Anti-CD22 antibodies and uses thereof

Abstract

Disclosed herein are high affinity anti-CD22 antibodies and methods of using them for therapeutic and / or diagnostic purposes. Also provided herein are methods of producing such anti-CD22 antibodies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 889,739, filed on August 21, 2019, the entire contents of which are incorporated herein by reference. Background of the Invention

[0004] Cluster of differentiation 22 (CD22) is a member of the SIGLEC lectin family. This molecule is expressed at high levels on the surface of mature B cells relative to immature B cells. As an inhibitory receptor for B cell receptor (BCR) signaling, it plays a regulatory role in preventing overactivation of the immune system.

[0005] CD22 has been shown to be a promising target for leukemia therapy, such as acute lymphoblastic leukemia therapy, and for the treatment of systemic autoimmune diseases. SUMMARY OF THE INVENTION

[0007] The present disclosure is based at least in part on the development of excellent anti-CD22 antibodies with high binding affinity and specificity for CD22 expressed on the cell surface. Anti-CD22 antibodies disclosed herein bind to different CD22 epitopes, such as the known anti-CD22 antibodies M971 and RFB4 (the source of BL22) in current preclinical and clinical studies. In addition, certain exemplary anti-CD22 antibodies (e.g., clone EP160-D02) in the form of IgG show high binding affinity and specificity for cell surface CD22, as well as higher ADCC activity relative to BL22 and M971. The results provided herein show that it is expected that the anti-CD22 antibodies disclosed herein have a high therapeutic effect on CD22+ disease cells such as cancer cells.

[0008] Thus, one aspect of the disclosure features an isolated antibody that binds to CD22. Such an anti-CD22 may bind to the same epitope as a reference antibody or compete with a reference antibody for binding to CD22. Exemplary reference antibodies include EP35-A7, EP35-B5, EP35-C6, EP35-C6, EP35-C8, EP35-D6, EP35-E6, EP35-E7, EP97-A01, EP97-A10, EP97-B03, EP97-F01, EP97-G05, EP160-C07, EP160-D02, EP160-E03, EP160-F04, EP160-F10, EP160-G04, EP160-G05, and EP160-H02, for which structural information is provided below. In a specific example, the reference antibody is EP160-D02. In other embodiments, the reference antibody is EP97-B03.

[0009] In some embodiments, the anti-CD22 antibodies disclosed herein may comprise: (a) a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3), wherein the HC CDR1, HC CDR2, and HC CDR3 are collectively at least 80% identical to the heavy chain CDRs of a reference antibody; and / or a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3), wherein the LC CDR1, LC CDR2, and LC CDR3 are collectively at least 80% identical to the light chain CDRs of a reference antibody. In some cases, the anti-CD22 antibody may have a binding affinity for CD22 expressed on the surface of a cell of less than 10 nM (e.g., less than 1 nM).

[0010] In some embodiments, the anti-CD22 antibodies disclosed herein may comprise HC CDRs that collectively comprise no more than 8 amino acid residue variations compared to the HC CDRs of a reference antibody; and / or the LC CDRs of the antibodies collectively comprise no more than 8 amino acid residue variations compared to the LC CDRs of a reference antibody.

[0011] Any of the anti-CD22 antibodies disclosed herein may comprise a V H At least 85% the same V H , and / or with the reference antibody V L At least 85% the same V L In some examples, the anti-CD22 antibody may comprise the same heavy chain complementary determining region (HC CDR) and the same light chain complementary determining region (LC CDR) as the reference antibody. In a specific example, the anti-CD22 antibody may comprise the same V CDR as the reference antibody. H and the same V L .

[0012] Any anti-CD22 antibody disclosed herein may be a human antibody or a humanized antibody. Alternatively or in addition, the anti-CD22 antibody may be a full-length antibody or an antigen-binding fragment thereof. In some examples, the anti-CD22 antibody is a single-chain antibody (scFv), for example comprising an amino acid sequence of any one of SEQ ID NOs: 40-59.

[0013] In another aspect, provided herein are nucleic acids or nucleic acid groups that collectively encode the heavy and / or light chains of any anti-CD22 antibody disclosed herein. In some embodiments, the nucleic acid or nucleic acid group may be a vector or vector group, such as an expression vector. Also within the scope of the present disclosure are host cells (e.g., mammalian cells or bacterial cells) comprising any nucleic acid or nucleic acid group disclosed herein, and pharmaceutical compositions comprising any anti-CD22 antibody, any nucleic acid encoding the same, and a host cell comprising the nucleic acid, a pharmaceutically acceptable carrier.

[0014] Moreover, the present disclosure provides a method of inhibiting CD22 in a subject, comprising administering to a subject in need thereof any effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the subject may be a human patient having CD22-positive disease cells. For example, the subject may be a human patient having cancer or an autoimmune disease or other disease / disorder involving CD22+ cells. Such human patients may be human patients having CD22-positive cancer cells (e.g., hematopoietic cancer cells) or CD22-positive autoreactive immune cells. Also within the scope of the present disclosure are pharmaceutical compositions disclosed herein for treating diseases comprising CD22 + Disease cells, such as those described herein, and use of any anti-CD22 antibody disclosed herein for the manufacture of a medicament for treating any target disease also disclosed herein.

[0015] In addition, the present disclosure provides a method for detecting the presence of CD22, comprising: (i) contacting an antibody according to any one of claims 1-12 with a sample suspected of containing CD22, and (ii) detecting binding of the antibody to CD22. The antibody may be conjugated to a detectable label. In some cases, CD22 is expressed on the surface of a cell. In some examples, the contacting step may be performed by administering the antibody to a subject.

[0016] In another aspect, the present disclosure provides a method of producing an antibody that binds to CD22, comprising: (i) culturing the host cell of claim 16 under conditions that allow expression of the antibody that binds to CD22; and (ii) harvesting the antibody thereby produced from the cell culture.

[0017] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will be apparent from the accompanying drawings and detailed description of several embodiments, as well as from the appended claims.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings constitute a part of this specification and are included to further illustrate certain aspects of the present disclosure, which aspects may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0020] Figure 1 It is shown to be useful for selecting antibody libraries such as scFv libraries and single heavy chain (V H ) Illustration of an exemplary strategy for library enrichment of high affinity CD22 binders.

[0021] Figure 2 is a graph showing exemplary single-chain (scFv) CD22 binders obtained from a scFv library by multiple rounds of mRNA display selection followed by ELISA screening of individual positive clones.

[0022] Figures 3A-3D Included are graphs showing titration curves of exemplary anti-CD22 antibodies against K562 cells expressing surface CD22. Figure 3A : Clones EP-84-A6, EP84-F6, EP84-H7 and EP84-G12. Figure 3B : Clones EP97-A10 and EP97-D06. Figure 3C : clones EP160-C04, EP160-F04, EP160-C07, EP160-H02, EP160-D02, EP97-A10, EP97-B03 and EP97-G05. Figure 3D :EP160-G04,EP160-G01,EP160-E03,EP160-F10 and EP160-G05.

[0023] Figure 4 is a graph showing the binding activity of exemplary anti-CD22 antibodies to CD22-expressing K562 cells in the presence or absence of the anti-CD22 antibody M971.

[0024] Figure 5 is a graph showing the binding activity of anti-CD22 antibodies to cells expressing recombinant or endogenous CD22. For each anti-CD22 scFv antibody tested, the bars from left to right correspond to K562 cells, CD22K562 cells, CD22HEK293 cells, Daudi cells, and Raji cells.

[0025] Figure 6 are photographs showing immunohistochemical (IHC) staining of endogenous CD22-positive cells using exemplary anti-CD22 scFv EP097-G05.

[0026] Fig. 7A and 7BIncluded are graphs showing epitope binning of exemplary anti-CD22 antibodies compared to known anti-CD22 antibodies M971 and RFB4. Fig. 7A : Epitope partitioning relative to the M971 antibody. Figure 7B : Epitope partitioning of BL22 relative to that from the RFB4 antibody.

[0027] Figures 8A-8C Included are graphs showing the binding activity and specificity of anti-CD22 antibodies in IgG format. Fig. 8A is a graph showing the results of a binding assay using HEK293 cells expressing surface CD22. Figure 8B is a graph showing the results of a binding assay using CHO-K1 cells expressing surface CD123. Figure 8C It is a graph showing the results of binding activity measured by ELISA.

[0028] Fig. 9 It is a graph showing the antibody-dependent cellular cytotoxicity (ADCC) activity of the indicated anti-CD22 IgG antibodies.

[0029] Fig.10 is a graph showing internalization of anti-CD22 IgG antibody after binding to cell surface CD22. DETAILED DESCRIPTION OF THE INVENTION

[0031] Provided herein are antibodies capable of binding to human CD22, particularly CD22 expressed on the surface of cells ("anti-CD22 antibodies"). Anti-CD22 antibodies disclosed herein show high binding affinity to CD22 (e.g., cell surface CD22), high stability, and / or bind to a CD22 epitope different from M971 (fully human anti-CD22 known in the art).

[0032] CD22 is a transmembrane glycoprotein expressed primarily on the surface of mature B cells. This cell surface receptor specifically binds sialic acid via an immunoglobulin (Ig) domain located at the N-terminus of the receptor. CD22 functions as an inhibitory receptor for the signal transduction pathway mediated by BCR. CD22 molecules from different species are well known in the art. For example, the amino acid sequence of human CD22 can be found in GenBank accession number NP_001762.2.

[0033] CD22 is present on malignant B cells and is therefore a promising target for the treatment of hematopoietic cancers, especially those of B cell origin, such as acute lymphoblastic leukemia (ALL), B cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). CD22 is also involved in autoimmunity and will therefore be a target for the treatment of autoimmune diseases.

[0034] Therefore, the anti-CD22 antibodies disclosed herein can be used as therapeutic agents for treating diseases with CD22+ disease cells, such as cancers of the B cell lineage or diseases caused by CD22. + Therapeutic agents for autoimmune diseases mediated by autoreactive immune cells. In addition, the anti-CD22 antibodies disclosed herein can be used as diagnostic agents for detecting the presence of CD22, such as CD22-positive cells. The antibodies disclosed herein can also be used for research purposes.

[0035] I. and CD22 Bound Antibodies

[0036] The present disclosure provides antibodies that bind to CD22, such as human CD22. In some embodiments, the anti-CD22 antibodies disclosed herein are capable of binding to CD22 expressed on the surface of a cell. Therefore, the antibodies disclosed herein can be used for therapeutic or diagnostic purposes to target CD22-positive cells (e.g., leukemia cells). As used herein, the term "anti-CD22 antibody" refers to any antibody that is capable of binding to a CD22 polypeptide (e.g., a CD22 polypeptide expressed on the surface of a cell), which can be of a suitable origin, such as a human or non-human mammal (e.g., mouse, rat, rabbit, primate such as monkey, etc.).

[0037] Antibodies (plural forms are used interchangeably) are immunoglobulin molecules that can specifically bind to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the antibody. Immunoglobulin molecules. As used herein, the term "antibody", such as anti-CD22 antibody, includes not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins comprising antibody portions (e.g., chimeric antigen receptors or CARs), humanized antibodies, chimeric antibodies, diabodies, single domain antibodies (e.g., V only) HAntibodies, such as nanobodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies, such as anti-Galectin-9 antibodies, include antibodies of any kind, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and the antibodies do not need to be of any particular kind. According to the amino acid sequence of the constant domain of the antibody heavy chain, immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0038] A typical antibody molecule contains a heavy chain variable region (V H ) and light chain variable region (V L ), which are usually involved in antigen binding. H and V L The V region can be further subdivided into regions of hypervariability, also called "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, called "framework regions" ("FR"). H and V LUsually composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Methods known in the art can be used, for example, by Kabat definition, Chothia definition, AbM definition and / or Contact definition to accurately identify the scope of the framework region and CDR, all of which are well known in the art. See, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0039] The anti-CD22 antibodies described herein may be full-length antibodies comprising two heavy chains and two light chains, each comprising a variable domain and a constant domain. Alternatively, the anti-CD22 antibodies may be antigen-binding fragments of full-length antibodies. Examples of binding fragments included in the term "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, which is composed of a V L 、V H , C L and C H 1 domain; (ii) F(ab')2 fragment, which is a bivalent fragment consisting of two Fab fragments connected by a disulfide bond at the hinge region; (iii) H and C H 1 domain; (iv) a V fragment consisting of a single arm of the antibody L and V H Fv fragments composed of V and B domains, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which are composed of V H (vi) separate complementary determining regions (CDRs) that retain function. In addition, although the two domains V L and V Hare encoded by different genes, but they can be joined using recombinant methods via synthetic linkers, allowing them to be made into a single protein chain, with V L and V H The regions pair to form a monovalent molecule known as a single-chain Fv (scFv). See, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0040] The antibodies described herein may be of a suitable origin, such as mouse, rat or human. Such antibodies are non-naturally occurring, i.e., not produced in animals without human behavior (e.g., immunized with a desired antigen or fragment thereof or isolated from an antibody library). Any antibody described herein, such as an anti-CD22 antibody, may be monoclonal or polyclonal. "Monoclonal antibody" refers to a homologous antibody population, and "polyclonal antibody" refers to a heterologous antibody population. These two terms do not limit the source of the antibody or how it is prepared.

[0041] In some embodiments, the anti-CD22 antibody is a human antibody that can be isolated from a human antibody library or produced in a transgenic mouse. For example, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulins. Transgenic animals designed to produce more ideal (e.g., fully human antibodies) or more robust immune responses can also be used to produce humanized or human antibodies. An example of such technology is the Xenomouse from Amgen, Inc. (Fremont, Calif.). TM and HuMAb-Mouse from Medarex, Inc. (Princeton, NJ) TM and TC Mouse TM In another alternative, antibodies can be produced recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150 and Winter et al. (1994) Annu. Rev. Immunol. 12: 433-455. Alternatively, antibody library display technologies, such as phage, yeast display, mammalian cell display or mRNA display technologies known in the art, can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0042] In other embodiments, anti-CD22 antibodies can be humanized antibodies or chimeric antibodies. Humanized antibodies refer to the form of non-human (e.g., mouse) antibodies, which are specific chimeric immunoglobulins, immunoglobulin chains or their antigen-binding fragments, which contain minimal sequences derived from non-human immunoglobulins. In general, humanized antibodies are human immunoglobulins (receptor antibodies), wherein residues from receptor CDRs are replaced by CDR residues from non-human species (donor antibodies) such as mice, rats or rabbits with desired specificity, affinity and ability. In some cases, one or more Fv framework regions (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are neither found in the receptor antibody nor in the imported CDR or framework sequences, but are included to further improve and optimize antibody performance. In some cases, humanized antibodies may include substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins and all or substantially all of the FR regions are regions of human immunoglobulin consensus sequences. The humanized antibody preferably also comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five or six) that are altered relative to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation. Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989).

[0043] In some embodiments, the anti-CD22 antibodies disclosed herein may be chimeric antibodies. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light chain and the heavy chain mimic the variable regions of an antibody derived from a mammal (e.g., a non-human mammal, such as a mouse, rabbit, and rat), while the constant portion is homologous to the sequence in an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or the constant region. The techniques developed for producing "chimeric antibodies" are well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314: 452.

[0044] In some embodiments, the anti-CD22 antibodies described herein specifically bind to the corresponding target antigen (e.g., CD22) or an epitope thereof. "Specific binding" to an antigen or epitope is a term well known in the art. If a molecule reacts more frequently, more rapidly, longer, and / or more strongly with a specific target antigen than it reacts with an alternative target antigen, it is said to exhibit "specific binding". If the antibody binds with greater affinity, avidity, more readily, and / or longer duration than other substances, the antibody "specifically binds" to the target antigen or epitope. For example, an antibody that specifically (or preferentially) binds to an antigen (CD22) or an antigenic epitope therein is an antibody that binds to other antigens or other epitopes in the same antigen with a higher affinity, avidity, more readily, and / or longer duration than it binds to. It can also be understood by this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or an epitope thereof may not bind to other antigens or other epitopes on the same antigen (i.e., only baseline binding activity can be detected in conventional methods). In some instances, the anti-CD22 antibodies disclosed herein do not bind to the same epitope as FMC63. In other instances, the anti-CD22 antibodies bind to a CD22 epitope that does not overlap with the CD22 epitope bound by M971. V H and V L The sequences are well known in the art and are provided below (CDRs in bold):

[0045] M971-V H (SEQ ID NO:1):

[0046]

[0047] M971-V L (SEQ ID NO:2):

[0048]

[0049] In some embodiments, the anti-CD22 antibodies described herein have a suitable binding affinity for the target antigen (e.g., CD22) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A . K A is the dissociation constant (K D The anti-CD22 antibodies described herein may have a binding affinity (K) of at least 100 nM, 10 nM, 1 nM, 0.1 nM or less for CD22. D ). Increased binding affinity corresponds to a decreased KD The higher affinity binding of an antibody to a first antigen relative to a second antigen can be explained by the K A (or value K D ) compared to a higher K for binding to the first antigen A (or a smaller value K D ). In this case, the antibody is specific for a first antigen (e.g., a first protein in a first conformation, or a mimetic thereof) relative to a second antigen (e.g., the same first protein in a second conformation, or a mimetic thereof; or a second protein). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 90, 100, 500, 1000, 10,000, or 10 5 In some embodiments, any anti-CD22 antibody can be further affinity matured to increase the binding affinity of the antibody to the target antigen or its antigenic epitope.

[0050] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (e.g., using fluorescence determination). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as the concentration of the target protein changes. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) as follows:

[0051] [Bound] = [Free] / (Kd + [Free])

[0052] However, it is not always necessary to precisely determine K A , because sometimes it is sufficient to obtain a quantitative measure of affinity, for example, the affinity determined using methods such as ELISA or FACS analysis is comparable to the K A is proportional and can therefore be used for comparison, e.g., to determine if a higher affinity is 2-fold higher, to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, such as an in vitro or in vivo assay.

[0053] In some embodiments, the anti-CD22 antibodies disclosed herein are effective for binding to ECs of CD22-positive cells. 50 As used herein, EC 50The values ​​are the lowest concentration of antibody required to bind to 50% of the CD22-positive cell population. 50 Values ​​can be determined using conventional assays and / or assays disclosed herein. See, e.g., the examples below.

[0054] A number of exemplary anti-CD22 antibodies are provided below (CDRs are in bold and were determined by the Chothia method (Chothia et al. (1992) J. Mol. Biol., 227, 776-798, Tomlinson et al. (1995) EMBO J., 14, 4628-4638 and Williams et al. (1996) J. Mol. Biol., 264, 220-232). See also www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php.

[0055] EP160-C07

[0056] V H (SEQ ID NO:3):

[0057]

[0058] V L (SEQ ID NO:4):

[0059]

[0060] EP160-E03

[0061] V H (SEQ ID NO:5):

[0062]

[0063] V L (SEQ ID NO:6)

[0064]

[0065] EP160-F10 (single domain antibody)

[0066] V H (SEQ ID NO:7)

[0067]

[0068] EP97-A10

[0069] V H(SEQ ID NO:8)

[0070]

[0071] V L (SEQ ID NO:9)

[0072]

[0073] EP97-B03

[0074] V H (SEQ ID NO:10)

[0075]

[0076] V L (SEQ ID NO:11)

[0077]

[0078] EP160-D02

[0079] V H (SEQ ID NO:12)

[0080]

[0081] V L (SEQ ID NO:13)

[0082]

[0083] EP160-G04

[0084] V H (SEQ ID NO:14)

[0085]

[0086] V L (SEQ ID NO:15)

[0087]

[0088] EP160-H02

[0089] V H (SEQ ID NO:16)

[0090]

[0091] V L (SEQ ID NO:17)

[0092]

[0093] EP160-G05

[0094] V H (SEQ ID NO:18)

[0095]

[0096] V L (SEQ ID NO:19)

[0097]

[0098] EP35-C6

[0099] V H (SEQ ID NO:20)

[0100]

[0101] V L (SEQ ID NO:21)

[0102]

[0103] EP35-A7

[0104] V H (SEQ ID NO:22)

[0105]

[0106] V L (SEQ ID NO:23)

[0107]

[0108] EP35-D6

[0109] V H (SEQ ID NO:24)

[0110]

[0111] V L (SEQ ID NO:25)

[0112]

[0113] EP35-E6

[0114] V H(SEQ ID NO:26)

[0115]

[0116] V L (SEQ ID NO:27)

[0117]

[0118] EP35-C8

[0119] V H (SEQ ID NO:28)

[0120]

[0121] V L (SEQ ID NO:29)

[0122]

[0123] EP160-F04

[0124] V H (SEQ ID NO:30)

[0125]

[0126] V L (SEQ ID NO:31)

[0127]

[0128] BP35-B05

[0129] V H (SEQ ID NO:32)

[0130]

[0131] V L (SEQ ID NO:33)

[0132]

[0133] EP97-G05

[0134] V H (SEQ ID NO:34)

[0135]

[0136] V L (SEQ ID NO:35)

[0137]

[0138] EP97-F01

[0139] V H (SEQ ID NO:36)

[0140]

[0141] V L (SEQ ID NO:37)

[0142]

[0143] EP97-A01

[0144] V H (SEQ ID NO:38)

[0145]

[0146] V L (SEQ ID NO:39)

[0147]

[0148] In some embodiments, the anti-CD22 antibodies described herein bind to the same CD22 polypeptide epitope as any of the exemplary antibodies described herein (e.g., EP35-A7, EP35-B5, EP35-C6, EP35-C8, EP35-D6, EP35-E6, EP35-E7, EP97-A01, EP97-A10, EP97-B03, EP97-F01, EP97-G05, EP160-C07, EP160-D02, EP160-E03, EP160-F04, EP160-F10, EP160-G04, EP160-G05, EP160-H02, and EP97-A01), or compete with the exemplary antibodies for binding to the CD22 antigen. In some examples, the anti-CD22 antibodies disclosed herein bind to the same CD22 polypeptide epitope as EP160-D02 or compete with the exemplary antibodies for binding to the CD22 antigen. In other examples, the anti-CD22 antibodies disclosed herein bind to the same CD22 polypeptide epitope as EP97-B03 or compete with the exemplary antibodies for binding to the CD22 antigen.

[0149] "Epitope" refers to the site on the target antigen that is recognized and bound by an antibody. The site can be composed entirely of amino acid components, composed entirely of chemical modifications of protein amino acids (e.g., glycosyl moieties), or composed of a combination thereof. Overlapping epitopes include at least one common amino acid residue. An epitope can be linear, typically 6-15 amino acids in length. Alternatively, an epitope can be conformational. The epitope to which an antibody binds can be determined by conventional techniques, e.g., epitope mapping methods (see, e.g., description below). An antibody that binds to the same epitope as an exemplary antibody described herein can bind to an epitope identical to the exemplary antibody or an epitope that substantially overlaps (e.g., containing less than 3 non-overlapping amino acid residues, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue). Whether two antibodies compete with each other to bind to a homologous antigen can be determined by a competition assay well known in the art.

[0150] In some examples, the anti-CD22 antibodies comprise the same V domains as the exemplary antibodies described herein. H and / or V L CDR. With the same V H and / or V L CDRs of two antibodies means that their CDRs are the same when determined by the same method (e.g., Kabat method, Chothia method, AbM method, Contact method, or IMGT method. See, e.g., bioinf.org.uk / abs / ). Such anti-CD22 antibodies may have the same V as compared to the exemplary antibodies described herein. H , the same V L or both.

[0151] Functional variants of any exemplary anti-CD22 antibody disclosed herein (e.g., EP160-D2 or EP97-B03) are also within the scope of the present disclosure. Such functional variants are substantially similar to the exemplary antibodies in structure and function. The functional variants comprise V sequences substantially identical to the exemplary antibodies. H and V L CDR. For example, it may comprise only up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue variations in the total CDR region of the antibody and bind to the same epitope of CD22 with substantially similar affinity (e.g., with a K of the same magnitude). D In some cases, the functional variant may have the same heavy chain CDR3 as the exemplary antibody, and optionally the same light chain CDR3 as the exemplary antibody. Alternatively or in addition, the functional variant may have the same heavy chain CDR2 as the exemplary antibody. Such an anti-CD22 antibody may comprise a V H Compared to V129 ...H In some examples, the anti-CD22 antibody may further comprise a fragment having the same V as the exemplary antibody. L CDR3 and optionally the same V L CDR1 or V L V of CDR2 L Fragment.

[0152] Alternatively or in addition, the amino acid residue variation can be a conservative amino acid residue replacement. As used herein, "conservative amino acid replacement" refers to an amino acid replacement that does not change the relative charge or size characteristics of the protein to which the amino acid replacement is made. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as those found in references compiling such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conservative replacements of amino acids include replacements between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0153] In some embodiments, an anti-CD22 antibody may comprise a V sequence that is identical to the exemplary antibodies described herein. H The heavy chain CDRs have at least 80% (e.g., 85%, 90%, 95% or 98%) sequence identity compared to the VL CDRs of the exemplary antibodies described herein, either individually or collectively. Alternatively or in addition, the anti-CD22 antibodies may comprise light chain CDRs that have at least 80% (e.g., 85%, 90%, 95% or 98%) sequence identity compared to the VL CDRs of the exemplary antibodies described herein, either individually or collectively. As used herein, "individually" means that one CDR of an antibody shares a specified sequence identity with respect to the corresponding CDR of the exemplary antibody. "Collectively" means that the VL CDRs of an antibody share a specified sequence identity with respect to the corresponding CDRs of the exemplary antibody. H or V L The CDRs are combined relative to the corresponding three V H or V L The CDRs share a specified sequence identity in combination.

[0154] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990 and modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is integrated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecule of interest. In the event that there is a gap between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25 (17): 3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0155] In some embodiments, the heavy chain of any anti-CD22 antibody described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be from any suitable source, such as human, mouse, rat, or rabbit. Alternatively or in addition, the light chain of the anti-CD22 antibody may also comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, CL is a kappa light chain. In other examples, CL is a lambda light chain. Antibody heavy and light chain constant regions are well known in the art, such as those provided in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0156] In some embodiments, the anti-CD22 antibodies disclosed herein may be single chain antibodies (scFv). ScFv antibodies may comprise V H Fragment and V L fragments, which can be connected by a flexible peptide linker. In some cases, scFv antibodies may be in the V H →V L direction (from N-terminus to C-terminus). In other cases, scFv antibodies may be in the V L →V H Direction (from N-terminus to C-terminus). Exemplary scFv anti-CD22 antibodies are provided below (CDRs are shown in bold, peptide linkers are shown in bold and underlined):

[0157] EP160-C07 (scFv, V H -V L Direction; SEQ ID NO:40)

[0158]

[0159] EP160-E03 (scFv, V H -V L Direction; SEQ ID NO:41)

[0160]

[0161] EP160-F10 (single chain antibody; SEQ ID NO: 42)

[0162]

[0163] EP97-A10 (scFv, V H -V L Direction; SEQ ID NO:43)

[0164]

[0165] EP97-B03 (scFv, V H -V L Direction; SEQ ID NO:44)

[0166]

[0167] EP160-D02 (scFv, V H -V L Direction; SEQ ID NO:45)

[0168]

[0169] EP160-G04 (scFv, V H -V L Direction; SEQ ID NO:46)

[0170]

[0171] EP160-H02 (scFv, V H -V L Direction; SEQ ID NO:47)

[0172]

[0173] EP160-G05 (scFv, V H -V L Direction; SEQ ID NO:48)

[0174]

[0175] EP35-F7 (same as EP97-A01, (scFv, V H -V L Direction; SEQ ID NO:49)

[0176]

[0177] EP35-C6 (scFv, V H -V L Direction; SEQ ID NO:50)

[0178]

[0179] EP35-A7 (scFv, V H -V L Direction; SEQ ID NO:51)

[0180]

[0181] EP35-D6 (scFv, V H -V L Direction; SEQ ID NO:52)

[0182]

[0183] EP35-E6 (scFv, V H -V L Direction; SEQ ID NO:53)

[0184]

[0185] EP35-C8 (scFv, V H -V L Direction; SEQ ID NO:54)

[0186]

[0187] EP160-F04 (scFv, V H -V L Direction; SEQ ID NO:55)

[0188]

[0189] EP35-B05 (scFv, V H -V L Direction; SEQ ID NO:56)

[0190]

[0191] EP97-G05 (scFv, V H -V L Direction; SEQ ID NO:57)

[0192]

[0193] EP97-F01 (scFv, V H -V L Direction; SEQ ID NO:58)

[0194]

[0195] EP97-A01 (scFv, V H -V L Direction; SEQ ID NO:59)

[0196]

[0197] Any anti-CD22 antibody as described herein, such as the exemplary anti-CD22 antibodies provided herein, such as EP160-D2 or EP97-B03, can bind to and inhibit (e.g., reduce or eliminate) the activity of CD22-positive cells (e.g., B cells). In some embodiments, the anti-CD22 antibodies described herein can bind to CD22-positive cells and inhibit their activity by at least 30% (e.g., 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). The inhibitory activity of the anti-CD22 antibodies described herein can be determined by conventional methods known in the art, such as by measuring K i, app Method for determining value.

[0198] In some examples, the K of an antibody can be determined by measuring the inhibitory effect of different concentrations of the antibody on the degree of a relevant reaction. i, app The change in pseudo-first-order rate constant (v) as a function of inhibitor concentration was fitted to the modified Morrison equation (Equation 1) to obtain an estimate of the apparent Ki value. For competitive inhibitors, Kiapp From K i, app Obtained from the y-intercept extracted from the linear regression analysis of the substrate concentration plot.

[0199]

[0200] Where A is equal to v o / E, the initial rate (v) of the enzymatic reaction in the absence of an inhibitor (I) o ) divided by the total enzyme concentration (E). In some embodiments, the anti-CD22 antibodies described herein may have a Ki of 1000, 500, 100, 50, 40, 30, 20, 10, 5 pM or less for the target antigen or antigenic epitope. app value.

[0201] II. Preparation of anti-CD22 antibody

[0202] Antibodies capable of binding to CD22 as described herein can be prepared by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, antibodies can be produced by conventional hybridoma technology. Alternatively, anti-CD22 antibodies can be identified from a suitable library (e.g., a human antibody library).

[0203] In some cases, you can Figure 1 The screening strategy shown obtains high affinity fully human CD22 binders from a human antibody library. See also Example 1 below. This strategy allows maximizing library diversity to cover a broad (board) and active epitopes on cells expressing CD22.

[0204] If necessary, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma cell line or isolated from an antibody library) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector of a host cell, which can then be amplified and frozen for future use. In an alternative, the polynucleotide sequence can be used for genetic manipulation to, for example, humanize the antibody or improve the affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is from a non-human source and will be used for clinical trials and treatments of people, the constant region can be designed to be more similar to a human constant region to avoid an immune response. Alternatively or in addition, it may be necessary to genetically manipulate the antibody sequence to obtain greater affinity and / or specificity to the target antigen and greater efficacy to enhance CD22 activity. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

[0205] Alternatively, antibodies capable of binding to a target antigen (CD22 molecule) as described herein can be isolated from a suitable antibody library by conventional practice. Antibody libraries can be used to identify proteins that bind to a target antigen (e.g., human CD22, such as cell surface CD22) by conventional screening processes. During the selection process, the polypeptide component is probed with the target antigen or a fragment thereof, and if the polypeptide component binds to the target, the antibody library member is identified, usually by being retained on a support. The retained display library members are recovered from the support and analyzed. Analysis can include amplification and subsequent selection under similar or different conditions. For example, positive and negative selections can be performed alternately. Analysis can also include determining the amino acid sequence of the polypeptide component and purifying the polypeptide component for detailed characterization.

[0206] There are many conventional methods known in the art to identify and isolate antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosome display or mammalian display technology.

[0207] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bonds of F(ab')2 fragments.

[0208] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies and bispecific antibodies, can be produced by, for example, conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific for a target antigen can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the heavy and light chains of the monoclonal antibody encoding the antibody). Once separated, DNA can be placed in one or more expression vectors and then transfected into a host cell, such as an Escherichia coli cell, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins in addition, to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87 / 04462. DNA can then be modified in this way, for example, by replacing homologous mouse sequences with coding sequences of human heavy and light chain constant domains, Morrison et al., (1984) Proc.Nat.Acad.Sci.81:6851, or by covalently linking all or part of the coding sequences of non-immunoglobulin polypeptides to immunoglobulin coding sequences. In this manner, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies, having binding specificity for a target antigen can be prepared.

[0209] Techniques developed for producing "chimeric antibodies" are well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604 and Takeda et al. (1984) Nature 314: 452.

[0210] Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of a parent non-human antibody is modified according to methods known in the art. H and V L The variable regions were subjected to three-dimensional molecular modeling analysis. Next, the same molecular modeling analysis was used to identify framework amino acid residues that were predicted to be important for the formation of the correct CDR structure. H and V L The sequence is used as a search request to identify human V with amino acid sequence homologous to the parent non-human antibody from any antibody gene database. H and V L Then select Person V H and V L Receptor gene.

[0211] The CDR region in the selected human receptor gene can be replaced with a CDR region from a parent non-human antibody or a functional variant thereof. If necessary, residues in the parent chain framework region that are predicted to be important in interacting with the CDR region (see the description above) can be used to replace the corresponding residues in the human receptor gene.

[0212] Single-chain antibodies can be prepared by connecting a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region by recombinant technology. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the techniques described for producing single-chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be applied to produce phage display, yeast display, mammalian cell display or mRNA display scFv libraries, and scFv clones specific for CD22 can be identified from the library according to conventional procedures. Positive clones can be further screened to identify those clones that enhance CD22 activity.

[0213] Antibodies obtained according to methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping". It is known that there are many methods in the art for locating and characterizing the position of epitopes on proteins, including crystal structures of parsing antibody-antigen complexes, competitive assays, gene fragment expression assays, and assays based on synthetic peptides, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In another example, epitope mapping can be used to determine the sequence to which the antibody binds. The epitope can be a linear epitope, i.e., contained in a single amino acid segment, or a conformational epitope formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single segment (primary structure linear sequence). Peptides of different lengths (e.g., at least 4-6 amino acids long) can be separated or synthesized (e.g., recombined) and used for binding assays with antibodies. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining the binding of the antibody. According to the gene fragment expression assay, the open reading frame encoding the target antigen is randomly or fragmented by a specific gene structure, and the reactivity of the expressed antigen fragment with the antibody to be tested is determined. For example, gene fragments can be produced by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of antibodies to radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries (phage libraries) of random peptide sequences displayed on the surface of phage particles.

[0214] Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In another example, mutagenesis of the antigen binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of CD22 have been replaced (exchanged) with sequences from closely related but antigenically distinct proteins, such as another member of the tumor necrosis factor receptor family. By evaluating the binding of the antibody to mutant CD22, the importance of specific antigen fragments for antibody binding can be assessed.

[0215] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibodies.Competition assays are well known to those skilled in the art.

[0216] In some examples, anti-CD22 antibodies are produced by recombinant techniques as exemplified below.

[0217] The nucleic acid of the heavy chain and light chain of the anti-CD22 antibody as described herein can be cloned into an expression vector, and each nucleotide sequence is operably connected to a suitable promoter. In an example, each nucleotide sequence encoding the heavy chain and light chain is operably connected to different promoters. Alternatively, the nucleotide sequence encoding the heavy chain and light chain can be operably connected to a single promoter so that the heavy chain and light chain are expressed by the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy chain and light chain coding sequence.

[0218] In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing them, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions that allow the formation of antibodies.

[0219] In general, methods known in the art can be used to clone the nucleic acid sequence of one or all chains of the encoding antibody into a suitable expression vector and operably connected to a suitable promoter. For example, the nucleotide sequence and the carrier can be contacted with a restriction endonuclease under suitable conditions to produce complementary ends on each molecule, and these ends can be paired with each other and connected together by a ligase. Alternatively, a synthetic nucleic acid linker can be connected to the end of the gene. These synthetic linkers include nucleic acid sequences corresponding to specific restriction sites in the carrier. The selection of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0220] A variety of promoters can be used to express the antibodies described herein, including but not limited to the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter, and herpes simplex virus tk viral promoter.

[0221] Regulatable promoters may also be used. Such regulatable promoters include those that use the lac repressor from E. coli as a transcriptional regulator to regulate transcription from a mammalian cell promoter with a lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astradiol, RU486, diphenolmurislerone or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0222] A regulatable promoter including a repressor with an operator can be used. In one embodiment, the lac repressor from Escherichia coli can be used as a transcriptional regulator to regulate transcription from a mammalian cell promoter with a lac operator [M. Brown et al., Cell, 49: 603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992)] The tetracycline repressor (tetR) is combined with a transcription activator (VP16) to produce the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP 16), which is combined with tetO-with a minimal promoter derived from the major immediate early promoter of human cytomegalovirus (hCMV) to produce the tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is located downstream of the TATA element of the CMVIE promoter, a single tetracycline repressor (tetR) rather than a tetR-mammalian cell transcription factor fusion derivative can be used as an effective trans-regulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10 (16): 1392-1399 (2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressors-mammalian cell transactivators or repressor fusion proteins that may be toxic to cells in some cases (Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)) to achieve its regulatable effect.

[0223] In addition, the vector may include, for example, some or all of the following: a selectable marker gene, such as a neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma replication origin and ColE1 for appropriate episomal replication; an internal ribosome binding site (IRES), a multifunctional multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0224] Examples of polyadenylation signals that can be used to practice the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0225] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any antibody can be introduced into suitable host cells to produce the antibody. Host cells can be cultured under conditions suitable for expressing the antibody or any polypeptide chain thereof. Such antibodies or their polypeptide chains can be recovered by cultured cells (e.g., from cells or culture supernatants) by conventional methods, such as affinity purification. If necessary, the polypeptide chains of the antibody can be incubated under suitable conditions for a suitable time to produce the antibody.

[0226] In some embodiments, the method for preparing the antibodies described herein involves a recombinant expression vector encoding both the heavy chain and the light chain of the anti-CD22 antibody, also as described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positively transformed host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains that form the antibody, and the two polypeptide chains can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under conditions suitable for forming the antibody.

[0227] In one embodiment, two recombinant expression vectors are provided, one encoding the heavy chain of anti-CD22 antibodies, and the other encoding the light chain of anti-CD22 antibodies. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods such as calcium phosphate-mediated transfection. Alternatively, each expression vector can be introduced into suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing the expression of antibody polypeptide chains. When two expression vectors are introduced into the same host cell, the antibodies produced therein can be recovered from the host cell or culture medium. If necessary, polypeptide chains can be recovered from the host cell or culture medium, and then hatched under suitable conditions allowing the formation of antibodies. When two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or from the corresponding culture medium. Then two polypeptide chains can be hatched under suitable conditions to form antibodies.

[0228] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells and recover antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with protein A or protein G coupled matrices.

[0229] Any nucleic acid encoding the heavy chain, light chain, or both of an anti-CD22 antibody as described herein, a vector comprising the same (eg, an expression vector); and a host cell comprising the vector are within the scope of the present disclosure.

[0230] III. Application of anti-CD22 antibodies

[0231] Any of the anti-CD22 antibodies disclosed herein may be used for therapeutic, diagnostic, and / or research purposes, all of which are within the scope of the present disclosure.

[0232] Pharmaceutical composition

[0233] Antibodies as described herein and encoding nucleic acids or nucleic acid groups, vectors comprising them, or host cells comprising vectors can be mixed with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions for treating target diseases. "Acceptable" means that the carrier must be compatible with the active ingredients of the composition (and preferably, capable of stabilizing the active ingredients) and harmless to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0234] The pharmaceutical composition used in the present method may include a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants, such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0235] In some embodiments, the pharmaceutical compositions described herein include liposomes containing antibodies (or encoding nucleic acids), which can be prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of defined pore size to produce liposomes with a desired diameter.

[0236] Antibodies or encoding nucleic acids can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0237] In other examples, the pharmaceutical compositions described herein can be formulated into sustained release forms. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of molded articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0238] Pharmaceutical compositions for in vivo administration must be sterile. This is easily achieved, for example, by filtering through a sterile filtration membrane. Therapeutic antibody compositions are typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

[0239] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0240] In order to prepare solid compositions such as tablets, the main active ingredient can be mixed with a drug carrier, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talcum, stearic acid, magnesium stearate, dicalcium phosphate or gum, and other drug diluents, such as water, to form a solid preformulation composition containing a uniform mixture of the compounds of the present invention or its non-toxic pharmaceutically acceptable salt. When referring to these preformulation compositions of homogeneity, it means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily subdivided into the same effective unit dosage form, such as tablets, pills and capsules. The solid preformulation composition is then subdivided into the unit dosage form of the above-mentioned type, which contains 0.1 to about 500mg of the active ingredient of the present invention. The tablet or pill of the new composition can be coated or otherwise compounded to provide a dosage form with the advantage of prolonged action. For example, a tablet or pill can include an internal dosage form and an external dosage form component, the latter being an envelope form on the former. The two components can be separated by an enteric layer, which is used to resist disintegration in the stomach and allow the inner component to enter the duodenum intact or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0241] Suitable surfactants include, inter alia, non-ionic agents such as polyoxyethylene sorbitan (e.g., Tween TM 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span TM 20, 40, 60, 80 or 85). Compositions with surfactant will conveniently contain between 0.05 and 5% surfactant, and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added if desired, such as mannitol or other pharmaceutically acceptable vehicles.

[0242] Suitable emulsions can be prepared using commercially available fat emulsions, such as Intralipid TM , Liposyn TM Infonutrol TM , Lipofundin TM Lipiphysan TMThe active ingredient may be dissolved in a premixed emulsion composition, or it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and mixed with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water to form an emulsion. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tension of the emulsion. Suitable emulsions will typically contain up to 20% oil, such as 5-20%. The fat emulsion may contain fat droplets between 0.1 and 1.0 μm, particularly between 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0.

[0243] The emulsion composition can be prepared by combining the antibody with Intralipid TM Or those prepared by mixing its components (soybean oil, lecithin, glycerin and water).

[0244] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0245] Preferably, the composition in a sterile pharmaceutically acceptable solvent can be atomized by using a gas. The atomized solution can be breathed directly from the atomizing device, or the atomizing device can be connected to a mask, an oxygen tent, or an intermittent positive pressure breathing machine. The solution, suspension, or powder composition can be administered from a device that delivers the formulation in an appropriate manner, preferably orally or nasally.

[0246] Therapeutic applications

[0247] In order to implement the methods disclosed herein, an effective amount of the pharmaceutical compositions described herein can be administered to a subject (e.g., a human) in need of treatment by a suitable route, such as intravenous administration, such as bolus injection or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid preparations, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid preparations can be directly atomized, and lyophilized powders can be atomized after reconstitution. Alternatively, antibodies as described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and ground powders.

[0248] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment can be a human subject that has, is at risk of, or is suspected of having a disease that carries CD22 +Examples of such target diseases / disorders include hematopoietic cancers, such as cancers of the B-cell lineage. Examples include, but are not limited to, hematological B-cell neoplasms, including lymphocytic leukemias, such as B-cell chronic lymphocytic leukemia (CLL); B-cell acute lymphoblastic leukemia (ALL) and B-cell non-Hodgkin lymphoma (NHL). Alternatively, CD22 + The disease cell may be an immune cell (eg, a B cell) specific for a self-antigen.

[0249] Subjects with target cancers can be identified by routine medical examinations, such as laboratory tests, organ function tests, CT scans, or ultrasound. In some embodiments, subjects to be treated by the methods described herein can be human cancer patients who have or are receiving anti-cancer therapy, such as chemotherapy, radiotherapy, immunotherapy, or surgery.

[0250] A subject with a target autoimmune disease can also be identified by routine medical examination. In some embodiments, the subject to be treated by the methods described herein can be a human patient with an autoimmune disease. Such a human patient may have or is receiving treatment for an autoimmune disease.

[0251] A subject suspected of having any such target disease / disorder may exhibit one or more symptoms of the disease / disorder. A subject at risk of the disease / disorder may be a subject having one or more risk factors for the disease / disorder.

[0252] As used herein, "effective amount" refers to the amount of each active agent required to impart a therapeutic effect to a subject alone or in combination with one or more other active agents. It is obvious to those skilled in the art to determine whether a certain amount of antibody achieves a therapeutic effect. As known to those skilled in the art, the effective amount depends on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, stature, gender and weight, the duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of health practitioners. These factors are well known to those of ordinary skill in the art and can be solved by experiments that do not exceed routine experiments. It is generally preferred to use the maximum dose of a single component or a combination thereof, i.e., the highest safe dose according to reasonable medical judgment.

[0253] Empirical considerations, such as half-life, often help determine dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during the course of treatment, and is usually but not necessarily based on the treatment and / or inhibition and / or improvement and / or delay of the target disease / disorder. Alternatively, a continuous sustained release formulation of the antibody may be suitable. Various preparations and devices for achieving sustained release are known in the art.

[0254] In one example, the dosage of an antibody as described herein can be determined empirically in an individual who has been given one or more administrations of the antibody. The individual is given increasing doses of the agonist. In order to assess the efficacy of the agonist, indicators of the disease / disorder can be followed.

[0255] Typically, for the administration of any antibody described herein, the initial candidate dose can be about 2 mg / kg. For the purposes of the present disclosure, depending on the above factors, a typical daily dose range can be from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, continued treatment occurs until the desired symptom suppression or until sufficient treatment levels are reached to alleviate the target disease or disorder or its symptoms. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a maintenance dose of about 1 mg / kg antibody administered weekly, or a maintenance dose of about 1 mg / kg administered every other week. However, other dosage regimens may be useful, depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, administration is considered once to four times a week. In some embodiments, the dosage range that can be used is about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg). In some embodiments, the dosage frequency is once a week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once a month, every 2 months, or every 3 months or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) will change over time.

[0256] In some embodiments, for adult patients of normal weight, a dose of about 0.3 to 5.00 mg / kg may be administered. In some instances, the dose of the anti-CD22 antibodies described herein may be 10 mg / kg. The specific dosing regimen, i.e., dose, timing, and repetition, will depend on the specific individual and the individual's medical history, as well as the characteristics of the individual agent (e.g., the half-life of the agent, and other considerations well known in the art).

[0257] For the purposes of the present disclosure, the appropriate dosage of an antibody as described herein will depend on the specific antibody, multiple antibodies and / or non-antibody peptides (or combinations thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to agonists, and the judgment of the attending physician. Typically, the clinician will administer the antibody until a dose that achieves the desired result is achieved. In some embodiments, the desired result is an increase in the anti-tumor immune response in the tumor microenvironment. Methods for determining whether a dose produces the desired result are apparent to those skilled in the art. Administration of one or more antibodies may be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to practitioners in the art. Administration of the antibody may be substantially continuous over a preselected time period, or may be, for example, a series of spaced doses before, during, or after the development of a target disease or disorder.

[0258] As used herein, the term "treat" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a target disease or condition, a symptom of the disease / condition, or a susceptibility to the disease / condition, with the purpose of curing, rehabilitating, alleviating, alleviating, altering, remedying, improving, ameliorating, or affecting the condition, symptoms of the disease, or susceptibility to the disease or condition.

[0259] Alleviation of the target disease / disorder includes delaying the development or progression of the disease, or reducing the severity of the disease or prolonging survival. Alleviation of the disease or prolonging survival does not necessarily require a curative outcome. As used herein, "delaying" the development of the target disease or disorder refers to postponing, hindering, slowing down, delaying, stabilizing and / or postponing the progression of the disease. This delay may have different lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the development of a disease or delaying the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of a disease within a given time frame and / or reduces the degree of symptoms within a given time frame compared to not using the method. This comparison is usually based on clinical studies, and the number of subjects used is sufficient to give statistically significant results.

[0260] "Development" or "progression" of a disease refers to the initial manifestation and / or subsequent progression of the disease. The development of a disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological course of symptoms. "Development" includes occurrence, recurrence, and onset. As used herein, "onset" or "occurrence" of a target disease or condition includes initial onset and / or recurrence.

[0261] Depending on the type of disease to be treated or the position of disease, the pharmaceutical composition can be applied to the subject using conventional methods known to those of ordinary skill in the medical field. The composition can also be applied by other conventional routes, such as oral, parenteral, by inhalation spray, local, rectal, nasal cavity, oral cavity, vaginal or by implanted reservoir application. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In addition, it can be applied to the subject by injectable reservoir administration route, such as using 1 month, 3 months or 6 months reservoir injectable or biodegradable materials and methods. In some instances, the pharmaceutical composition is intraocular or intravitreal administration.

[0262] Injectable compositions can include various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by drip infusion, thereby infusing a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, such as sterile preparations in the form of suitable soluble salts of antibodies, can be dissolved and administered in pharmaceutical excipients, such as water for injection, 0.9% saline or 5% glucose solution.

[0263] In one embodiment, the antibody is administered by site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable reservoir sources or local delivery catheters of the antibody, such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection or direct application. See, for example, PCT Publication No. WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0264] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263: 621; Wu et al., J. Biol. Chem. (1994) 269: 542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87: 3655; Wu et al., J. Biol. Chem. (1991) 266: 338.

[0265] Therapeutic compositions containing polynucleotides (e.g., those encoding antibodies described herein) are administered in the range of about 100 ng to about 200 mg DNA for local administration in gene therapy protocols. In some embodiments, DNA in a concentration range of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg or more can also be used in gene therapy protocols.

[0266] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vectors. Gene delivery vectors can be viral or non-viral sources (see generally Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148). Endogenous mammalian or heterologous promoters and / or enhancers can be used to induce the expression of such coding sequences. The expression of coding sequences can be constitutive or regulated.

[0267] Viral-based vectors for delivery of desired polynucleotides and expression in desired cells are well known in the art. Exemplary viral-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651 and European Patent No. 0 345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-1250). VR-532)) and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[0268] Non-viral delivery vectors and methods can also be used, including but not limited to polycation condensed DNA linked or unlinked to killed adenovirus only (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985); eukaryotic cell delivery vector cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication No. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO90 / 11092 and U.S. Pat. No. 5,580,859. Liposomes that can be used as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and European Patent No. 0524968. Other methods are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0269] The specific dosing regimen, ie, dosage, timing, and repetition, used in the methods described herein will depend on the particular subject and that subject's medical history.

[0270] In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, may be administered to a subject in need of treatment.Antibodies may also be used in combination with other agents that enhance and / or supplement the effectiveness of the agents.

[0271] The efficacy of treatment for a target disease / disorder can be assessed by methods well known in the art.

[0272] Kits for treating diseases

[0273] The present disclosure also provides kits for treating or alleviating a target disease, such as a hematopoietic cancer as described herein. Such kits may include one or more containers comprising an anti-CD22 antibody, such as any of those described herein. In some cases, the anti-CD22 antibody may be used in conjunction with a second therapeutic agent.

[0274] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of administering an anti-CD22 antibody and an optional second therapeutic agent to treat, delay the onset of, or alleviate a target disease such as those described herein. The kit may also include a description of selecting an individual suitable for treatment based on identifying whether the individual suffers from the target disease, for example, using a diagnostic method as described herein. In other embodiments, the instructions include a description of administering the antibody to an individual at risk for the target disease.

[0275] Instructions for use of anti-CD22 antibodies typically include information about the dosage, dosing regimen, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multiple dose package), or subunit dose. The instructions provided in the kit of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0276] The label or package insert indicates that the composition is used to treat, delay the onset of, and / or alleviate a disease, such as cancer or an immune disease (eg, an autoimmune disease).Instructions for practicing any of the methods described herein may be provided.

[0277] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packaging used in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices such as miniature pumps. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an anti-CD22 antibody as described herein.

[0278] The kit may optionally provide additional components, such as buffer and interpretative information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article comprising the contents of the above-mentioned kit.

[0279] General techniques

[0280] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press; Animal Cell Culture(RIFreshney,ed.1987);Introduction to Cell and Tissue Culture(JPMather and P.E.Roberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.1993-8)J.Wiley and Sons;Methods in Enzymology(Academic Press, Inc.);Handbook of Experimental Immunology(DMWeirand CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds. 1994); Current Protocols in Immunology (JEColigan et al., eds. 1994);, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (DN Glover ed.1985); NucleicAcid Hybridization (BD Hames & S.J. Higgins, eds. (1985); Transcription and Translation (BD Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells and Enzymes (lRL Press, (1986)); and B. Perbal, A practical Guide To Molecular Cloning (1984); FMAusubel et al. (eds.).

[0281] Without further elaboration, it is believed that those skilled in the art can make full use of the present invention based on the above description. Therefore, the following specific examples should be interpreted as merely illustrative and not limiting the remainder of the present disclosure in any way. For the purposes or themes cited herein, all publications cited herein are incorporated herein by reference.

[0282] Example 1. Generation of fully human anti-CD22 antibodies

[0283] Fully human antibodies with binding specificity for cell surface human CD22 were identified from a human antibody library as follows.

[0284] Generation of CD22-overexpressing recombinant cell lines

[0285] HEK293 and K562 cells (ATCC) were transfected with pCMV6-Entry vectors, which carry nucleotide sequences encoding full-length human CD22 and are fused with flag and Myc tags at the C-terminus. The G418 drug selection process produced a polyclonal, drug-resistant CD22-expressing cell library. At the same time, a parental cell line transferred with an empty pCMV6-Entry vector was used as a negative control. Cells expressing CD22 were sorted by FACS to produce a cell library expressing CD22. The library was amplified under G418 drug selection. Single cell sorting was then performed, followed by further drug selection to produce a clonal cell line. Clonal lines expressing CD22 were screened by FACS. Cell lines showing high expression levels of CD22 were selected for selection, screening and determination disclosed herein.

[0286] Screening of anti-CD22 antibodies from human antibody libraries

[0287] From multiple initial Natural human antibody libraries were constructed from bone marrow MNCs and PBMCs from healthy donors and autoimmune disease patients. RT-PCR was performed to capture V H and V L The complete immunoglobulin repertoire of domains (generating V H and V L library). Then, through V H and V L The library size is expected to be 10 12-13 . V H The scFv and scFv libraries have been further modified to insert in vitro transcription and translation signals at the N-terminus of the antibody fragments and a flag tag at the C-terminus of the antibody fragments for mRNA display selection, respectively.

[0288] Then, mRNA display technology was used according to conventional practice to generate V H CD22 binders were identified from scFv and scFv libraries (see, e.g., US6258558B1, the relevant disclosure of which is incorporated herein by reference for the subject matter or purpose cited herein). Briefly, the DNA library was first transcribed into an mRNA library, which was then covalently coupled to an mRNA-V library via a puromycin linker. Hor scFv fusion library. The library was then purified and converted to an mRNA / cDNA fusion library. The fusion library was first counter-selected with human IgG (negative selection) or K562 cells to remove non-specific binders, and then selected for recombinant CD22-Fc fusion proteins captured on protein G magnetic beads (rounds 1-3) or CD22 overexpressing recombinant K562 cells (round 4). CD22 binders were recovered and enriched by PCR amplification. In round 3, the initial V L The library was reorganized and the enriched V H The library was converted to a scFv library and further enriched for 3 rounds. A total of 4 rounds of selection were performed to generate a highly enriched anti-CD22 antibody library, such as Figure 1 shown.

[0289] The enriched anti-CD22 antibody library was cloned into the bacterial periplasmic expression vector pET22b and transformed into TOP 10 competent cells. Each scFv molecule was engineered to have a C-terminal flag and 6xHIS tag for purification and assay detection. Clones from TOP 10 cells were pooled, miniprep DNA was prepared, and then transformed into the bacterial Rosetta II strain for expression. Single clones were selected, grown and induced with 0.1mM IPTG in 96-well plates for expression. Supernatants were collected after 16-24 hours of induction at 30°C for determination of anti-CD22 antibodies.

[0290] Supernatant samples were evaluated by sandwich ELISA assay to determine the presence / level of anti-CD22 scFv antibodies contained therein. Briefly, 96-well plates were fixed with anti-HIS tag antibodies (R&D Systems) at a final concentration of 2 μg / mL in 1x PBS, with a total volume of 50 μL per well. The plates were incubated overnight at 4°C and then blocked with 200 μL of super blocking buffer per well for 1 hour. 100 μL of supernatant diluted 1:101XPBST was added to each well and incubated with shaking for 1 hour. The expression level of CD22 scFv was detected by incubating the mixture in the plate with 50 μL of HRP-conjugated anti-Flag antibody for one hour, which was diluted 1:5000 in 1x PBST. Between each step, the plate was washed 3 times with 1XPBST in a plate washer. The plate was then developed for 5 minutes with 50 μL of TMB substrate and development was stopped by adding 50 μL of 2N sulfuric acid. Plates were read at OD450nm in a Biotek microplate reader and data were analyzed using Excel bar graphs.

[0291] A CD22 binding screening ELISA was developed to identify individual CD22 binders. Briefly, 96-well plates were immobilized with human Fc or human CD22-Fc proteins as controls at a final concentration of 2 μg / mL in 1xPBS in a total volume of 50 μL per well. The plates were incubated overnight at 4°C and then blocked with 200 μL per well of Super Blocking Buffer for 1 hour. 100 μL of supernatant was added to each well immobilized with Fc and CD22-Fc fusion proteins and incubated with shaking for 1 hour. CD22 binding was detected by adding 50 μL of HRP-conjugated anti-Flag antibody diluted 1:5000 in 1x PBST. Between each step, the plates were washed 3 times with 1XPBST in a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and development was stopped by adding 50 μL of 2N sulfuric acid. Plates were read at OD450nm in a Biotek microplate reader and binding and selectivity were analyzed using Excel bar graphs.

[0292] like Figure 2 As shown, a number of positive anti-CD22 clones were identified during the screening process disclosed herein.

[0293] Example 2. Identification of exemplary anti-CD22 clones capable of binding to cell surface expressed CD22

[0294] Production and purification of anti-CD22 antibodies in E. coli cells

[0295] From the glycerol stock plates, the expressing V genes identified in the screening process disclosed in Example 1 above were selected. H Cells expressing anti-CD22 scFv antibodies or anti-CD22 scFv antibodies were grown overnight in 5 mL cultures in Thomson 24-well plates with gas permeable membranes. Bacterial cells as described in the Examples herein were grown at 37°C and shaken at 225 RPM in Terrific Broth Complete, with 100 μg / mL carbenicillin and 34 μg / mL chloramphenicol, and antifoam 204 was added at a 1:5,000 dilution, unless otherwise specifically indicated. The overnight starter culture was then used to inoculate a larger culture into a designated production culture (e.g., 50 mL culture in a 125 mL Thomson Ultra Yield flask, 100 mL culture in a 250 mL UltraYield Thomson flask, or 250 mL culture in a 500 mL Ultra Yield Thomson flask) at the appropriate starter culture dilution ratio and grown until OD 600 At this time, the final concentration is 0.5mM (V HThe culture was induced with 0.1 mM IPTG (scFv) and incubated overnight at 30° C. The culture was then centrifuged at 5,000×g for 30 minutes to pellet the cells, and the supernatant was sterilized by filtration through a 0.2 μm sterile PES membrane for further analysis.

[0296] For purifying antibody fragment, 3 μ l GE Ni Sepharose Excel resin is mixed with 1 mL filtered supernatant and loaded onto 10 mL or 20 mL BioRad Econo-Pac column. Before loading, the resin of the column is balanced with at least 20 column volumes (CV) buffer A (1xPBS, pH 7.4, adding extra NaCl to 500 mM). The supernatant of filtration sterilization is purified on the same packed resin bed by gravity flow, by controlling the flow rate to 1 mL / min or pouring twice. The column is then washed with the following buffer: 10 CV buffer A, 20 CV buffer B (1xPBS, pH 7.4, containing extra NaCl to 500 mM, and 30 mM imidazole). If necessary, two Detox buffers are used to remove endotoxin. To purify antibody fragments from 250 mL expression culture, the antibody binding column was washed sequentially with 20 CV buffer C (1xPBS pH 7.4 with additional NaCl to 500 mM, 1% Tx114), 20 CV buffer D (1xPBS pH 7.4 with additional NaCl to 500 mM, 1% Tx100 + 0.2% TNBP), and 40 CV buffer E (1xPBS pH 7.4 with additional NaCl to 500 mM).

[0297] The protein was eluted with elution buffer F (1xPBS pH 7.4, containing additional NaCl to 500 mM, and 500 mM imidazole) in a total of six fractions (0.5 CV pre-elution, 5x 1 CV elution). The fractions were run on the Bradford assay (100 ul diluted Bradford solution + 10 ul sample). Fractions with bright blue were combined and their protein concentrations were measured by A280 extension coefficient. SDS-PAGE gel assay was performed to analyze the purity of the purified antibodies.

[0298] In most cases, a Tm shift thermostability assay was performed to measure the thermal stability of purified antibodies.

[0299] FACS analysis of cell surface binding activity of anti-CD22 ScFv antibodies

[0300] To determine the binding capacity of each anti-CD22 antibody to CD22 expressed on the cell surface, 50Value, starting from 100nM, serially diluted 2 times in complete medium to titrate each purified scFv protein. The diluted samples were incubated on ice for 1 hour with HEK293 cells expressing CD22 (CD22 / HEK293 cells) in 96-well plates. The cells were centrifuged at 1200rpm for 5 minutes at 4°C to remove unbound antibodies. The cells were then washed once with 200μL of complete medium per well. The samples were mixed with anti-His antibodies conjugated to Alexafluor 488 (secondary antibodies, 100μL, 1:1000 dilution) and incubated at 4°C in the dark for 30 minutes. The samples were then centrifuged at 1200rpm for 5 minutes at 4°C and washed twice with 200uL of 1x PBS per well. The resulting samples were re-dissolved in 200uL of 1x PBS and read on Guava EasyCyte. Analyzed by counting only Alexa Fluor 488 positive cells, and then plotted in Prism 8.1 software.

[0301] As determined in this study, the exemplary anti-CD22 clones were able to bind to cell surface CD22. Figures 3A-3D Binding curves for several exemplary anti-CD22 clones at different concentrations as indicated are shown.

[0302] The binding affinities of various anti-CD22 antibodies disclosed herein to CD22 / K562 cells are provided in Table 1 below:

[0303] Table 1

[0304] Binding affinity of exemplary anti-CD22 antibodies to cell surface CD22

[0305] Clone Name: <![CDATA[EC 50 (nM)]]> EP160-D02 0.24 EP160-H02 0.68 EP97-B03 0.7 EP97-A10 1 EP160-G04 1.1 EP160-F04 2.79 EP160-G05 2.9 EP97-G05 3.3 EP35-C8 4.6 EP160-C07 5.2 EP160-E03 6.8 EP160-F10 9 EP97-F01 10 EP35-A7 10.38 EP35-E7 11 EP35-E6 14.18 EP35-F6 15 EP35-C6 19.31 EP35-D6 47 EP35-B5 77

[0306] Example 3. Epitope partitioning of anti-CD22 molecules using M971 and / or BL22

[0307] Epitope partitioning using M971 anti-CD22 scFv antibody

[0308] Epitope partitioning assays were performed to investigate whether any CD22 binding agent identified herein as disclosed in the above examples can compete with the anti-CD22 antibody M971 for binding to CD22. Briefly, recombinant K562 cells overexpressing CD22 were incubated on ice for 1 hour with 200 nM of purified anti-CD22 scFv antibodies disclosed herein or a premix containing 200 nM purified anti-CD22 scFv and 20 nM M971 IgG antibodies. The cells were centrifuged at 1200 rpm for 5 minutes at 4°C. The binding activity of anti-CD22 scFv to K562 cells expressing CD22 included anti-His antibodies (100 uL, 1:1000 dilution) conjugated to Alexa fluor 647, which were protected from light at 4°C for 30 minutes. The mixture thus formed was centrifuged at 1200 rpm for 5 minutes at 4°C, and each well was washed twice with 200 uL 1x PBS. The cells so collected were reconstituted in 200uL of 1x PBS and read on an Attune flow cytometer. Analysis was performed by overlaying the binding histogram of 200nM anti-CD22 scFv antibody to recombinant K562 cells overexpressing CD22 relative to the binding histogram of premixed 200nM anti-CD22 scFv antibody and 20nM M971 IgG antibody to the same recombinant cells. The results thus obtained indicated that none of the scFv antibodies tested, including EP160-G04, EP97-B03, EP160-H02, EP97-A10, EP160-E03, EP160-F04, EP97-A01, EP35-C6, EP160-F10, EP160-G05, EP160-C07, EP35-E6, EP35-C8 and EP35-F07, competed with M971 for binding to cell surface CD22.

[0309] Epitope partitioning with M971 was further confirmed by ELISA assay. In short, 384-well plates were coated with 2 μg / mL recombinant human CD22 or recombinant human Fc overnight at 4 ° C. The plate was then blocked with Pierce super blocking buffer for 1 hour at room temperature. 200nM purified anti-CD22 scFv antibodies or a premix containing 200nM purified anti-CD22 scFv and 100nM M971 IgG antibodies as disclosed herein were loaded into a plate pre-coated with recombinant human Fc or recombinant human CD22. The plate was then shaken and incubated at room temperature for 1 hour. Afterwards, 25uL of HRP-conjugated anti-labeled antibodies (diluted at 1:5000) were added to each well, and the plate was incubated at room temperature in the dark for 1 hour. The plate was washed 3 times with 80uL 1x PBST between each step. Then, the plate was developed with 20uL 1-step ultra TMB-ELISA substrate solution for 5 minutes, and then 20μL 2N sulfuric acid was added to terminate the reaction. 450 Read the plate. The analysis was performed by plotting on an Excel bar graph comparing the binding of 200 nM anti-CD22 scFv antibody alone versus premixed 200 nM anti-CD22 scFv with 100 nM IgG M971 antibody on the recombinant human CD22 protein plate.

[0310] like Figure 4 As shown, none of the exemplary anti-CD22 scFv antibodies shown competed with M971 for binding to CD22.

[0311] Anti-CD22 antibody binding epitopes compared with M971 and BL22

[0312] BL22 (also known as CAT-3888) is a recombinant anti-CD22 immunotoxin proposed for use as a therapeutic agent for the treatment of B cell malignancies and is known in the art. BL22 is a recombinant fusion protein comprising the disulfide-linked VH and VL chains of the mouse anti-CD22 monoclonal antibody RFB4 fused to a truncated form of Pseudomonas exotoxin A called PE38. The epitope specificity and tissue reactivity of RFB4 were reported in Li et al., Cell Immunol. 118(1):85-99(1989).

[0313] Epitope partitioning of CD22EP160-D02 antibody with M971 and BL22 was completed by FACS analysis using EP160-D02 scFv and CD22 overexpressing recombinant K562 cell lines. Purified anti-CD22 scFv was serially diluted 2-fold on ice for one hour from a premix of 200nM and 5.13nM, 1.77nM M971 or 0.7nM, 0.175nM BL22 mAbs, respectively. Cells were centrifuged at 1200rpm for 5 minutes at 4°C. Anti-His Alexa fluor 647 was used to detect the binding activity of anti-CD22 scFv by adding 100uL of 1:1000 diluted secondary antibody and incubating at 4°C in the dark for 30 minutes. Samples were centrifuged at 1200rpm for 5 minutes at 4°C and washed twice with 200uL 1x PBS per well. Cells were reconstituted in 200uL of 1x PBS and read on an Attune flow cytometer. Analysis was performed by counting anti-CD22 scFv positively stained cells on recombinant K562 cells overexpressing CD22 in the presence and absence of M971 and BL22 mAbs. EC50 was calculated using Prism 8.0.

[0314] like Fig. 7A and 7B As shown, the presence of M971 and BL22 had no significant effect on the binding activity of clone EP160-D02 to K562 cells expressing CD22, indicating that M971 and BL22 do not compete with EP160-D02 for binding to cell surface CD22. In other words, the results show that EP160-D02 does not bind to the same epitope as M971 or BL22. The EC values ​​of EP160-D02 determined in this assay are 50 and IC 50 The values ​​are provided in Tables 2 and 3 below:

[0315] Table 2. EC of EP160-D02 in the presence or absence of M971 50 value

[0316] <![CDATA[EC 50 (nM)]]> EP160-D02 scFv 0.1246 EP160-D02 scFv+1.77nM M971 0.09457 EP160-D02 scFv+5.13nM M971 0.1436 EP160-D02 scFv,K562 N / D

[0317] Table 3. IC of EP160-D02 in the presence or absence of BL22 50 value

[0318] <![CDATA[IC 50 (nM)]]> EP160-D02 scFv 0.08216 EP160-D02 scFv, 0.175nM BL22 0.0811 EP160-D02 scFv, 0.77nM BL22 0.08978 EP160-D02 scFv,K562 N / D

[0319] In summary, the results from these epitope partitioning assays indicate that the exemplary anti-CD22 antibodies reported herein (e.g., EP160-D02) do not bind to the same CD22 epitope as the known anti-CD22 antibodies M971 and RFB4. Therefore, it is expected that the exemplary anti-CD22 antibodies disclosed herein have different biological activities relative to known anti-CD22 antibodies in at least some aspects.

[0320] Example 4. Binding kinetics of anti-CD22 scFv antibodies

[0321] The kinetic analysis of the binding of anti-CD22 scFv to CD22 has been evaluated with Biacore T200 by SPR technology. The assay was run using Biacore T200 control software version 2.0. For each cycle, 1 μg / mL human CD22-Fc fusion protein was captured on flow cell 2 at a flow rate of 10ul / min in 1xHBST buffer on a protein G sensor chip for 60 seconds. The 2-fold serial dilution of HIS-tag purified anti-CD22 scFv was injected into reference flow cell 1 and CD22-captured flow cell 2 at a flow rate of 30ul / min for 150 seconds, followed by washing for 300 seconds. The flow cell was then regenerated at a flow rate of 30ul / min with glycine pH 2 for 60 seconds. Eight concentration points of each anti-CD22 scFv from 100-0nM were measured in a 96-well plate. The kinetics of scFv binding to CD22 protein were analyzed using Biacore T200 evaluation software 3000. Specific binding response units were derived from subtracting binding to reference flow cell 1 from CD22 captured flow cell 2. The results are provided in Table 4 below.

[0322] Table 4. Binding kinetics of exemplary anti-CD22 scFv antibodies

[0323]

[0324]

[0325] Example 5. Thermal stability evaluation of exemplary anti-CD22 scFv antibodies

[0326] In this example, each sample and control was prepared in at least duplicate to ensure reproducible results.A plate map was first designed in Excel so that the exact location of each sample could match the software used to run and analyze the samples.

[0327] Protein thermal shift dye (1000x) was freshly diluted to 8x in water. LifeTech MicroAmpOptical 96-well plates or 8cap strips were used for the experiment. The following reagents were added in the order listed:

[0328] - First sample: 5ul protein thermal migration buffer,

[0329] - Second sample: 12.5ul sample diluted to 0.4mg / mL in water,

[0330] - Third sample: 2.5ul of thermal shift dye diluted 8x, total volume 20ul / well.

[0331] -Negative control sample: 12.5ul protein-free buffer

[0332] - Positive control sample: 10.5ul water and 2.0uL protein thermal shift control protein.

[0333] After adding the thermal migration dye, pipette up and down 10 times. Then, once sealed with the MicroAmp optical film of the cap, spin the plate or strip at 1000 RPM for 1 minute. After that, place the plate or strip in the Thermo Fisher Quant Studio 3 instrument and run the method as follows.

[0334] - Step 1: Rise to 25.0° at 100% slope in 2 minutes

[0335] - Step 2: Ramp up to 99.0°C at a 1% ramp rate over 2 minutes

[0336] The samples and subsequent Tm (and Tm calculation) were then analyzed using QuantStudio Design and Analysis Software and Protein ThermalShift Software 1.3. The results are shown in Table 5 below:

[0337] Table 5. Thermal shift assays for exemplary anti-CD22 antibodies

[0338] scFv Tm℃ EP160-D02 57.5 EP97-G05 56.6 EP97-F01 59.8 EP160-G04 52.0 EP97-B03 54.4 EP160-H02 57.7 EP97-A10 61.8 EP160-E03 71.4 EP160-F04 47.2 EP35-F07 56.3 EP97-A01 72.3 EP35-C06 71.2 EP35-B05 66.7 EP160-F10 69.7 EP160-G05 59.7 EP160-C07 48.5 EP35-C08 52

[0339] Example 6: Anti-CD22 antibodies bind to endogenous CD22 and recombinant CD22 on the cell surface

[0340] Exemplary anti-CD22 scFv antibodies, including EP97-G05, EP97-A10, EP160-E03, and EP160-H02, were tested for their ability to bind to endogenous CD22 expressed on the cell surface and recombinant CD22 expressed on the cell surface using FACS analysis.

[0341] Briefly, 200 nM of each purified CD22 scFv antibody (containing a HIS tag) was diluted in complete medium and incubated with Daudi and Raji, CD22 / HEK293, CD22 / K562, and K562 cell lines in a 96-well plate on ice for 1 hour. The cells were centrifuged at 1200 rpm for 5 minutes at 4°C to remove unbound scFv. The cells were then washed once with 200 uL of complete medium per well. Anti-HIS biotin / streptavidin was used by adding 100 uL of diluted secondary antibody and incubating at 4°C in the dark for 30 minutes. Samples were detected with fluor 647. Samples were centrifuged at 1200 rpm for 5 minutes at 4°C and washed twice with 200uL 1x PBS per well. Samples were reconstituted in 200uL of 1x PBS and read on the Attune NxT cytometer. Overlay histograms of CD22 protein binding to negative and target cell lines were plotted for analysis by Attune NxT software. Anti-CD22 mouse antibody and anti-HIS biotin / streptavidin secondary Alexafluor 647 served as positive and negative (background) controls for the assay.

[0342] like Figure 5 As shown, at the antibody concentrations tested, all four anti-CD22 scFv antibodies bound to HEK and K562 cells expressing recombinant CD22 on the cell surface. Anti-CD22 scFv was also found to bind to Daudi and Raji expressing endogenous CD22 on the cell surface.

[0343] In addition, immunohistochemistry (IHC) studies were performed on 5 mm sections of formalin-fixed, paraffin-embedded diffuse large B-cell lymphoma (DLBCL) FFPE tissue blocks on the Ventana Ultra automated platform using an IHC staining protocol. Briefly, after dewaxing and rehydration, antigen retrieval was performed using standard CC1 antibody retrieval (EDTA-based antigen retrieval buffer, pH 9.0, Cat#950-500). Permeabilization and washing were performed with Ventana Discovery wash buffer Cat#905-510 and Discovery reaction buffer Cat#950-300 between staining steps. Discovery inhibitor CM Cat#764-4307 and IHC / ICCIHC protein blocking agent (Invitrogen Cat#00-4952-54) were applied during staining for pretreatment of nonspecific staining.

[0344] The exemplary anti-CD22 scFv EP97-G05 fused to the human Fc polypeptide was incubated with the above tissue samples at a concentration of 10ug / ml at 37°C for 60 minutes, and then incubated with anti-human IgG FC HRP antibody (Abcam Cat#ab98624) diluted 1 / 250. Ventana ChromapDAB kit (Cat#760-159) was used for the final IHC step. All sections were counterstained with hematoxylin, and the entire slide was imaged by Aperio AT2 scanning mirror, and image analysis was performed using Indica labs CytoNuclear v1.6 algorithm.

[0345] As shown in Figure 7, EP97-G05 was found to bind to CD22-positive DLBCL tissue in the IHC studies described herein, indicating that the antibody is able to bind to endogenous CD22 that may be expressed on disease cells.

[0346] Example 7: Preparation and characterization of anti-CD22 IgG antibodies

[0347] (i) Recombinant production of anti-CD22 IgG antibodies

[0348] According to conventional practice, anti-CD22 ScFv antibodies are converted to IgG form. In short, VH and VL sequences are fused to the constant domains of the human IgG1k main chain. The gene is codon-optimized for mammalian expression, synthesized by Life Technologies and subcloned into pCDNA3.4 expression vectors. According to standard protocols, antibodies are transiently expressed in ExpiHEK293-F cells in the Freestyle system (Invitrogen). Cells are grown for five days before harvest. The supernatant is collected by centrifugation and filtered through a 0.2 μm PES membrane.

[0349] The Fc-fused agonist was first purified by MabSelect PrismA Protein A resin (GE Health). The protein was eluted with 100 mM Gly pH 2.5 plus 150 mM NaCl and rapidly neutralized with 20 mM Tris-HCl pH 8.0 plus 300 mM NaCl.

[0350] The antibody was then further purified by Superdex 200 Increase 10 / 300 GL column. The monomer peak fractions were pooled and concentrated. The endotoxin content of the final purified protein was less than 10EU / mg and was stored in 1xPBS buffer.

[0351] (ii) Anti-CD22 IgG Antibody Cell Binding Activity

[0352] Determination of EC of anti-CD22 IgG on CD22-overexpressing recombinant cell lines by FACS binding assay 50 . The purified IgG was serially diluted 2-fold in complete medium for a total of 12 dilutions. The diluted IgG was incubated with 100,000 CD22K562 cells per well in a 96-well plate on ice for 1 hour. The cells were centrifuged at 1200 rpm for 5 minutes at 4°C to remove unbound antibodies. The cells were then washed once with 200uL complete medium per well. BL22 was used as a positive control for anti-CD22 antibodies, and CHO-K1 cells expressing CD123 (but not CD22) were used as negative controls.

[0353] Samples were detected with anti-hFc Alexa fluor 488 by adding 100uL of 1:1000 diluted secondary antibody and incubated at 4°C in the dark for 30 minutes. Samples were centrifuged at 1200rpm for 5 minutes at 4°C and washed twice with 200uL1x PBS per well. Samples were reconstituted in 200uL of 1x PBS and read on Guava EasyCyte. Analysis was performed by counting only positive Alexa Fluor 488 cells and then plotted in Prism 8.1 software.

[0354] like Fig. 8A and 8B As shown, the IgG form of clone EP160-D02 showed strong binding to cell surface CD22, but not to cell surface CD123. 50 The values ​​are provided in Tables 6 and 7 below.

[0355] Table 6. ECs that bind to cell surface CD22 50 value

[0356]

[0357] Table 7. ECs that bind to cell surface CD123 50 value

[0358]

[0359] Binding of anti-CD22 IgG antibodies to cell surface CD22 was also measured by ELISA, and similar results were observed. Figure 8C and Table 8 below.

[0360] Table 8. EC of anti-CD22 IgG antibodies determined by ELISA 50 value

[0361] <![CDATA[EC 50 (nM)]]> EP160-D02 0.039 EP97-B03 1.82 BL22 0.004 M971 0.059

[0362] (iii) Anti-CD22 IgG Antibody ADCC Activity

[0363] Antibody-dependent cellular cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a cell-mediated cytotoxic mechanism whereby effector cells of the immune system engage antibodies and actively lyse the target cells to which the antibody is bound.

[0364] ADCC activity of anti-CD22 IgG antibodies was tested using the Promega ADCC Bioreporter Assay Kit. Briefly, 30,000 CD22 / HEK293 target cells were plated in a white flat-bottom 96-well assay plate and incubated overnight at 37°C. Following the manufacturer's protocol, antibodies were serially diluted 3-fold from 200 nM in ADCC assay buffer. The supernatant of target cells was removed. 25 μL of ADCC assay buffer was mixed with 25 μL of antibody dilution into each well of cells. Cells were incubated at room temperature for one hour before adding effector cells.

[0365] Thaw effector cells following the manufacturer's protocol and seed 25 μL of effector cells onto each target cell / antibody mixture. Incubate the plates at 37 °C for 16 h.

[0366] The next day, samples were equilibrated at room temperature for 30 minutes, then 75 μL of room temperature Bio-glow reagent was added and incubated at room temperature in the dark for 30 minutes. Bioluminescent reagent was prepared according to the manufacturer's protocol. Plates were read by luminescence on a Biotek Neo2 microplate reader and data were plotted on Prism 8.0.

[0367] The results obtained from this assay showed that exemplary anti-CD22 IgG antibodies, including EP97-B03 and EP160-D02, exhibited ADCC activity, while the control antibody M971 exhibited little or no ADCC activity. At least clone EP160-D02 exhibited better ADCC activity relative to BL22. The EC values ​​of the tested antibodies were 50 The values ​​are provided in Table 9 below:

[0368] Table 9. EC of anti-CD22 antibodies in ADCC assay 50

[0369] <![CDATA[EC 50 (nM)]]> EP97-B03 3.714 EP160-D02 1.947 EP160-H02 ～73.80 BL22 3.314 M971 ～

[0370] (iv) Anti-CD22 IgG Antibody Internalization Activity

[0371] The kinetics of anti-CD22 antibody internalization was determined using an image-based fluorescence assay. Briefly, 30,000 CD22 / HEK293 target cells were plated in 96-well black-bottom plates treated with poly-L-lysine and incubated overnight at 37°C. CD22 IgG and secondary antibody pHrodo were diluted to a final concentration of 4 nM and 120 nM, respectively, in 10% RPMI without phenol red and incubated at room temperature for at least 5 minutes in the dark.

[0372] The media was then removed from the target cells and 100 μL of the antibody / secondary pHrodo mix was added to the cells. The cells were imaged immediately using RFP and brightfield on a Cytation 5 and every two hours at 37°C. The internalization rate was quantified by the Cytation 5 analysis software and analyzed by Prism 8.0.

[0373] like Fig.10 As shown, clones EP160-D02 and EP97-B03 showed cellular internalization, albeit slower than that of the BL22 molecule. See also Table 10 below.

[0374] Table 10. Internalization of anti-CD22 IgG antibodies

[0375] T1 / 2(hours) BL22 3.95 M971 6.07 EP160-D02 5.06 EP97-B03 5.26

[0376] Other Implementations

[0377] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0378] Through the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0379] Equivalent

[0380] Although several invention embodiments have been described and illustrated herein, a person of ordinary skill in the art will readily envision various other devices and / or structures for performing functions and / or obtaining results and / or one or more advantages described herein, and each such variation and / or modification is considered to be within the scope of the invention embodiments described herein. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application or multiple applications for which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine many equivalents of the specific invention embodiments described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments are presented only as examples, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be implemented in a manner different from that specifically described and claimed. The invention embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, if such features, systems, articles, materials, kits, and / or methods do not contradict each other, any combination of two or more such features, systems, articles, materials, kits, and / or methods is also included within the scope of the invention of the present disclosure.

[0381] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms as defined and used herein.

[0382] All references, patents, and patent applications disclosed herein are incorporated by reference into the entirety of each cited subject matter, which in some cases may be incorporated into the entirety of the document.

[0383] Unless expressly indicated to the contrary, the indefinite articles "a" and "an" used in the specification and claims should be understood to mean "at least one".

[0384] The phrase "and / or" as used herein in the specification and claims should be understood to mean "one or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether or not these other elements are related or unrelated to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising", a reference to "A and / or B" may refer to only A (optionally including elements other than B) in one embodiment; to only B (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; etc.

[0385] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one, but also including more than one element of a number of elements or a series of elements, and (optionally) other unlisted items. Only when the contrary term is clearly indicated, such as "only one of" or "exactly one of", or when used in the claims, "consisting of..." will refer to containing exactly one element of a number of elements or a series of elements. In general, the term "or" used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") if it is preceded by an exclusive term, such as "any", "one of", "only one of" or "exactly one of". "Substantially consisting of...", as used in the claims, shall have the ordinary meaning used in the field of patent law.

[0386] As used herein in the specification and claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B"), in one embodiment, may refer to at least one, optionally including more than one A without B (and optionally including elements other than B); in another embodiment, may refer to at least one, optionally including more than one B without A (and optionally including elements other than A); in yet another embodiment, may refer to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); etc.

[0387] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described unless explicitly indicated to the contrary. SEQUENCE LISTING <110> ELPIS BIOPHARMACEUTICALS <120> ANTI-CD22 ANTIBODIES AND USES THEREOF <130> 083661-8002CN01 <140> PCT / US2020 / 047479 <141> 2020-08-21 <150> 62 / 889,739 <151> 2019-08-21 <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Trp Tyr Asn Asp Tyr Ala Val 50 55 60 Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn Gln 65 70 75 80 Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Val Ala Gly Ser Arg Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 4 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Glu Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Trp Thr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 6 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30 Gly Met Gln Trp Val Arg Gln Thr Pro Asp Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ala His Asp Gly Thr Val Gln Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ala Thr Pro Arg Ala Ala Asp His Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Gly Thr Val Ser Ser 115 120 <210> 8 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Gly Ile Ala Val Ala Gly Thr Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Thr Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Gly Ile 20 25 30 Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln Gly 50 55 60 Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Ile Ala Tyr Met Glu 65 70 75 80 Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Thr 85 90 95 Gly Gly Gln Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 11 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Leu Glu Pro Leu Glu Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 13 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Ser Leu Ser Ile Thr Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Ile Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Gln Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 15 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Asn Trp Ala Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Gly Ile Lys 100 105 <210> 16 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Pro Glu Tyr Ser Ser Ser Ala Gly Thr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Thr Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Ser Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 19 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 20 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Arg Phe 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ala Phe Ile Arg Thr Asp Gly Gly Ser Gln His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Asp Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Pro Arg Val Thr Gly Asn Thr Gly Tyr Asp Tyr Asp 100 105 110 Trp Gly Gln Gly Val Gln Val Thr Val Ser Ser 115 120 <210> 21 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ala Asn Asn Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Pro Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 22 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 Glu Val Gln Leu Val Glu Ser Arg Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Thr Val Thr Thr Asn Tyr Tyr Tyr Tyr Met Asp Val Trp 100 105 110 Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 23 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Glu Tyr Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe His Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Asp Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 24 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 Gln Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Asn 20 25 30 Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ser Thr Ser Ser Asp Asn Ile Asn Tyr Ala Gln Lys Phe 50 55 60 Arg Gly Arg Leu Thr Leu Thr Thr Asp Thr Ser Thr Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Phe Gly Gly Arg Asp Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 25 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Ile Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Arg 100 105 <210> 26 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 26 Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Pro Phe Ser Thr Ala 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Lys Ser Glu Ala His Gly Gly Thr Thr His Tyr Ala Pro 50 55 60 Pro Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Ser Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Gly Asp Phe Gln Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Gly Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Ser Ser Asp Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 29 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 29 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Met 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 30 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Ser Ile Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 31 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 31 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Glu Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Asn Ser Pro Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 33 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 33 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 34 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 34 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Asp Pro Ser Gly Asp Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 35 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 35 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 36 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ile Ala Ala Ala Gly Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 37 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Thr Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Trp Lys Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 39 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 39 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr Trp Pro Pro 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 40 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 40 Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Val Ala Gly Ser Arg Gly Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln 145 150 155 160 Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala 165 170 175 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro 180 185 190 Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile 195 200 205 Ser Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 210 215 220 His Thr Trp Pro Pro Val Thr Phe Gly Glu Gly Thr Lys Val Glu Ile 225 230 235 240 Lys <210> 41 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 41 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Trp Thr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 130 135 140 Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser 145 150 155 160 Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro 165 170 175 Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp 180 185 190 Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser 195 200 205 Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His 210 215 220 Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 225 230 235 240 <210> 42 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30 Gly Met Gln Trp Val Arg Gln Thr Pro Asp Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ala His Asp Gly Thr Val Gln Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ala Thr Pro Arg Ala Ala Asp His Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Gly Thr Val Ser Ser 115 120 <210> 43 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 43 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Gly Ile Ala Val Ala Gly Thr Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr 180 185 190 Asp Val Pro Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Ser Ile Ser Asn Val Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr His Thr Trp Thr Pro Val Thr Phe Gly Gly Gly Thr Lys 225 230 235 240 Val Glu Ile Lys <210> 44 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Gly Ile 20 25 30 Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp 35 40 45 Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln Gly 50 55 60 Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Ile Ala Tyr Met Glu 65 70 75 80 Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Thr 85 90 95 Gly Gly Gln Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 145 150 155 160 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 165 170 175 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 180 185 190 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 195 200 205 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp Pro Pro 210 215 220 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 45 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Leu Glu Pro Leu Glu Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr 130 135 140 Leu Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser 145 150 155 160 Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val 180 185 190 Pro Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Ser Leu Ser 195 200 205 Ile Thr Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 210 215 220 Tyr His Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys <210> 46 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Ile Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Gln Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 130 135 140 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 145 150 155 160 Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 165 170 175 Leu Ile Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe 180 185 190 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 195 200 205 Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Asn Trp 210 215 220 Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Gly Ile Lys 225 230 235 <210> 47 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Pro Glu Tyr Ser Ser Ser Ala Gly Thr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser 130 135 140 Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys 145 150 155 160 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg 165 170 175 Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala 180 185 190 Thr Asp Val Pro Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Ser Ile Thr Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr 210 215 220 Cys Gln Gln Tyr His Thr Trp Ser Pro Val Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys Val Glu Ile Lys 245 <210> 48 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 48 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 130 135 140 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 145 150 155 160 Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 165 170 175 Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe 180 185 190 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 195 200 205 Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp 210 215 220 Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 225 230 235 <210> 49 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Trp Lys Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 130 135 140 Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser 145 150 155 160 Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro 165 170 175 Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp 180 185 190 Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser 195 200 205 Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His 210 215 220 Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 225 230 235 240 <210> 50 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 50 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Arg Phe 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ala Phe Ile Arg Thr Asp Gly Gly Ser Gln His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Val Asp Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Pro Arg Val Thr Gly Asn Thr Gly Tyr Asp Tyr Asp 100 105 110 Trp Gly Gln Gly Val Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 130 135 140 Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn 145 150 155 160 Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ala Asn Asn Lys Asn Cys 165 170 175 Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro Lys Leu Leu Ile 180 185 190 Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Gly Arg Phe Ser Gly 195 200 205 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 210 215 220 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Pro Pro Arg 225 230 235 240 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 245 250 <210> 51 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 51 Glu Val Gln Leu Val Glu Ser Arg Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Thr Val Thr Thr Asn Tyr Tyr Tyr Tyr Met Asp Val Trp 100 105 110 Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser 130 135 140 Pro Leu Ser Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys 145 150 155 160 Arg Ser Ser Arg Ser Leu Glu Tyr Asn Asp Gly Asn Thr Tyr Leu Asn 165 170 175 Trp Phe His Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Lys 180 185 190 Val Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 195 200 205 Ser Asp Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 210 215 220 Val Gly Ile Tyr Tyr Cys Met Gln Gly Thr His Trp Pro Leu Thr Phe 225 230 235 240 Gly Gln Gly Thr Arg Leu Glu Ile Lys 245 <210> 52 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 52 Gln Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Asn 20 25 30 Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ser Thr Ser Ser Asp Asn Ile Asn Tyr Ala Gln Lys Phe 50 55 60 Arg Gly Arg Leu Thr Leu Thr Thr Asp Thr Ser Thr Gly Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Phe Gly Gly Arg Asp Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser 145 150 155 160 Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Ile Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Tyr Asp Asn Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Arg 225 230 235 <210> 53 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Pro Phe Ser Thr Ala 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Lys Ser Glu Ala His Gly Gly Thr Thr His Tyr Ala Pro 50 55 60 Pro Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Ser Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Gly Asp Phe Gln Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 130 135 140 Asp Arg Ile Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Phe 145 150 155 160 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Leu 165 170 175 Tyr Asp Ala Ser Asn Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly 180 185 190 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 195 200 205 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asp Asn Leu Pro Leu 210 215 220 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 54 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 54 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Gly Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Ser Ser Asp Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln 145 150 155 160 Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 165 170 175 Pro Arg Leu Leu Met Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro 180 185 190 Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile 195 200 205 Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 210 215 220 His Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 225 230 235 240 Lys <210> 55 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Ser Ile Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val 130 135 140 Ser Pro Gly Glu Glu Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 145 150 155 160 Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg 165 170 175 Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp Arg 180 185 190 Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn 195 200 205 Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Thr 210 215 220 Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 56 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 56 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Gly Asn Ser Pro Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln 145 150 155 160 Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala 165 170 175 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro 180 185 190 Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile 195 200 205 Ser Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 210 215 220 His Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 225 230 235 240 Lys <210> 57 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 57 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Gly Asp Pro Ser Gly Asp Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr 130 135 140 Leu Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser 145 150 155 160 Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val 180 185 190 Pro Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser 195 200 205 Ile Ser Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 210 215 220 Tyr His Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys <210> 58 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 58 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ile Ala Ala Ala Gly Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln 145 150 155 160 Ser Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala 165 170 175 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro 180 185 190 Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile 195 200 205 Thr Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 210 215 220 His Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 225 230 235 240 Lys <210> 59 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 59 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Trp Lys Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 130 135 140 Val Ser Pro Gly Glu Gly Val Thr Leu Ser Cys Arg Ala Ser Gln Ser 145 150 155 160 Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro 165 170 175 Arg Leu Leu Ile Tyr Gly Ala Ser Ile Lys Ala Thr Asp Val Pro Asp 180 185 190 Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser 195 200 205 Asn Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His 210 215 220 Thr Trp Pro Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 225 230 235 240 <210> 60 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 60 His His His His His His 1 5

Claims

1. An isolated antibody that binds to CD22, wherein the antibody comprises (i) a heavy chain variable region (V H ), wherein the heavy chain variable region comprises a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2) and a heavy chain complementary determining region 3 (HC CDR3); and (ii) a light chain variable region (V L ), the light chain variable region comprises a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2) and a light chain complementary determining region 3 (LC CDR3), wherein the antibody comprises: (a)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 34, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 34, and HC CDR3 as shown in amino acid residues 99-108 of SEQ ID NO: 34, wherein the V L comprising the LC CDR1 as set forth at amino acid residues 24-34 of SEQ ID NO:35, the LC CDR2 as set forth at amino acid residues 50-56 of SEQ ID NO:35, and the LC CDR3 as set forth at amino acid residues 89-98 of SEQ ID NO:35; or (b)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 3, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 3, and HC CDR3 as shown in amino acid residues 99-107 of SEQ ID NO: 3, wherein the V L comprising the LC CDR1 as shown in amino acid residues 24-34 of SEQ ID NO:4, the LC CDR2 as shown in amino acid residues 50-56 of SEQ ID NO:4, and the LC CDR3 as shown in amino acid residues 89-98 of SEQ ID NO:4; or (c)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO:5, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO:5, and HC CDR3 as shown in amino acid residues 99-106 of SEQ ID NO:5, wherein the V L comprising the LC CDR1 as shown in amino acid residues 24-34 of SEQ ID NO:6, the LC CDR2 as shown in amino acid residues 50-56 of SEQ ID NO:6, and the LC CDR3 as shown in amino acid residues 89-98 of SEQ ID NO:6; or (d)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 8, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 8, and HC CDR3 as shown in amino acid residues 99-110 of SEQ ID NO: 8, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO:9, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO:9, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO:9; or (e)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 29-33 of SEQ ID NO: 10, HC CDR2 as shown in amino acid residues 48-64 of SEQ ID NO: 10, and HC CDR3 as shown in amino acid residues 97-102 of SEQ ID NO: 10, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO: 11, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO: 11, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO: 11; or (f)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 12, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 12, and HC CDR3 as shown in amino acid residues 99-108 of SEQ ID NO: 12, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO: 13, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO: 13, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO: 13; or (g)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 14, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 14, and HC CDR3 as shown in amino acid residues 99-104 of SEQ ID NO: 14, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO: 15, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO: 15, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO: 15; or (h)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 16, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 16, and HC CDR3 as shown in amino acid residues 99-111 of SEQ ID NO: 16, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO: 17, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO: 17, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO: 17; or (i)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 18, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 18, and HC CDR3 as shown in amino acid residues 99-104 of SEQ ID NO: 18, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO: 19, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO: 19, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO: 19; or (j)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 28, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 28, and HC CDR3 as shown in amino acid residues 99-107 of SEQ ID NO: 28, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO:29, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO:29, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO:29; or (k)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 30, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 30, and HC CDR3 as shown in amino acid residues 99-105 of SEQ ID NO: 30, wherein the V L comprising the LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO:31, the LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO:31, and the LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO:31; or (l)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 32, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 32, and HC CDR3 as shown in amino acid residues 99-107 of SEQ ID NO: 32, wherein the V L comprising the LC CDR1 as set forth at amino acid residues 24-34 of SEQ ID NO:33, the LC CDR2 as set forth at amino acid residues 50-56 of SEQ ID NO:33, and the LC CDR3 as set forth at amino acid residues 89-98 of SEQ ID NO:33; or (m)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 36, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 36, and HC CDR3 as shown in amino acid residues 99-107 of SEQ ID NO: 36, wherein the V L comprising the LC CDR1 as set forth at amino acid residues 24-34 of SEQ ID NO:37, the LC CDR2 as set forth at amino acid residues 50-56 of SEQ ID NO:37, and the LC CDR3 as set forth at amino acid residues 89-98 of SEQ ID NO:37; or (n)V H and V L , the V H comprising HC CDR1 as shown in amino acid residues 31-35 of SEQ ID NO: 38, HC CDR2 as shown in amino acid residues 50-66 of SEQ ID NO: 38, and HC CDR3 as shown in amino acid residues 99-106 of SEQ ID NO: 38, wherein the V L comprising LC CDR1 as set forth in amino acid residues 24-34 of SEQ ID NO:39, LC CDR2 as set forth in amino acid residues 50-56 of SEQ ID NO:39, and LC CDR3 as set forth in amino acid residues 89-98 of SEQ ID NO:39; The CDRs are determined by the Chothia method.

2. The isolated antibody of claim 1, wherein the antibody comprises: (a) V as shown in SEQ ID NO: 34 H and V as shown in SEQ ID NO:35 L ; (b) V as shown in SEQ ID NO: 3 H and V as shown in SEQ ID NO:4 L ; (c) V as shown in SEQ ID NO: 5 H and V as shown in SEQ ID NO:6 L ; (d) V as shown in SEQ ID NO: 8 H and V as shown in SEQ ID NO:9 L ; (e) V as shown in SEQ ID NO: 10 H and V as shown in SEQ ID NO:11 L ; (f) V as shown in SEQ ID NO: 12 H and V as shown in SEQ ID NO: 13 L ; (g) V as shown in SEQ ID NO: 14 H and V as shown in SEQ ID NO: 15 L ; (h) V as shown in SEQ ID NO: 16 H and V as shown in SEQ ID NO: 17 L ; (i) V as shown in SEQ ID NO: 18 H and V as shown in SEQ ID NO: 19 L ; (j) V as shown in SEQ ID NO: 28 H and V as shown in SEQ ID NO: 29 L ; (k) V as shown in SEQ ID NO: 30 H and V as shown in SEQ ID NO:31 L ; (1) V as shown in SEQ ID NO: 32 H and V as shown in SEQ ID NO:33 L ; (m) V as shown in SEQ ID NO: 36 H and V as shown in SEQ ID NO:37 L ; (n) V as shown in SEQ ID NO: 38 H and V as shown in SEQ ID NO:39 L .

3. The isolated antibody of claim 1 or claim 2, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

4. The isolated antibody of claim 1 or claim 2, wherein the antibody is a single chain antibody (scFv).

5. The isolated antibody according to claim 1, wherein the antibody is a single-chain antibody, and the single-chain antibody is represented by the amino acid sequence of any one of SEQ ID NOs: 40-41, 43-49, 54-59.

6. A nucleic acid or a group of nucleic acids which collectively encode the antibody according to claim 1.

7. The nucleic acid or nucleic acid set according to claim 6, wherein the antibody comprises: (a) V as shown in SEQ ID NO: 34 H and V as shown in SEQ ID NO:35 L ; (b) V as shown in SEQ ID NO: 3 H and V as shown in SEQ ID NO:4 L ; (c) V as shown in SEQ ID NO: 5 H and V as shown in SEQ ID NO:6 L ; (d) V as shown in SEQ ID NO: 8 H and V as shown in SEQ ID NO:9 L ; (e) V as shown in SEQ ID NO: 10 H and V as shown in SEQ ID NO:11 L ; (f) V as shown in SEQ ID NO: 12 H and V as shown in SEQ ID NO: 13 L ; (g) V as shown in SEQ ID NO: 14 H and V as shown in SEQ ID NO: 15 L ; (h) V as shown in SEQ ID NO: 16 H and V as shown in SEQ ID NO: 17 L ; (i) V as shown in SEQ ID NO: 18 H and V as shown in SEQ ID NO: 19 L ; (j) V as shown in SEQ ID NO: 28 H and V as shown in SEQ ID NO: 29 L ; (k) V as shown in SEQ ID NO: 30 H and V as shown in SEQ ID NO:31 L ; (1) V as shown in SEQ ID NO: 32 H and V as shown in SEQ ID NO:33 L ; (m) V as shown in SEQ ID NO: 36 H and V as shown in SEQ ID NO:37 L ; (n) V as shown in SEQ ID NO: 38 H and V as shown in SEQ ID NO:39 L .

8. The nucleic acid according to claim 7, wherein the nucleic acid encodes a scFv, wherein the scFv is represented by the amino acid sequence of any one of SEQ ID NOs: 40-41, 43-49, 54-59.

9. The nucleic acid or nucleic acid set according to any one of claims 6 to 8, which is a vector or a vector set.

10. The nucleic acid or nucleic acid set according to claim 9, wherein the vector is an expression vector.

11. A host cell comprising the nucleic acid or nucleic acid group according to any one of claims 6 to 10.

12. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5, a nucleic acid or a nucleic acid group encoding the antibody, or a host cell comprising the nucleic acid or a nucleic acid group, and a pharmaceutically acceptable carrier.

13. Use of an antibody according to any one of claims 1 to 5, a nucleic acid or a nucleic acid group encoding the antibody, or a pharmaceutical composition comprising the antibody in the preparation of a medicament for cancer in a subject, wherein the subject is a human patient with CD22-positive leukemia cells or lymphoma, and the lymphoma is a B-cell non-Hodgkin lymphoma (NHL).

14. The use according to claim 13, wherein the subject is a human patient suffering from leukemia or lymphoma, the leukemia is acute lymphocytic leukemia or chronic lymphocytic leukemia (CLL), and the lymphoma is B-cell non-Hodgkin lymphoma (NHL).

15. Use of the antibody according to any one of claims 1 to 5 in the preparation of a kit for in vitro detection of the presence of CD22, wherein the detection comprises: (i) contacting the antibody with a sample suspected of containing CD22, and (ii) detecting the binding of the antibody to CD22.

16. The use according to claim 15, wherein the antibody is conjugated to a detectable label.

17. The use according to claim 15, wherein the CD22 is expressed on the cell surface.

18. A method for producing an antibody that binds to CD22, comprising: (i) culturing the host cell according to claim 11 under conditions that allow expression of the antibody that binds to CD22; and (ii) harvesting the antibodies thus produced from the cell culture.

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