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="787" />

&lt;210&gt; 125

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 125<img alt="" file="TWI596115B_D0181.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1608" />

&lt;210&gt; 126

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 126<img alt="" file="TWI596115B_D0182.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="678" />

&lt;210&gt; 127

&lt;211&gt; 66

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 127<img alt="" file="TWI596115B_D0183.tif" he="99" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1659" />

&lt;210&gt; 128

&lt;211&gt; 22

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 128<img alt="" file="TWI596115B_D0184.tif" he="199" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1575" />

&lt;210&gt; 129

&lt;211&gt; 324

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 129<img alt="" file="TWI596115B_D0185.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1659" />

&lt;210&gt; 130

&lt;211&gt; 108

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 130<img alt="" file="TWI596115B_D0186.tif" he="675" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1582" />

&lt;210&gt; 131

&lt;211&gt; 18

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 131<img alt="" file="TWI596115B_D0187.tif" he="54" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1647" />

&lt;210&gt; 132

&lt;211&gt; 6

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 132<img alt="" file="TWI596115B_D0188.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="636" />

&lt;210&gt; 133

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 133<img alt="" file="TWI596115B_D0189.tif" he="54" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1586" />

&lt;210&gt; 134

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 134<img alt="" file="TWI596115B_D0190.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="350" />

&lt;210&gt; 135

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 135<img alt="" file="TWI596115B_D0191.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1670" />

&lt;210&gt; 136

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 136<img alt="" file="TWI596115B_D0192.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="851" /><img alt="" file="TWI596115B_D0193.tif" he="93" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="456" />

&lt;210&gt; 137

&lt;211&gt; 384

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 137<img alt="" file="TWI596115B_D0194.tif" he="334" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1703" />

&lt;210&gt; 138

&lt;211&gt; 128

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 138<img alt="" file="TWI596115B_D0195.tif" he="761" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1577" />

&lt;210&gt; 139

&lt;211&gt; 321

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 139<img alt="" file="TWI596115B_D0196.tif" he="250" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1674" /><img alt="" file="TWI596115B_D0197.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1638" />

&lt;210&gt; 140

&lt;211&gt; 107

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 140<img alt="" file="TWI596115B_D0198.tif" he="668" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1602" />

&lt;210&gt; 141

&lt;211&gt; 384

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 141<img alt="" file="TWI596115B_D0199.tif" he="353" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1647" />

&lt;210&gt; 142

&lt;211&gt; 128

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 142<img alt="" file="TWI596115B_D0200.tif" he="395" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1529" /><img alt="" file="TWI596115B_D0201.tif" he="392" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1587" />

&lt;210&gt; 143

&lt;211&gt; 321

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 143<img alt="" file="TWI596115B_D0202.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1674" />

&lt;210&gt; 144

&lt;211&gt; 107

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 144<img alt="" file="TWI596115B_D0203.tif" he="668" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1552" />

&lt;210&gt; 145

&lt;211&gt; 378

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 145<img alt="" file="TWI596115B_D0204.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1642" /><img alt="" file="TWI596115B_D0205.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1678" />

&lt;210&gt; 146

&lt;211&gt; 126

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 146<img alt="" file="TWI596115B_D0206.tif" he="758" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1568" />

&lt;210&gt; 147

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 147<img alt="" file="TWI596115B_D0207.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1617" />

&lt;210&gt; 148

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 148<img alt="" file="TWI596115B_D0208.tif" he="102" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="835" />

&lt;210&gt; 149

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 149<img alt="" file="TWI596115B_D0209.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1607" />

&lt;210&gt; 150

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 150<img alt="" file="TWI596115B_D0210.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="800" />

&lt;210&gt; 151

&lt;211&gt; 57

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 151<img alt="" file="TWI596115B_D0211.tif" he="54" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1668" />

&lt;210&gt; 152

&lt;211&gt; 19

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 152<img alt="" file="TWI596115B_D0212.tif" he="157" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1536" />

&lt;210&gt; 153

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 153<img alt="" file="TWI596115B_D0213.tif" he="257" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1697" /><img alt="" file="TWI596115B_D0214.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1648" />

&lt;210&gt; 154

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 154<img alt="" file="TWI596115B_D0215.tif" he="719" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1542" />

&lt;210&gt; 155

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 155<img alt="" file="TWI596115B_D0216.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1603" />

&lt;210&gt; 156

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 156<img alt="" file="TWI596115B_D0217.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1079" />

&lt;210&gt; 157

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 157<img alt="" file="TWI596115B_D0218.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1626" />

&lt;210&gt; 158

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 158<img alt="" file="TWI596115B_D0219.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="343" />

&lt;210&gt; 159

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 159<img alt="" file="TWI596115B_D0220.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1626" />

&lt;210&gt; 160

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 160<img alt="" file="TWI596115B_D0221.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="880" />

&lt;210&gt; 161

&lt;211&gt; 378

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 161<img alt="" file="TWI596115B_D0222.tif" he="343" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" />

&lt;210&gt; 162

&lt;211&gt; 126

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 162<img alt="" file="TWI596115B_D0223.tif" he="768" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1587" />

&lt;210&gt; 163

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 163<img alt="" file="TWI596115B_D0224.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1648" />

&lt;210&gt; 164

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 164<img alt="" file="TWI596115B_D0225.tif" he="633" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1539" /><img alt="" file="TWI596115B_D0226.tif" he="93" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1125" />

&lt;210&gt; 165

&lt;211&gt; 378

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 165<img alt="" file="TWI596115B_D0227.tif" he="340" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1678" />

&lt;210&gt; 166

&lt;211&gt; 126

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 166<img alt="" file="TWI596115B_D0228.tif" he="755" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1633" />

&lt;210&gt; 167

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 167<img alt="" file="TWI596115B_D0229.tif" he="247" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1682" /><img alt="" file="TWI596115B_D0230.tif" he="69" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1659" />

&lt;210&gt; 168

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 168<img alt="" file="TWI596115B_D0231.tif" he="674" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1551" />

&lt;210&gt; 169

&lt;211&gt; 375

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 169<img alt="" file="TWI596115B_D0232.tif" he="337" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1647" />

&lt;210&gt; 170

&lt;211&gt; 125

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 170<img alt="" file="TWI596115B_D0233.tif" he="395" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1570" /><img alt="" file="TWI596115B_D0234.tif" he="392" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1600" />

&lt;210&gt; 171

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 171<img alt="" file="TWI596115B_D0235.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1616" />

&lt;210&gt; 172

&lt;211&gt; 10

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 172<img alt="" file="TWI596115B_D0236.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1002" />

&lt;210&gt; 173

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 173<img alt="" file="TWI596115B_D0237.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1642" />

&lt;210&gt; 174

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 174<img alt="" file="TWI596115B_D0238.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="694" />

&lt;210&gt; 175

&lt;211&gt; 51

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 175<img alt="" file="TWI596115B_D0239.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1667" />

&lt;210&gt; 176

&lt;211&gt; 17

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 176<img alt="" file="TWI596115B_D0240.tif" he="163" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1551" />

&lt;210&gt; 177

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 177<img alt="" file="TWI596115B_D0241.tif" he="311" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1676" />

&lt;210&gt; 178

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 178<img alt="" file="TWI596115B_D0242.tif" he="645" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1545" /><img alt="" file="TWI596115B_D0243.tif" he="125" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1366" />

&lt;210&gt; 179

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 179<img alt="" file="TWI596115B_D0244.tif" he="73" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1626" />

&lt;210&gt; 180

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 180<img alt="" file="TWI596115B_D0245.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1057" />

&lt;210&gt; 181

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 181<img alt="" file="TWI596115B_D0246.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1597" />

&lt;210&gt; 182

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 182<img alt="" file="TWI596115B_D0247.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="327" />

&lt;210&gt; 183

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 183<img alt="" file="TWI596115B_D0248.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1604" />

&lt;210&gt; 184

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 184<img alt="" file="TWI596115B_D0249.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="881" />

&lt;210&gt; 185

&lt;211&gt; 375

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 185<img alt="" file="TWI596115B_D0250.tif" he="340" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1633" />

&lt;210&gt; 186

&lt;211&gt; 125

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 186<img alt="" file="TWI596115B_D0251.tif" he="778" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1585" />

&lt;210&gt; 187

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 187<img alt="" file="TWI596115B_D0252.tif" he="311" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1658" />

&lt;210&gt; 188

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 188<img alt="" file="TWI596115B_D0253.tif" he="671" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1562" />

&lt;210&gt; 189

&lt;211&gt; 375

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 189<img alt="" file="TWI596115B_D0254.tif" he="340" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1687" />

&lt;210&gt; 190

&lt;211&gt; 125

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 190<img alt="" file="TWI596115B_D0255.tif" he="765" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1533" />

&lt;210&gt; 191

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 191<img alt="" file="TWI596115B_D0256.tif" he="305" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" />

&lt;210&gt; 192

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 192<img alt="" file="TWI596115B_D0257.tif" he="636" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1549" /><img alt="" file="TWI596115B_D0258.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1073" />

&lt;210&gt; 193

&lt;211&gt; 375

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 193<img alt="" file="TWI596115B_D0259.tif" he="343" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1703" />

&lt;210&gt; 194

&lt;211&gt; 125

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 194<img alt="" file="TWI596115B_D0260.tif" he="758" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1594" />

&lt;210&gt; 195

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 195<img alt="" file="TWI596115B_D0261.tif" he="80" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1623" />

&lt;210&gt; 196

&lt;211&gt; 10

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 196<img alt="" file="TWI596115B_D0262.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="987" />

&lt;210&gt; 197

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 197<img alt="" file="TWI596115B_D0263.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1601" />

&lt;210&gt; 198

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 198<img alt="" file="TWI596115B_D0264.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="697" />

&lt;210&gt; 199

&lt;211&gt; 51

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 199<img alt="" file="TWI596115B_D0265.tif" he="83" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1611" />

&lt;210&gt; 200

&lt;211&gt; 17

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 200<img alt="" file="TWI596115B_D0266.tif" he="164" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1534" />

&lt;210&gt; 201

&lt;211&gt; 339

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 201<img alt="" file="TWI596115B_D0267.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1643" />

&lt;210&gt; 202

&lt;211&gt; 113

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 202<img alt="" file="TWI596115B_D0268.tif" he="720" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1588" />

&lt;210&gt; 203

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 203<img alt="" file="TWI596115B_D0269.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1633" />

&lt;210&gt; 204

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 204<img alt="" file="TWI596115B_D0270.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1137" />

&lt;210&gt; 205

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 205<img alt="" file="TWI596115B_D0271.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1599" />

&lt;210&gt; 206

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 206<img alt="" file="TWI596115B_D0272.tif" he="102" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="314" />

&lt;210&gt; 207

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 207<img alt="" file="TWI596115B_D0273.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1628" />

&lt;210&gt; 208

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 208<img alt="" file="TWI596115B_D0274.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="880" />

&lt;210&gt; 209

&lt;211&gt; 375

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 209<img alt="" file="TWI596115B_D0275.tif" he="350" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1628" />

&lt;210&gt; 210

&lt;211&gt; 125

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 210<img alt="" file="TWI596115B_D0276.tif" he="761" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1545" />

&lt;210&gt; 211

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 211<img alt="" file="TWI596115B_D0277.tif" he="301" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1683" />

&lt;210&gt; 212

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 212<img alt="" file="TWI596115B_D0278.tif" he="668" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1586" />

&lt;210&gt; 213

&lt;211&gt; 375

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 213<img alt="" file="TWI596115B_D0279.tif" he="340" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1680" />

&lt;210&gt; 214

&lt;211&gt; 125

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 214<img alt="" file="TWI596115B_D0280.tif" he="790" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1567" />

&lt;210&gt; 215

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 215<img alt="" file="TWI596115B_D0281.tif" he="292" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1696" />

&lt;210&gt; 216

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 216<img alt="" file="TWI596115B_D0282.tif" he="658" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1581" />

&lt;210&gt; 217

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 217<img alt="" file="TWI596115B_D0283.tif" he="337" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1683" />

&lt;210&gt; 218

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 218<img alt="" file="TWI596115B_D0284.tif" he="771" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1523" />

&lt;210&gt; 219

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 219<img alt="" file="TWI596115B_D0285.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1645" />

&lt;210&gt; 220

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 220<img alt="" file="TWI596115B_D0286.tif" he="134" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="806" />

&lt;210&gt; 221

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 221<img alt="" file="TWI596115B_D0287.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1638" />

&lt;210&gt; 222

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 222<img alt="" file="TWI596115B_D0288.tif" he="96" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="761" />

&lt;210&gt; 223

&lt;211&gt; 60

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 223<img alt="" file="TWI596115B_D0289.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1604" />

&lt;210&gt; 224

&lt;211&gt; 20

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 224<img alt="" file="TWI596115B_D0290.tif" he="205" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1546" />

&lt;210&gt; 225

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 225<img alt="" file="TWI596115B_D0291.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1691" />

&lt;210&gt; 226

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 226<img alt="" file="TWI596115B_D0292.tif" he="678" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1581" />

&lt;210&gt; 227

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 227<img alt="" file="TWI596115B_D0293.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1645" />

&lt;210&gt; 228

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 228<img alt="" file="TWI596115B_D0294.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1105" />

&lt;210&gt; 229

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 229<img alt="" file="TWI596115B_D0295.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1590" />

&lt;210&gt; 230

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 230<img alt="" file="TWI596115B_D0296.tif" he="141" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="340" />

&lt;210&gt; 231

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 231<img alt="" file="TWI596115B_D0297.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1620" />

&lt;210&gt; 232

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 232<img alt="" file="TWI596115B_D0298.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="868" />

&lt;210&gt; 233

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 233<img alt="" file="TWI596115B_D0299.tif" he="347" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1662" />

&lt;210&gt; 234

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 234<img alt="" file="TWI596115B_D0300.tif" he="350" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1546" /><img alt="" file="TWI596115B_D0301.tif" he="446" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1536" />

&lt;210&gt; 235

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 235<img alt="" file="TWI596115B_D0302.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1662" />

&lt;210&gt; 236

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 236<img alt="" file="TWI596115B_D0303.tif" he="665" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1565" />

&lt;210&gt; 237

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 237<img alt="" file="TWI596115B_D0304.tif" he="340" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1675" />

&lt;210&gt; 238

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 238<img alt="" file="TWI596115B_D0305.tif" he="749" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1559" />

&lt;210&gt; 239

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 239<img alt="" file="TWI596115B_D0306.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1672" />

&lt;210&gt; 240

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 240<img alt="" file="TWI596115B_D0307.tif" he="54" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1537" /><img alt="" file="TWI596115B_D0308.tif" he="629" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1567" />

&lt;210&gt; 241

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 241<img alt="" file="TWI596115B_D0309.tif" he="334" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1660" />

&lt;210&gt; 242

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 242<img alt="" file="TWI596115B_D0310.tif" he="767" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1573" />

&lt;210&gt; 243

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 243<img alt="" file="TWI596115B_D0311.tif" he="51" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1626" />

&lt;210&gt; 244

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 244<img alt="" file="TWI596115B_D0312.tif" he="102" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="806" />

&lt;210&gt; 245

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 245<img alt="" file="TWI596115B_D0313.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1600" />

&lt;210&gt; 246

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 246<img alt="" file="TWI596115B_D0314.tif" he="106" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="793" />

&lt;210&gt; 247

&lt;211&gt; 60

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 247<img alt="" file="TWI596115B_D0315.tif" he="77" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1619" />

&lt;210&gt; 248

&lt;211&gt; 20

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 248<img alt="" file="TWI596115B_D0316.tif" he="208" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1525" />

&lt;210&gt; 249

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 249<img alt="" file="TWI596115B_D0317.tif" he="301" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1683" />

&lt;210&gt; 250

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 250<img alt="" file="TWI596115B_D0318.tif" he="661" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1583" />

&lt;210&gt; 251

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 251<img alt="" file="TWI596115B_D0319.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1618" />

&lt;210&gt; 252

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 252<img alt="" file="TWI596115B_D0320.tif" he="128" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1078" />

&lt;210&gt; 253

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 253<img alt="" file="TWI596115B_D0321.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1611" />

&lt;210&gt; 254

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 254<img alt="" file="TWI596115B_D0322.tif" he="93" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="346" />

&lt;210&gt; 255

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 255<img alt="" file="TWI596115B_D0323.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1640" />

&lt;210&gt; 256

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 256<img alt="" file="TWI596115B_D0324.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="828" /><img alt="" file="TWI596115B_D0325.tif" he="86" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="504" />

&lt;210&gt; 257

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 257<img alt="" file="TWI596115B_D0326.tif" he="347" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1633" />

&lt;210&gt; 258

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 258<img alt="" file="TWI596115B_D0327.tif" he="768" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1555" />

&lt;210&gt; 259

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 259<img alt="" file="TWI596115B_D0328.tif" he="257" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1687" /><img alt="" file="TWI596115B_D0329.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1635" />

&lt;210&gt; 260

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 260<img alt="" file="TWI596115B_D0330.tif" he="668" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1516" />

&lt;210&gt; 261

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 261<img alt="" file="TWI596115B_D0331.tif" he="347" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1677" />

&lt;210&gt; 262

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 262<img alt="" file="TWI596115B_D0332.tif" he="385" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1581" /><img alt="" file="TWI596115B_D0333.tif" he="388" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1581" />

&lt;210&gt; 263

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 263<img alt="" file="TWI596115B_D0334.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1678" />

&lt;210&gt; 264

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 264<img alt="" file="TWI596115B_D0335.tif" he="665" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1568" />

&lt;210&gt; 265

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 265<img alt="" file="TWI596115B_D0336.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1642" /><img alt="" file="TWI596115B_D0337.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" />

&lt;210&gt; 266

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 266<img alt="" file="TWI596115B_D0338.tif" he="777" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1587" />

&lt;210&gt; 267

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 267<img alt="" file="TWI596115B_D0339.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" />

&lt;210&gt; 268

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 268<img alt="" file="TWI596115B_D0340.tif" he="118" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="796" />

&lt;210&gt; 269

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 269<img alt="" file="TWI596115B_D0341.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1617" />

&lt;210&gt; 270

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 270<img alt="" file="TWI596115B_D0342.tif" he="118" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="816" />

&lt;210&gt; 271

&lt;211&gt; 60

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 271<img alt="" file="TWI596115B_D0343.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1623" />

&lt;210&gt; 272

&lt;211&gt; 20

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 272<img alt="" file="TWI596115B_D0344.tif" he="199" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1520" />

&lt;210&gt; 273

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 273<img alt="" file="TWI596115B_D0345.tif" he="205" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" /><img alt="" file="TWI596115B_D0346.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1707" />

&lt;210&gt; 274

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 274<img alt="" file="TWI596115B_D0347.tif" he="675" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1543" />

&lt;210&gt; 275

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 275<img alt="" file="TWI596115B_D0348.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1604" />

&lt;210&gt; 276

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 276<img alt="" file="TWI596115B_D0349.tif" he="118" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1070" />

&lt;210&gt; 277

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 277<img alt="" file="TWI596115B_D0350.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1580" />

&lt;210&gt; 278

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 278<img alt="" file="TWI596115B_D0351.tif" he="102" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="327" />

&lt;210&gt; 279

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 279<img alt="" file="TWI596115B_D0352.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1622" />

&lt;210&gt; 280

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 280<img alt="" file="TWI596115B_D0353.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="883" />

&lt;210&gt; 281

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 281<img alt="" file="TWI596115B_D0354.tif" he="350" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1667" />

&lt;210&gt; 282

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 282<img alt="" file="TWI596115B_D0355.tif" he="777" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1593" />

&lt;210&gt; 283

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 283<img alt="" file="TWI596115B_D0356.tif" he="292" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1667" />

&lt;210&gt; 284

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 284<img alt="" file="TWI596115B_D0357.tif" he="664" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1554" />

&lt;210&gt; 285

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 285<img alt="" file="TWI596115B_D0358.tif" he="337" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1654" />

&lt;210&gt; 286

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 286<img alt="" file="TWI596115B_D0359.tif" he="764" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1564" />

&lt;210&gt; 287

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 287<img alt="" file="TWI596115B_D0360.tif" he="301" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1667" />

&lt;210&gt; 288

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 288<img alt="" file="TWI596115B_D0361.tif" he="668" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1536" />

&lt;210&gt; 289

&lt;211&gt; 372

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 289<img alt="" file="TWI596115B_D0362.tif" he="337" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1693" />

&lt;210&gt; 290

&lt;211&gt; 124

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 290<img alt="" file="TWI596115B_D0363.tif" he="446" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1548" /><img alt="" file="TWI596115B_D0364.tif" he="343" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1545" />

&lt;210&gt; 291

&lt;211&gt; 30

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 291<img alt="" file="TWI596115B_D0365.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1596" />

&lt;210&gt; 292

&lt;211&gt; 10

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 292<img alt="" file="TWI596115B_D0366.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="986" />

&lt;210&gt; 293

&lt;211&gt; 21

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 293<img alt="" file="TWI596115B_D0367.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1625" />

&lt;210&gt; 294

&lt;211&gt; 7

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 294<img alt="" file="TWI596115B_D0368.tif" he="122" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="706" />

&lt;210&gt; 295

&lt;211&gt; 48

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 295<img alt="" file="TWI596115B_D0369.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1629" />

&lt;210&gt; 296

&lt;211&gt; 16

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 296<img alt="" file="TWI596115B_D0370.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1533" />

&lt;210&gt; 297

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 297<img alt="" file="TWI596115B_D0371.tif" he="302" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1732" />

&lt;210&gt; 298

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 298<img alt="" file="TWI596115B_D0372.tif" he="678" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1591" />

&lt;210&gt; 299

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 299<img alt="" file="TWI596115B_D0373.tif" he="51" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1648" />

&lt;210&gt; 300

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 300<img alt="" file="TWI596115B_D0374.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1079" />

&lt;210&gt; 301

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 301<img alt="" file="TWI596115B_D0375.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1600" />

&lt;210&gt; 302

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 302<img alt="" file="TWI596115B_D0376.tif" he="99" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="327" />

&lt;210&gt; 303

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 303<img alt="" file="TWI596115B_D0377.tif" he="61" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1626" />

&lt;210&gt; 304

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 304<img alt="" file="TWI596115B_D0378.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="903" />

&lt;210&gt; 305

&lt;211&gt; 372

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 305<img alt="" file="TWI596115B_D0379.tif" he="347" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1668" />

&lt;210&gt; 306

&lt;211&gt; 124

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 306<img alt="" file="TWI596115B_D0380.tif" he="765" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1539" />

&lt;210&gt; 307

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 307<img alt="" file="TWI596115B_D0381.tif" he="296" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1679" />

&lt;210&gt; 308

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 308<img alt="" file="TWI596115B_D0382.tif" he="666" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1566" />

&lt;210&gt; 309

&lt;211&gt; 372

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 309<img alt="" file="TWI596115B_D0383.tif" he="337" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1685" />

&lt;210&gt; 310

&lt;211&gt; 124

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 310<img alt="" file="TWI596115B_D0384.tif" he="778" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1579" />

&lt;210&gt; 311

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 311<img alt="" file="TWI596115B_D0385.tif" he="299" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1637" />

&lt;210&gt; 312

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 312<img alt="" file="TWI596115B_D0386.tif" he="652" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1598" />

&lt;210&gt; 313

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 313<img alt="" file="TWI596115B_D0387.tif" he="350" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1661" />

&lt;210&gt; 314

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 314<img alt="" file="TWI596115B_D0388.tif" he="768" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1581" />

&lt;210&gt; 315

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 315<img alt="" file="TWI596115B_D0389.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1632" />

&lt;210&gt; 316

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 316<img alt="" file="TWI596115B_D0390.tif" he="115" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="784" />

&lt;210&gt; 317

&lt;211&gt; 24

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 317<img alt="" file="TWI596115B_D0391.tif" he="57" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1635" />

&lt;210&gt; 318

&lt;211&gt; 8

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 318<img alt="" file="TWI596115B_D0392.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="806" />

&lt;210&gt; 319

&lt;211&gt; 60

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 319<img alt="" file="TWI596115B_D0393.tif" he="70" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1632" />

&lt;210&gt; 320

&lt;211&gt; 20

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 320<img alt="" file="TWI596115B_D0394.tif" he="212" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1542" />

&lt;210&gt; 321

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 321<img alt="" file="TWI596115B_D0395.tif" he="289" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1648" />

&lt;210&gt; 322

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 322<img alt="" file="TWI596115B_D0396.tif" he="671" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1532" />

&lt;210&gt; 323

&lt;211&gt; 33

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 323<img alt="" file="TWI596115B_D0397.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1597" />

&lt;210&gt; 324

&lt;211&gt; 11

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 324<img alt="" file="TWI596115B_D0398.tif" he="128" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1047" />

&lt;210&gt; 325

&lt;211&gt; 9

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 325<img alt="" file="TWI596115B_D0399.tif" he="67" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1593" />

&lt;210&gt; 326

&lt;211&gt; 3

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 326<img alt="" file="TWI596115B_D0400.tif" he="99" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="337" />

&lt;210&gt; 327

&lt;211&gt; 27

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 327<img alt="" file="TWI596115B_D0401.tif" he="64" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1629" />

&lt;210&gt; 328

&lt;211&gt; 9

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 328<img alt="" file="TWI596115B_D0402.tif" he="109" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="912" />

&lt;210&gt; 329

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 329<img alt="" file="TWI596115B_D0403.tif" he="250" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1699" /><img alt="" file="TWI596115B_D0404.tif" he="112" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1642" />

&lt;210&gt; 330

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 330<img alt="" file="TWI596115B_D0405.tif" he="777" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1558" />

&lt;210&gt; 331

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 331<img alt="" file="TWI596115B_D0406.tif" he="298" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1671" />

&lt;210&gt; 332

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 332<img alt="" file="TWI596115B_D0407.tif" he="286" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1587" /><img alt="" file="TWI596115B_D0408.tif" he="411" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1564" />

&lt;210&gt; 333

&lt;211&gt; 381

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 333<img alt="" file="TWI596115B_D0409.tif" he="350" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1674" />

&lt;210&gt; 334

&lt;211&gt; 127

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 334<img alt="" file="TWI596115B_D0410.tif" he="774" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1584" />

&lt;210&gt; 335

&lt;211&gt; 336

&lt;212&gt; DNA

&lt;213&gt; 人工序列

&lt;220&gt;

&lt;223&gt; 合成的

&lt;400&gt; 335<img alt="" file="TWI596115B_D0411.tif" he="295" id="" img-content="drawing" img-format="tif" inline="no" orientation="portrait" wi="1693" />

&lt;210&gt; 336

&lt;211&gt; 112

&lt;212&gt; PRT

&lt;213&gt; 人工序列

&lt;220&gt;