Anti-cd3 antibodies and uses thereof

Abstract

Novel antibodies and antibody fragments that specifically bind CD3 and compositions containing the antibodies or antibody fragments. Nucleic acids encoding the antibodies or antibody fragments thereof and host cells containing the same, and related uses, and therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

TECHNICAL FIELD

[0001] The present application relates to novel humanized antibodies and antibody fragments that specifically bind to CD3 and compositions containing the same. Furthermore, the present application also relates to bispecific antibodies against CD3 and other antigens. Further, the present application relates to nucleic acids encoding the antibodies or antibody fragments thereof and host cells comprising the same, and related uses. Furthermore, the present application relates to therapeutic and diagnostic uses of these antibodies and antibody fragments. BACKGROUND

[0002] CD3 (cluster of differentiation 3) is a protein complex that, together with T cell antigen receptor alpha, T cell antigen receptor beta and two zeta chains, forms the T cell receptor complex and is involved in the activation of cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells).

[0003] The CD3 protein complex is a definitive marker of the T cell lineage, and thus anti-CD3 antibodies can be effectively used as T cell markers.

[0004] CD3 antibodies recognize all T cells, and react with 70-80% of human peripheral blood lymphocytes and 65-85% of thymocytes. Upon activation, CD3 antibodies direct T cells to the vicinity of tumor cells, and the contact between the two types of cells forms a synapse, triggering the activation of the T cell receptor (TCR) signaling pathway, the expression and release of granzymes, and the resulting perforation of the tumor cell membrane, leading to lysis and apoptosis of the latter. The activation of the TCR signaling pathway also causes the expression and release of a series of cytokines, such as IL-2, which stimulates the proliferation of T cells, amplifying the immune killing effect. Preclinical data have shown that CD3 bispecific antibody molecules targeting tumor-associated antigens can effectively activate T cells in the presence of target cells, stimulate their proliferation, and cause the death of target cells.

[0005] A variety of antibody molecules that bind to CD3 are now known, particularly bispecific antibody molecules comprising a CD3 binding specificity. The currently available published CD3 antibodies are all from hybridoma platform murine antibodies in the 1980s, including the following: OKT3, TR66, UCHT1, L2K and SP34, etc. The species cross-reactivity of CD3 monoclonal antibodies is crucial for the development of CD3 bispecific antibodies.

[0006] The affinity of the CD3 antibody to the CD3 complex is the first key factor for the success of the CD3 -related bispecific antibody. A CD3 antibody with too high affinity can cause non-specific activation of T cells and thus unnecessary cytokine release syndrome on the one hand, and on the other hand, in the body, it can preferentially target peripheral T cells and less affect tumor cells, thus leading to a decrease in drug efficacy. However, a CD3 antibody with too low affinity is not enough to activate T cells to kill tumor cells. It is necessary to adjust the CD3 affinity of the CD3 -related bispecific antibody according to the molecular weight, expression level, antibody epitope and tissue distribution characteristics of different tumor-associated antigens.

[0007] Therefore, there is a need in the art to develop anti-CD3 monoclonal antibodies with different binding affinities, and thus different T cell activation abilities, which can be used to develop multispecific antibodies, such as bispecific antibodies or trispecific antibodies, etc. that meet different tumor-associated antigens. SUMMARY

[0008] In some aspects, the present application relates to an antibody or antigen binding fragment thereof that binds CD3, comprising 3 heavy chain variable region CDRs and 3 light chain variable region CDRs as described herein.

[0009] In some aspects, the antibody or antigen binding fragment thereof that binds CD3 of the present application comprises a heavy chain variable region and / or a light chain variable region as described herein.

[0010] In some aspects, the antibody or antigen binding fragment thereof that binds CD3 of the present application further comprises a heavy chain constant region and / or a light chain constant region as described herein.

[0011] In some embodiments, the antibody or antigen binding fragment thereof that binds CD3 of the present application binds to a CD3 antigen, such as human or cynomolgus CD3, with a plurality of binding affinities, such as with a lower binding affinity, such as with a non-detectable binding affinity. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 : Figure 1 A-1C shows the binding affinity of the sp34 humanized antibody to human CD3 at the cellular level, Figure 1 D shows the cellular level binding of humanized antibodies with different CD3 affinities.

[0013] Figure 2 : sp34 humanized antibody T cell activation experiment

[0014] Figure 3 : Figure 3 A and Figure 3B shows the binding affinity of sp34 humanized antibody CDR region mutants to human CD3 at the cellular level, Figure 3 C further shows the cellular level affinity of partial CDR mutants to CD3.

[0015] Figure 4 T cell activation experiment of sp34 humanized antibody CDR mutants.

[0016] Figure 5 Figure 5 A is a schematic diagram of Her2 / CD3 bispecific antibody molecule; Figure 5 B shows the T cell activation ability of the bispecific antibody molecule.

[0017] Figure 6 Figure 6 A is a schematic diagram of CD70 / CD3 bispecific antibody molecule; Figure 6 B shows the T cell activation ability of the bispecific antibody molecule.

[0018] Figure 7 A schematic diagram of CD3 / Claudin18.2 bispecific antibody structure is shown.

[0019] Figure 8 The specific killing of CLDN18.2-positive gastric cancer cells NUGC-4 by the bispecific antibody of the present application is shown.

[0020] Figure 9 The specific killing of CLDN18.2-positive pancreatic cancer cells DAN-G-CLDN18.2 by the bispecific antibody is shown.

[0021] Figure 10 The non-specific killing of cells negative for CLDN18.2 expression by the bispecific antibody is shown.

[0022] Figure 11 The bispecific antibody-dependent T cell-mediated cytokine release in NUGC-4 is shown.

[0023] Figure 12 The bispecific antibody-dependent T cell-mediated cytokine release in DAN-G-CLDN18.2 is shown.

[0024] Figure 13 The CLDN18.2 expression-dependent bispecific antibody-mediated T cell activation is shown.

[0025] Figure 14 The in vivo efficacy results of the bispecific antibody in the NUGC-4 gastric cancer humanized model are shown.

[0026] Figure 15 ​​In vivo efficacy results of bispecific antibodies in a DAN-G-CLDN18.2 pancreatic cancer humanized model are shown.

[0027] Figure 16 PK of bispecific antibodies in mice is shown. DETAILED DESCRIPTION

[0028] I. DEFINITIONS

[0029] Before the present application is described in detail below, it is to be understood that this application is not limited to the particular methodology, protocols and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0030] To interpret the description, the following definitions will apply and whenever appropriate, terms used in the singular will include the plural and vice versa. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0031] The term "about" when used in connection with a numerical value means a range of numerical values that has a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0032] As used herein, the term "and / or" means either or both of the items it connects.

[0033] As used herein, the term "comprising" or "including," means including, but not limited to, whatever follows the term. In this document, when we refer to something being "coupled" to something else, it means that the two are attached to each other. Although the use of "coupled" is intended to refer to an indirect connection, where the intervening component(s) do not materially modify the informational or electrical connection between the enabling component(s) and the disabled component(s), the term "connected" is used to refer to a direct connection between the enabling component(s) and the disabled component(s), where it is understood that using "connected" also means "coupled".

[0034] The term "CD3" as used herein refers to an antigen expressed on T cells as part of the multi-molecular T cell receptor (TCR) and is composed of a homodimer or heterodimer of two chains of the following four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon n (hCD3s) comprises the amino acid sequence set forth in UniProtKB / Swiss-Prot: P07766.2. Human CD3-delta (hCD3s) comprises the amino acid sequence set forth in UniProtKB / Swiss-Prot: P04234.1. In some embodiments, CD3 as described herein refers to CD3 from a human or cynomolgus monkey.

[0035] The term "antibody that binds to CD3" or "anti-CD3 antibody" as used herein includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta) or bind thereto, as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). Antibodies and antigen-binding fragments of the application can bind to soluble CD3, to CD3 bound and / or cell surface-expressed CD3. Soluble CD3 includes native CD3 proteins as well as recombinant CD3 protein variants, e.g., monomeric and dimeric CD3 structures that lack a transmembrane region or otherwise do not bind to a cell membrane. The present application provides antibodies that bind to human and cynomolgus monkey CD3 with low or undetectable binding affinity, thereby enabling activation of human and cynomolgus monkey T cells. In some embodiments, the binding is measured, e.g., by radioimmunoassay (RIA), Bio-Layer Interferometry (BLI), MSD assay, or surface plasmon resonance (SPR) or flow cytometry.

[0036] The term "cell surface-expressed CD3" refers to one or more CD3 proteins expressed on the surface of a cell, either in vivo or in vitro, such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and accessible to an antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes CD3 proteins that are included in the context of a functional T cell receptor in a cell membrane. The term "cell surface-expressed CD3" includes CD3 proteins expressed as a homodimer or heterodimer (e.g., delta / epsilon, gamma / epsilon, and zeta / zeta CD3 dimers) portion on the surface of a cell.

[0037] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Thl, Th2, and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes).

[0038] An "anti-CD3 antibody" or "antibody that binds CD3" includes monovalent antibodies with a single specificity, as well as bispecific antibodies comprising a first antigen binding domain that binds CD3 and a second antigen binding domain that binds a second (target) antigen, and also includes multispecific antibodies that bind CD3 and one or more (e.g. two) other targets.

[0039] The term "multispecific antibody" refers to an antibody that is at least bispecific, i.e. the antibody comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds one target or antigen and the second binding domain binds another antigen or target. Thus, an antibody according to the application comprises a specificity for at least two different antigens or targets. An antibody according to the application also encompasses multispecific antibodies comprising multiple binding domains / binding sites, such as trispecific antibodies, wherein the antibody comprises three binding domains.

[0040] The term "linker" as used herein refers to any molecule that enables the direct linkage of different parts of a bispecific antibody. Examples of linkers that establish a covalent linkage between the different antibody parts include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or copolymers of polyethylene glycol, polypropylene glycol.

[0041] The term "peptide linker" according to the application refers to a sequence of amino acids, wherein the sequence links the amino acid sequence of a first part of an antibody to a second part of an antibody. For example, a peptide linker can link a first (variable and / or binding) domain of an antibody to a second variable and / or binding) domain. For example, a peptide linker can also link a part of an antibody to another part of an antibody, such as an antigen binding domain to an Fc domain or fragment thereof. Preferably, the peptide linker has a length that is sufficient to link two entities in such a way that they maintain their conformation relative to each other in a way that the desired activity is not hampered.

[0042] The term "valency" according to the application denotes the presence of a specified number of binding sites in an antibody molecule. Thus, the terms bivalent, trivalent, tetravalent, respectively, denote the presence of two, three or four binding sites in an antibody construct. Bispecific antibodies according to the application are at least bivalent and can be multivalent, e.g. bivalent, trivalent, tetravalent or hexavalent.

[0043] The term "binding region" as used herein refers to any part of a bispecific antibody that binds a specific target or antigen. A binding region is an antigen binding site. A binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a binding region can or can not have a tertiary structure independent of the remainder of the BsAB, and can or can not bind its target as a separate entity.

[0044] The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen binding fragment.

[0045] An "antigen binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibody (e.g., scFv); single domain antibodies such as VHH; diabodies or fragments thereof; or a camelid antibody.

[0046] The term "antigen" refers to a molecule that elicits an immune response. This immune response can involve either antibody production or the activation of specific immune cells, or both. The skilled artisan will understand that any macromolecule, including essentially any protein or peptide, can serve as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA. The term "epitope" as used herein refers to the portion of an antigen (e.g., CD3) that specifically interacts with an antibody molecule.

[0047] An antibody that "binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay, and vice versa.

[0048] An antibody that "competes with" a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. And vice versa, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay.

[0049] An antibody that "inhibits" (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen. And vice versa, the reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen. Binding of an antibody to its antigen can be measured in terms of affinity (e.g., the equilibrium dissociation constant). Methods of determining affinity are known in the art.

[0050] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0051] A "complementarity determining region" or "CDR region" or "CDR" is a region of an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of a heavy chain and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering with a large number of crystal structures.

[0052] For example, depending on the different CDR determination scheme, the residues of each CDR are as follows.

[0053]

[0054] CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence, such as any of the exemplary CDRs of the application.

[0055] Unless otherwise indicated, in the application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above.

[0056] Unless otherwise indicated, in the application, when referring to residue positions in an antibody variable region, including heavy chain variable region residues and light chain variable region residues, reference is made to the numbering positions according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0057] In one embodiment, the CDRs of the heavy chain variable region of an antibody of the application are determined according to the following rules

[0058] VH CDR1 is determined according to the AbM rules; and both VH CDR2 and 3 are determined according to the Kabat rules.

[0059] In one embodiment, the CDRs of the light chain variable region of an antibody of the application are determined according to the Kabat rules.

[0060] In one embodiment, the CDRs of the heavy chain variable region of an antibody of the application are determined according to the following rules: VH CDR1 is determined according to the AbM rules; and both VH CDR2 and 3 are determined according to the Kabat rules; and the CDRs of the light chain variable region are determined according to the Kabat rules.

[0061] It should be noted that the boundaries of the CDRs of the variable region of the same antibody can vary depending on the different assignment system. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Therefore, when referring to an antibody defined by a specific CDR sequence defined in the application, the scope of the antibody also encompasses an antibody whose variable region sequence contains the specific CDR sequence, but the claimed CDR boundaries thereof are different from the specific CDR boundaries defined in the application due to the application of different schemes (e.g., different assignment system rules or combinations).

[0062] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although the CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlap region can be determined, providing a "minimal binding unit" for antigen binding. The minimal binding unit can be a sub-portion of a CDR. As will be apparent to those in the art, by the structure and protein folding of an antibody, the identity of the residues of the remainder of the CDR sequence can be determined. Thus, variants of any of the CDRs given herein are also contemplated by the application. For example, in a variant of one CDR, the amino acid residues of the minimal binding unit can remain unchanged, while the remainder of the CDR residues according to the Kabat or Chothia definition can be replaced with conservative amino acid residues.

[0063] The term "Fc region" is used herein to define the constant regions of an immunoglobulin heavy chain that comprise the CH2 and CH3 domains. The term includes native-sequence Fc regions and variant Fc regions. Native-sequence Fc regions are capable of binding to Fc receptors on the surface of immune cells, thereby enabling CDC\ADCC\ADCP effector functions. Such effector functions generally require the Fc region to be associated with a binding domain (e.g., an antibody variable domain). In some embodiments, the Fc region is mutated to enhance its CDC\ADCC\ADCP effector functions. In some embodiments, the Fc region is mutated to impair or delete its CDC\ADCC\ADCP effector functions.

[0064] An "IgG form of an antibody" refers to the IgG form to which the heavy chain constant region of the antibody belongs. The heavy chain constant region of all antibodies of the same type is identical, and differs between antibodies of different types. For example, an antibody of IgG4 form refers to an antibody whose heavy chain constant region is from IgG4, or an antibody of IgG1 form refers to an antibody whose heavy chain constant region is from IgG1.

[0065] A "humanized" antibody refers to an antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0066] The "knobs-into-holes" technology is described in, for example, US 5,731,168; US 7,695,936. Generally, the method involves introducing a knob at the interface of a first polypeptide and a corresponding hole in the interface of a second polypeptide, such that the protuberance can fit into the socket, thereby promoting heterodimer formation and impeding homodimer formation. Knobs are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains, such as tyrosine or tryptophan. A compensatory hole of the same or similar size as the knob is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones, such as alanine or threonine. The knobs and holes can be generated by altering the nucleic acid encoding the polypeptides, for example, by site-specific mutagenesis, or by peptide synthesis.

[0067] The term "binds" or "binds specifically" as used herein means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or biolayer interferometry assay or MSD assay or surface plasmon resonance (SPR).

[0068] An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a cytotoxic agent or a label.

[0069] The term "therapeutic agent" as described herein encompasses any substance that is effective in the prevention or treatment of a tumor, e.g., a cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0070] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0071] A "chemotherapeutic agent" includes a chemical compound useful in the treatment of diseases of the immune system.

[0072] The term "small molecule drug" refers to an organic compound of low molecular weight that is capable of modulating a biological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can penetrate cells more readily than macromolecules, be less susceptible to degradation, and be less likely to elicit an immune response.

[0073] The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response can be a humoral response or a cellular response. Immune modulators include immunosuppressants.

[0074] An "immunosuppressant," "immunosuppressive drug," or "immunosuppressive agent" as used herein is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

[0075] The term "effective amount" refers to the amount or dose of an antibody or fragment or conjugate or composition or combination of the application, which elicits the intended effect in a patient in need of treatment or prevention, either as a single dose or as repeated doses.

[0076] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition or combination thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70% relative to an untreated subject.

[0077] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Since the prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

[0078] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which an exogenous nucleic acid has been introduced, including the progeny of the cell that has been transformed. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny can not be completely identical to the parent cell both in nucleic acid content and in physical identity, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein.

[0079] The term "label" as used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or antibody, and that facilitates detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze chemical changes to a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody and indirect labeling by conjugating the probe or antibody to a secondary reagent which itself is labeled with a detectable substance.

[0080] An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0081] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).

[0082] An "isolated nucleic acid encoding an anti-CD3 antibody or fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy chain or light chain (or fragment thereof, e.g., a heavy chain variable region or a light chain variable region), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0083] Calculations of sequence identity between sequences are performed as follows.

[0084] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first and second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one preferred embodiment, for comparison purposes, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence then the molecules are identical at that position.

[0085] Sequence comparisons and percent identity calculations between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) as implemented in the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap length penalty of 1, 2, 3, 4, 5, or 6, is used to determine percent identity between two amino acid sequences. In another preferred embodiment, the GAP program (available at http: / / www.gcg.com) in the GCG software package is used to determine percent identity between two nucleotide sequences using a NWSgapdna.CMP matrix and a gap penalty of 40, 50, 60, 70, or 80 and a gap length penalty of 1, 2, 3, 4, 5, or 6. An especially preferred set of parameters (and the set that should be used unless otherwise indicated) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Additionally or alternatively, nucleic acid sequences and protein sequences described herein can be further used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.

[0086] As used herein, the term "hybridize under stringent conditions" (e.g., under low stringency, medium stringency, high stringency, or very high stringency conditions) describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. Both aqueous and nonaqueous methods are described in the reference and either method can be used. Preferred hybridization conditions mentioned herein are as follows: 1) low stringency hybridization conditions are in about 45°C in 6X sodium chloride / sodium citrate (SSC), followed by at least one wash in 0.2X SSC, 0.1% SDS at 50°C (for low stringency conditions, the temperature can be increased to 55°C); 2) medium stringency hybridization conditions are in about 45°C in 6X SSC, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions are in about 45°C in 6X SSC, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are in about 65°C in 0.5M sodium phosphate, 7% SDS, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Very high stringency conditions (4) are preferred conditions and the conditions that should be used unless otherwise indicated.

[0087] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0088] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and liquid tumors.

[0089] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the application include gastric or pancreatic cancer, including metastatic forms of those cancers.

[0090] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive as referred to herein.

[0091] As used herein, a "tumor-associated antigen" refers to an antigenic determinant presented on the surface of a target cell, wherein the target cell is a cell in a tumor, such as a cancer cell, a cell of the tumor stroma. In certain aspects, the tumor-associated antigen is HER2 or CD70 or CLAUDIN18.2.

[0092] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active agent is administered.

[0093] The term "pharmaceutical composition" refers to a composition that is in a form that is effective for the biological activity of the active ingredient contained therein, and that contains no additional ingredients that are unacceptable to the subject to which the composition is administered.

[0094] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an anti-CD3 antibody or fragment thereof, and (ii) another therapeutic agent) are administered to a patient as separate entities either simultaneously, without specific time limitations, or sequentially with no specific time limitations, wherein such administration provides therapeutically effective levels of the two or more active agents in the body of the patient. In some embodiments, the anti-CD3 antibody or fragment thereof and the other therapeutic agent used in the pharmaceutical combination are administered at levels that would not be effective in the absence of the other. The term "fixed combination" means that the two or more active agents are administered to a patient as a single entity. Preferably, the dosage of each active agent and / or the time interval of administration is / are selected so that the use of the combination results in a synergistic effect in treating the disease or condition. The individual components can each be present in a separate formulation that can or can not be identical.

[0095] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities (e.g., radiotherapy or surgery), to treat a disease described herein. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses use of a multiple- or separate- container kit, wherein each active ingredient is administered contiguously or sequentially with the other. The powdered and / or liquid containers can be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent at approximately the same time, or at different sequential times. In any case, the treatment regimen will provide therapeutically effective levels of the drugs in the body of the patient over the course of treatment.

[0096] As used herein, "treat," ...

[0097] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0098] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0099] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0100] II. Antibodies

[0101] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention bind to CD3 (e.g., human CD3 or cynomolgus CD3) with a desired affinity. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both human CD3 and cynomolgus CD3. In some embodiments, the affinity of the antibodies is determined by thin-layer interferometry or surface plasmon resonance.

[0102] In some embodiments, the anti-CD3 antibodies of the present invention have an equilibrium dissociation constant (K D ) binds to human CD3 or cynomolgus monkey CD3, the K Dbetween 0.5 nM and 200 nM, preferably between 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, or 50 nM to 180 nM, 190 nM, or 200 nM, for example between 100 nM and 200 nM. In some embodiments, the anti-CD3 antibody of the application has a Kd between 10 nM and 150 nM, or between 10 nM and 120 nM, or between 10 nM and 100 nM. D binds to a human CD3E&G complex or a human CD3E&D complex. In some embodiments, the anti-CD3 antibody of the application binds to human or cynomolgus CD3 with an affinity that is not detectable.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof of the application binds to CD3 on the surface of an effector cell. In some embodiments, the antibody or antigen-binding fragment thereof of the application is capable of activating an effector cell. In some embodiments, the effector cell is a T cell, for example a T lymphocyte, or a CD4+ T cell, or a CD8+ T cell. In some embodiments, the binding is detected using flow cytometry.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof of the application is capable of activating an effector cell to induce killing of a tumor cell.

[0105] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the application comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3.

[0106] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the application comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3.

[0107] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the application comprises 3 complementarity determining regions from a heavy chain variable region (HCDR) and 3 complementarity determining regions from a light chain variable region (LCDR).

[0108] In some aspects, the anti-CD3 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH). In some aspects, the anti-CD3 antibody or antigen-binding fragment thereof of the present application comprises a light chain variable region (VL). In some aspects, the anti-CD3 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions (CDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementarity determining regions (CDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3.

[0109] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present application further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present application further comprises an antibody light chain constant region LC. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present application further comprises a heavy chain constant region HC and a light chain constant region LC.

[0110] In some embodiments, the heavy chain variable region of the present application

[0111] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-75; or

[0112] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-75; or

[0113] (iii) comprises or consists of an amino acid sequence that has 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-75, preferably the amino acid alterations do not occur in the CDR regions.

[0114] In some embodiments, the light chain variable region of the present application

[0115] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-99; or

[0116] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-99; or

[0117] (iii) comprises or consists of an amino acid sequence which has 1 or more, preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1, amino acid alterations, preferably amino acid substitutions, more preferably amino acid conservative substitutions, compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-99, preferably the amino acid alterations do not occur in the CDR regions.

[0118] In some embodiments, the 3 complementarity determining regions from the heavy chain variable region (HCDRs), HCDR1, HCDR2 and HCDR3 of the present application are selected from the group consisting of

[0119] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as shown in any one of SEQ ID NOs: 50-75, or

[0120] (ii) a sequence which comprises, in total, at least one and not more than 5, 4, 3, 2 or 1 amino acid alterations, preferably amino acid substitutions, preferably conservative substitutions, in the three HCDR regions, relative to the sequence of any one of (i).

[0121] In some embodiments, the 3 complementarity determining regions from the light chain variable region (LCDRs), LCDR1, LCDR2 and LCDR3 of the present application are selected from the group consisting of

[0122] (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as shown in any one of SEQ ID NOs: 85-99, or

[0123] (ii) a sequence which comprises, in total, at least one and not more than 5, 4, 3, 2 or 1 amino acid alterations, preferably amino acid substitutions, preferably conservative substitutions, in the three LCDR regions, relative to the sequence of any one of (i).

[0124] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 22, or the HCDR1 comprises an amino acid sequence which has one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 22.

[0125] In some embodiments, the HCDR1 of the present application comprises or consists of the amino acid sequence of SEQ ID NO: 103, wherein the amino acid sequence of SEQ ID NO: 103 is as shown below:

[0126] GFTFX1X2X3AMN (SEQ ID NO: 103), wherein

[0127] X1 is selected from N, G, S, D or E, preferably N, G or S;

[0128] X2 is selected from T, G, L or R, preferably T or G;

[0129] X3 is selected from Y, G, A or S, preferably Y, A or S,

[0130] SEQ ID NO: 103 differs from SEQ ID NO: 1 at 1, 2 or 3 amino acids.

[0131] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 7, 9, 10, 11, 12, 23 or 24, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2, 7, 9, 10, 11, 12, 23 or 24.

[0132] In some embodiments, the HCDR2 of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 104, wherein the amino acid sequence of SEQ ID NO: 104 is as follows:

[0133] RIX1X2KX3X4X5YATYYADSVKD (SEQ ID NO: 104), wherein

[0134] X1 is selected from R, G, A or S, preferably R or S;

[0135] X2 is selected from S, G, L or R, preferably S or L;

[0136] X3 is selected from Y, G, A or S, preferably Y or A;

[0137] X4 is selected from N, G, S, D or E, preferably N or G;

[0138] X5 is selected from N, G, S, D or E, preferably N or G,

[0139] SEQ ID NO: 104 differs from SEQ ID NO: 2 at 1, 2 or 3 amino acids.

[0140] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13-21, 25-28, or the HCDR3 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13-21, 25-28.

[0141] In some embodiments, the HCDR3 of the present application comprises or consists of the amino acid sequence of SEQ ID NO: 105, wherein the amino acid sequence of SEQ ID NO: 105 is as follows:

[0142] X1X2X3X4X5X6X7X8X9SWFAY (SEQ ID NO: 105), - wherein

[0143] X1is selected from H, G, A or S, preferably H or A;

[0144] X2is G or Y;

[0145] X3is selected from N, G, S, D or E, preferably N or G;

[0146] X4is selected from F, G, A or S, preferably F or A;

[0147] X5is G or Y;

[0148] X6is selected from N, G, S, D or E, preferably N, Q or G;

[0149] X7is selected from S, G, L or R, preferably S or R;

[0150] X8is selected from Y, G, A or S, preferably Y or A;

[0151] X9is selected from V or A;

[0152] and SEQ ID NO: 105 differs from SEQ ID NO: 3 in 1, 2 or 3 amino acids.

[0153] In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 29, 32-36, 41 and 42, or the LCDR1 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of any one of SEQ ID NOs: 29, 32-36, 41 and 42.

[0154] In some embodiments, the LCDR1 of the application comprises or consists of the amino acid sequence of SEQ ID NO: 106, wherein the amino acid sequence of SEQ ID NO: 106 is shown below:

[0155] X1 SSTGAV X2X3X4YAN (SEQ ID NO: 106), - wherein

[0156] X1 is selected from R, G, A or S, preferably R or G;

[0157] X2 is selected from T, G, L or R, preferably T or G;

[0158] X3 is selected from T, G, L or R, preferably T or G;

[0159] X4 is selected from S, G, L or R, preferably S or R;

[0160] and SEQ ID NO: 106 differs from SEQ ID NO: 29 in 1, 2 or 3 amino acids.

[0161] In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 30 or 45, or the LCDR2 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 30 or 45.

[0162] In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 31, 37, 38, 39, 40, 43, 44 or 46, or the LCDR3 comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 31, 37, 38, 39, 40, 43, 44 or 46.

[0163] In some embodiments, the LCDR3 of the application comprises or consists of the amino acid sequence of SEQ ID NO: 107, wherein the amino acid sequence of SEQ ID NO: 107 is shown below:

[0164] ALX1 X2X3X4LWV (SEQ ID NO: 107), - wherein

[0165] X1 is selected from W, G, A or S, preferably W or A;

[0166] X2 is selected from Y, G, A or S, preferably Y or A;

[0167] X3 is selected from S, G, L or R, preferably S or R;

[0168] X4 is selected from N, G, S, D or E, preferably N, G or D;

[0169] and SEQ ID NO: 107 differs from SEQ ID NO: 31 at 1, 2 or 3 amino acids.

[0170] In some embodiments, the antibody heavy chain constant region HC of the present application is a heavy chain constant region of IgGl, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgGl, for example an IgGl constant region with LALA mutations. In some embodiments, the antibody light chain constant region LC of the present application is a Lambda or Kappa light chain constant region, preferably a Lambda light chain constant region.

[0171] In some preferred embodiments, the antibody heavy chain constant region HC of the present application is

[0172] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 100;

[0173] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 100; or

[0174] (iii) comprises or consists of an amino acid sequence which has 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 100.

[0175] In some embodiments, the antibody light chain constant region LC of the present application is

[0176] (i) comprises or consists of an amino acid sequence which has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 101 or 102;

[0177] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 101 or 102; or

[0178] (iii) comprises or consists of an amino acid sequence which has 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 101 or 102.

[0179] In some embodiments of the application, the anti-CD3 antibody or antigen binding fragment thereof of the application comprises:

[0180] (i) a heavy chain variable region VH which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 47-51; and

[0181] (ii) a light chain variable region VL which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 76-84.

[0182] In some embodiments of the application, the anti-CD3 antibody or antigen binding fragment thereof of the application comprises:

[0183] (i) a VH which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47 or an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a VL which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76 or an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0184] (ii) a VH which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48 or an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a VL which comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 77-84 or an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0185] (iii) a VH comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 49 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 77-84 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0186] (iv) a VH comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 50 or an amino acid sequence that is at least 90% identical thereto, and a VL comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 77-84 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0187] (v) a VH comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 51 or an amino acid sequence that is at least 90% identical thereto, and a VL comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 77-84 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0188] In some embodiments of the application, the anti-CD3 antibody or antigen binding fragment thereof of the application comprises:

[0189] (i) a heavy chain variable region VH comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 47-51, wherein the amino acid sequence has 1, 2, or 3 mutations at positions H31, H32, H33, H52, H52A, H52C, H53, H54, H95, H96, H97, H98, H99, H100, H100A, H100B, H100C (Kabat number) that are selected from the following mutations: amino acid Y, W, or F mutated to amino acid G, A, S; amino acid R, K, or H mutated to amino acid G, A, or S; amino acid G mutated to amino acid Y; amino acid N or Q mutated to amino acid G, S, D, or E; and / or amino acid T or S mutated to G, L, R, as compared to the amino acid sequence of any one of SEQ ID NOs: 47-51;

[0190] (ii) a light chain variable region VL comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 76-84, wherein 1, 2, or 3 of L24, L28, L29, L30, L31, L53, L91, L92, L93, L94 (Kabat number) have a mutation selected from the group consisting of:

[0191] amino acid Y, W, or F is mutated to amino acid G, A, S; amino acid R, K, or H is mutated to amino acid G, A, or S; amino acid G is mutated to amino acid Y; amino acid N or Q is mutated to amino acid G, S, D, or E; and / or amino acid T or S is mutated to G, L, R.

[0192] In some embodiments of the application, the anti-CD3 antibody or antigen binding fragment thereof of the application comprises:

[0193] (i) a heavy chain variable region VH comprising or consisting of an amino acid sequence of SEQ ID NO: 50, and wherein 1, 2, or 3 of H31, H32, H33, H52, H52A, H52C, H53, H54, H95, H96, H97, H98, H99, H100, H100A, H100B, H100C (Kabat number) have a mutation selected from the group consisting of:

[0194] amino acid Y, W, or F is mutated to amino acid G, A, S; amino acid R, K, or H is mutated to amino acid G, A, or S; amino acid G is mutated to amino acid Y; amino acid N or Q is mutated to amino acid G, S, D, or E; and / or amino acid T or S is mutated to G, L, R;

[0195] (ii) a light chain variable region VL comprising or consisting of an amino acid sequence of SEQ ID NO: 80, and wherein 1, 2, or 3 of L24, L28, L29, L30, L31, L53, L91, L92, L93, L94 (Kabat number) have a mutation selected from the group consisting of:

[0196] amino acid Y, W, or F is mutated to amino acid G, A, S; amino acid R, K, or H is mutated to amino acid G, A, or S; amino acid G is mutated to amino acid Y; amino acid N or Q is mutated to amino acid G, S, D, or E; and / or amino acid T or S is mutated to G, L, R.

[0197] In some embodiments of the application, the anti-CD3 antibody or antigen binding fragment thereof of the application comprises:

[0198] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in any one of SEQ ID NOs: 52-75, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 80;

[0199] (ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 50, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in any one of SEQ ID NOs: 85-99.

[0200] In some embodiments of the application, the anti-CD3 antibody or antigen-binding fragment thereof of the application comprises:

[0201] (1) HCDR1 as set forth in any one of SEQ ID NOs: 4-6, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31;

[0202] (2) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 7, 9, 10, 11 or 12, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31;

[0203] (3) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in any one of SEQ ID NOs: 13-21; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31;

[0204] (4) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 23 or 24, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31;

[0205] (5) HCDR1 as set forth in SEQ ID NO: 22, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31 ;

[0206] (6) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in any one of SEQ ID NOs: 25-28; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31 ;

[0207] (7) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in any one of SEQ ID NOs: 32-36, 41, and 42, LCDR2 as set forth in any one of SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31 ;

[0208] (8) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in any one of SEQ ID NO: 30, and LCDR3 as set forth in any one of SEQ ID NOs: 37-40, 43, and 44; or

[0209] (9) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in any one of SEQ ID NO: 45, and LCDR3 as set forth in SEQ ID NO: 31 or 46.

[0210] In some embodiments of the application, the anti-CD3 antibody or antigen-binding fragment thereof of the application comprises:

[0211] (1) HCDR1 as depicted in SEQ ID NO: 103, HCDR2 as depicted in SEQ ID NO: 104, HCDR3 as depicted in SEQ ID NO: 105; LCDR1 as depicted in SEQ ID NO: 29, LCDR2 as depicted in SEQ ID NO: 30, and LCDR3 as depicted in SEQ ID NO: 31 ;

[0212] (2) HCDR1 as depicted in SEQ ID NO: 103, HCDR2 as depicted in SEQ ID NO: 2, HCDR3 as depicted in SEQ ID NO: 3 or 8; LCDR1 as depicted in SEQ ID NO: 29, LCDR2 as depicted in SEQ ID NO: 30, and LCDR3 as depicted in SEQ ID NO: 31 ;

[0213] (3) HCDR1 as depicted in SEQ ID NO: 1, HCDR2 as depicted in SEQ ID NO: 104, HCDR3 as depicted in SEQ ID NO: 3 or 8; LCDR1 as depicted in SEQ ID NO: 29, LCDR2 as depicted in SEQ ID NO: 30, and LCDR3 as depicted in SEQ ID NO: 31 ;

[0214] (4) HCDR1 as depicted in SEQ ID NO: 1, HCDR2 as depicted in SEQ ID NO: 2, HCDR3 as depicted in SEQ ID NO: 105; LCDR1 as depicted in SEQ ID NO: 29, LCDR2 as depicted in SEQ ID NO: 30, and LCDR3 as depicted in SEQ ID NO: 31 ;

[0215] (5) HCDR1 as depicted in SEQ ID NO: 103, HCDR2 as depicted in SEQ ID NO: 104, HCDR3 as depicted in SEQ ID NO: 3 or 8; LCDR1 as depicted in SEQ ID NO: 29, LCDR2 as depicted in SEQ ID NO: 30, and LCDR3 as depicted in SEQ ID NO: 31 ;

[0216] (6) HCDR1 as depicted in SEQ ID NO: 103, HCDR2 as depicted in SEQ ID NO: 2, HCDR3 as depicted in SEQ ID NO: 105; LCDR1 as depicted in SEQ ID NO: 29, LCDR2 as depicted in SEQ ID NO: 30, and LCDR3 as depicted in SEQ ID NO: 31 ;

[0217] (7) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 104, HCDR3 as set forth in SEQ ID NO: 105; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30, and LCDR3 as set forth in SEQ ID NO: 31;

[0218] (8) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3 or 8; LCDR1 as set forth in SEQ ID NO: 106, LCDR2 as set forth in SEQ ID NO: 30 or 45, and LCDR3 as set forth in SEQ ID NO: 107;

[0219] (9) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3 or 8; LCDR1 as set forth in SEQ ID NO: 106, LCDR2 as set forth in SEQ ID NO: 30 or 45, and LCDR3 as set forth in SEQ ID NO: 31;

[0220] (10) HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3 or 8; LCDR1 as set forth in SEQ ID NO: 29, LCDR2 as set forth in SEQ ID NO: 30 or 45, and LCDR3 as set forth in SEQ ID NO: 107;

[0221] (11) HCDR1 as set forth in SEQ ID NO: 103, HCDR2 as set forth in SEQ ID NO: 104, HCDR3 as set forth in SEQ ID NO: 105; LCDR1 as set forth in SEQ ID NO: 106, LCDR2 as set forth in SEQ ID NO: 30 or 45, and LCDR3 as set forth in SEQ ID NO: 107.

[0222] In some embodiments of the application, the anti-CD3 antibody or antigen-binding fragment thereof of the application comprises:

[0223] (i) a VH comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 52-75, or an amino acid sequence at least 90% identical thereto, and a VL comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 80;

[0224] (ii) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence at least 90% identical thereto, and a VL comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 85-99.

[0225] In an embodiment of the application, the amino acid alterations described herein include substitution, insertion, or deletion of an amino acid.

[0226] In preferred embodiments, the amino acid changes described herein occur in the CDR regions, such that the affinity of the antibodies of the application for CD3 is adjusted to the desired extent, particularly the extent required for the construction of a multispecific antibody, by amino acid changes in the CDR regions. In some embodiments, the number of amino acid changes in each CDR is no more than 3, 2, or 1. In some embodiments, the number of amino acid changes in the combination of heavy chain HCDRs is no more than 3, 2, or 1. In some embodiments, the number of amino acid changes in the combination of light chain HCDRs is no more than 3, 2, or 1. In some embodiments, the amino acid positions at which the above-described amino acid changes occur are selected from one or more (preferably no more than 6, more preferably no more than 3 in the combination of heavy chain CDRs, and / or no more than 3 in the combination of light chain CDRs) of the following: H31, H32, H33, H52, H52A, H52C, H53, H54, H95, H96, H97, H98, H99, H100, H100A, H100B, H100C (Kabat numbering), and L24, L28, L29, L30, L31, L53, L91, L92, L93, L94 (Kabat numbering). In preferred embodiments, the amino acid changes are amino acid substitutions, in which an aromatic amino acid Y, W, and / or F is mutated to a smaller side chain amino acid G, A, and / or S; a positively charged amino acid R, K, and / or H is mutated to a smaller side chain amino acid G, A, and / or S; an amino acid G with a hydrogen side chain is mutated to an aromatic amino acid Y; an amino acid N and / or Q with an amide side chain is mutated to an amino acid G, S, D, and / or E; a non-aromatic amino acid T and / or S with a hydroxyl side chain is mutated to G, L, R.

[0227] In preferred embodiments, the amino acid changes described herein occur outside the CDR regions (e.g., in the FRs). More preferably, the amino acid changes described herein occur outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0228] In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid for another amino acid within the same class, e.g., the substitution of one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. Exemplary substitutions are shown in the following table:

[0229]

[0230] In certain embodiments, substitutions occur in the CDR regions of the antibody. In general, the variants obtained are modifications (e.g., improvements) in certain biological properties with respect to the parent antibody (e.g., increased affinity) and / or will have certain biological properties substantially retained from the parent antibody. An exemplary substitutional variant is an affinity matured antibody.

[0231] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. When the antibody comprises an Fc region, carbohydrate attached to the Fc region can be altered. In some applications, it can be useful to remove a glycosylation site that is not desired, for example to remove a fucosyl motif to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement dependent cytotoxicity (CDC).

[0232] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to thereby generate Fc region variants, in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, complement dependent cytotoxicity, Fc receptor binding, and / or antibody dependent cellular cytotoxicity. Fc region variants can include human Fc region sequences (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid alteration (e.g., a substitution) at one or more amino acid positions.

[0233] In one embodiment of the application, an antibody described herein is altered in the Fc region to increase the ADCC activity or CDC activity of the antibody.

[0234] In certain embodiments, it can be desirable to create a cysteine engineered antibody, e.g., a "thioMAb," in which one or more residues of the antibody are substituted with cysteine residues.

[0235] In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-l,3-dioxan, poly-l,3,6-trioxan, ethylene / maleic acid copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0236] In some embodiments, the anti-CD3 antibody of the application or antigen-binding fragment thereof has one or more of the following properties:

[0237] (i) exhibits the same or similar binding affinity and / or specificity for CD3 as an antibody of the application;

[0238] (ii) inhibits (e.g., competitively inhibits) binding of an antibody of the application to CD3;

[0239] (iii) binds to the same or overlapping epitope as an antibody of the application;

[0240] (iv) competes with an antibody of the application for binding to CD3;

[0241] (v) has one or more of the biological properties of an antibody of the application.

[0242] In some embodiments, the anti-CD3 antibody of the application is an antibody of the IgGl class or an antibody of the IgG2 class or an antibody of the IgG3 class or an antibody of the IgG4 class, preferably an antibody of the IgGl class.

[0243] In some embodiments, the anti-CD3 antibody is a monoclonal antibody.

[0244] In some embodiments, the anti-CD3 antibody is humanized.

[0245] In some embodiments, at least part of the framework sequences of the anti-CD3 antibody are human consensus framework sequences.

[0246] In one embodiment, the anti-CD3 antibody of the application also encompasses antibody fragments (e.g., antigen-binding fragments) thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single domain antibody such as VHH, dAb (domain antibody), or linear antibody.

[0247] In one embodiment, the antibody fragment of the application is a scFv, comprising a VH and a VL as described herein, and a linker sequence, wherein the linker is (GGGGS) n wherein n = 1, 2, 3, 4 or 5, for example n = 4.

[0248] III. Multispecific antibodies

[0249] The CD3 antibodies described herein encompass multispecific antibodies, such as bispecific antibodies or trispecific antibodies, that bind to CD3 and another antigen or target. In one embodiment, the multispecific antibody comprises a first antigen binding domain that specifically binds to CD3, and a second antigen binding domain that specifically binds to another antigen, and optionally a third or more antigen binding domain that specifically binds to another antigen.

[0250] In some embodiments, the other antigen is a tumor associated antigen. In some embodiments, the tumor associated antigen is selected from HER2 or CD70 or CLAUDIN18.2.

[0251] In one embodiment, the antibody of the application is a bispecific antibody, comprising a first antigen binding region that specifically binds to CD3 and a second antigen binding region. In some embodiments, the second antigen binding region binds to a tumor associated antigen. In some embodiments, the tumor associated antigen is HER2 or CD70 or CLAUDIN18.2.

[0252] In some embodiments, the first antigen binding region that specifically binds to CD3 comprises a VH and / or VL as described above. In some embodiments, the first antigen binding region that specifically binds to CD3 comprises HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3 as described above.

[0253] In some embodiments, the multispecific antibody of the application further comprises a heavy chain constant region. In some embodiments, the multispecific antibody of the application further comprises a light chain constant region. In some embodiments, the multispecific antibody of the application further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions described above. In some embodiments, the light chain constant region is selected from the light chain constant regions described above. In some embodiments, the light chain constant region in the multispecific antibody of the application comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consisting of said amino acid sequence. In some embodiments, the heavy chain constant region in the multispecific antibody of the application comprises the amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or consisting of said amino acid sequence. In some embodiments, the heavy chain constant regions linked to different antigen binding regions can be the same or different. In some embodiments, the light chain constant regions linked to different antigen binding regions can be the same or different.

[0254] Any multispecific antibody format or technology can be used to make the multispecific antibodies of the application. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent associations, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a different antigen binding specificity(ies), to produce a multispecific antigen binding molecule.

[0255] In some embodiments, the bispecific antibody formats of the application comprise IgG-like and non-IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common IgG-like antibody types comprise two Fab regions and one Fc region, the heavy and light chains of each Fab can be from separate monoclonal antibodies. Non-IgG-like bispecific antibodies lack the Fc region, each of the antigen or target binding domains can be a Fab, can be a single-chain variable fragment (scFv), and can be a fusion protein mimicking the variable domains of two antibodies, the different binding domains are linked together by a peptide linker, chemical coupling, noncovalent bond, or otherwise.

[0256] Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, bispecific antibodies based on platforms such as TrioMab, CrossMab / KiH, KiH, DVD-Ig, IgG-scFv, FIT-Ig, mAb-Trap, BiTE, DART, TandAb, ImmTAC, TriKE, etc.

[0257] In some embodiments, the bispecific antibody of the present invention is a KiH-format bispecific antibody. In some embodiments, the bispecific antibody of the present invention comprises two Fabs and one Fc, wherein the first Fab comprises a first antigen-binding region that specifically binds to CD3, and the second Fab comprises a second antigen-binding region that specifically binds to a tumor-associated antigen, for example, Figure 5 The form shown in A.

[0258] In another embodiment, the bispecific antibody of the present invention comprises one Fab, one Fc, and one scFv, wherein the Fab or scFv comprises a second antigen-binding region that specifically binds to a tumor-associated antigen, and the scFv or Fab comprises a first antigen-binding region that specifically binds to CD3, e.g., Figure 6 The format shown in A. In some embodiments, the Fab comprises a second antigen-binding region that specifically binds to a tumor-associated antigen, and the scFv comprises a first antigen-binding region that specifically binds to CD3. In some embodiments, the scFv comprises a second antigen-binding region that specifically binds to a tumor-associated antigen, and the Fab comprises a first antigen-binding region that specifically binds to CD3. In some embodiments, the scFv may comprise a VH-linker-VL, or a VL-linker-VH. In some embodiments, the scFv is linked to the Fc via the VH to form a bispecific antibody. In some embodiments, the scFv is linked to the Fc via the VL to form a bispecific antibody.

[0259] In some embodiments, the linker is a peptide linker. Peptide linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful peptide linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the linker is (GGGGS)4.

[0260] In some embodiments, the Fc is from an IgG1 LALA sequence.In some embodiments, the CL comprises or consists of the amino acid sequence of SEQ ID NO: 116.

[0261] In some embodiments, the tumor-associated antigen is HER2. In some embodiments, the second antigen binding region that specifically binds HER2 is from trastuzumab.

[0262] In some embodiments, the tumor-associated antigen is CD70. In some embodiments, the second antigen binding region that specifically binds CD70 is from SGN70 of WO2004073656.

[0263] In some embodiments, the tumor-associated antigen is CLAUDIN18.2. In some embodiments, the second antigen binding region that specifically binds CLAUDIN18.2 is from CN202010570517.X.

[0264] IV. Nucleic acids of the application and host cells comprising the same

[0265] In one aspect, the application provides a nucleic acid encoding any of the anti-CD3 antibodies or fragments thereof above. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as pcDNA3.1. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell (e.g., CHO-S) or a 293 cell (e.g., 293F, e.g., Expi293F)) or other cell suitable for making an antibody or fragment thereof. In another embodiment, the host cell is prokaryotic.

[0266] In one aspect, the application provides a nucleic acid encoding any of the anti-CD3 antibodies or fragments thereof described herein. The nucleic acid can comprise a nucleic acid encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody, or a nucleic acid encoding the amino acid sequence of the light chain and / or the heavy chain of the antibody.

[0267] For example, the nucleic acid of the application comprises a nucleic acid encoding an amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 48-75 and 77-99, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 48-75 and 77-99.

[0268] The present application also encompasses nucleic acids that hybridize under stringent conditions to a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of 48-75 and 77-99; or a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs: 48-75 and 77-99, or nucleic acids having one or more substitutions (e.g., conservative substitutions), deletions, or insertions from the aforementioned nucleic acids.

[0269] In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, e.g., a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In one embodiment, the vector is pcDNA3.1.

[0270] In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria.

[0271] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for making an antibody or fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been “humanized” result in the production of antibodies having a partial or complete human glycosylation pattern. Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (HEK293, 293F, or 293T cells, e.g., Expi293F cells); etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. Mammalian host cell lines suitable for production of antibodies are known in the art.

[0272] V. Production and purification of antibody molecules of the present application

[0273] In one aspect, the present application provides methods of modulating the binding affinity of an anti-CD3 antibody or fragment thereof, comprising introducing an amino acid change to the heavy chain variable region CDR and / or light chain variable region CDR of the antibody molecule.

[0274] In one embodiment, the present application provides a method of making an antibody molecule of the present application or a fragment thereof (preferably an antigen binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding the antibody molecule of the present application or a fragment thereof (preferably an antigen binding fragment), and optionally isolating the antibody or fragment thereof (e.g. antigen binding fragment). In certain embodiments, the method further comprises recovering the antibody molecule of the present application or a fragment thereof (e.g. antigen binding fragment) from the host cell.

[0275] In one embodiment, a method of making an antibody molecule of the present application is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody (e.g. any one polypeptide chain and / or a plurality of polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for expression of the antibody, as provided herein above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0276] To recombinantly produce the antibody molecules of the present application, nucleic acid encoding the antibodies (e.g. any one polypeptide chain and / or a plurality of polypeptide chains as described herein above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide probes that are specific to genes encoding the heavy and light chains of the antibodies).

[0277] In one embodiment, the antibody molecule of the present application is a multispecific antibody molecule, e.g. a bispecific antibody molecule. Accordingly, the present application also provides a method of making a multispecific antibody molecule (e.g. a bispecific antibody molecule) that binds CD3 and another cancer-associated antigen, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody (e.g. any one polypeptide chain and / or a plurality of polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for expression of the multispecific antibody, as provided herein above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0278] Antibody molecules prepared as described herein can be purified by known art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the application can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0279] VI. Assays

[0280] The anti-CD3 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art. In one aspect, the antibodies of the application are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like. Methods known in the art can be used to determine binding to CD3, exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA) or Bio-Layer Interferometry or MSD assay or surface plasmon resonance (SPR) or flow cytometry measurements are used.

[0281] In another aspect, competition assays can be used to identify antibodies that compete for binding to CD3 with any of the anti-CD3 antibodies disclosed herein. In certain embodiments, such competing antibodies bind to the same or overlapping epitopes (e.g., linear or conformational epitopes) as any of the anti-CD3 antibodies disclosed herein.

[0282] The application also provides assays for identifying anti-CD3 antibodies having biological activities. Biological activities can include, for example, binding to CD3 (e.g., binding to human CD3 or cynomolgus CD3), binding to cells expressing CD3 (e.g., T cells, e.g., human T lymphocytes, e.g., Jurkat cells), activation of T cells, and the like. Antibodies having such biological activities in vivo and / or in vitro are also provided.

[0283] In certain embodiments, the antibodies of the application are tested for such biological activities.

[0284] Cells for use in any of the above-described in vitro assays include cell lines that naturally express CD3 or that have been engineered to express or overexpress CD3. Such cells also include cell lines that express CD3 and that have been transfected with DNA encoding CD3 that is not normally expressed. In some embodiments, such cells are T cells, e.g., human T lymphocytes, e.g., Jurkat cells.

[0285] It will be appreciated that the immunoconjugates of the application can be used to replace or supplement anti-CD3 antibodies in any of the above-described assays.

[0286] It is understood that any of the above assays can be performed using a combination of an anti-CD3 antibody and another active agent.

[0287] VII. Immunoconjugates

[0288] In some embodiments, the present application provides immunoconjugates comprising any of the anti-CD3 antibodies provided herein and another agent, e.g., a therapeutic agent, including a chemotherapeutic agent, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an anti-inflammatory agent or an immunosuppressive agent). In one embodiment, the other agent is, e.g., a cytotoxic agent, which includes any agent that is detrimental to a cell.

[0289] In some embodiments, the immunoconjugate is used to prevent or treat cancer.

[0290] VIII. Pharmaceutical compositions and pharmaceutical formulations

[0291] In some embodiments, the present application provides a composition comprising any of the anti-CD3 antibodies or fragments thereof (preferably antigen binding fragments thereof) or immunoconjugates thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises an anti-CD3 antibody or fragment thereof or immunoconjugate thereof of the present application in combination with one or more other therapeutic agents.

[0292] The present application also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an anti-CD3 antibody or immunoconjugate thereof, or compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding an anti-CD3 antibody. In certain embodiments, the composition comprises one or more antibodies or fragments thereof that bind CD3, or one or more polynucleotides encoding one or more antibodies or fragments thereof that bind CD3. These compositions can further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.

[0293] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0294] For the use and purposes of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", 8thEdition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0295] The compositions of the present application can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage

[0296] Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing an antibody of the present application having the desired degree of purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of a lyophilized formulation or aqueous solution.

[0297] The pharmaceutical compositions or formulations of the present application can also contain more than one active ingredient desired for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to provide other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulatory agents, etc. The active ingredients are suitably combined in amounts that are effective for the purpose intended.

[0298] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0299] IX. Pharmaceutical Combinations and Kits

[0300] In some embodiments, the present application also provides a pharmaceutical combination or combination product comprising an anti-CD3 antibody or fragment thereof, preferably an antigen binding fragment thereof, or an immunoconjugate thereof, of the present application, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents, etc.).

[0301] It is a further object of the present application to provide a pharmaceutical kit of parts comprising a pharmaceutical combination of the present application, preferably in the form of pharmaceutical dosage units. Thereby dosage units can be provided according to a dosing regimen or pharmaceutical administration interval.

[0302] In one embodiment, the kit of parts of the present application comprises in the same package:

[0303] - a first container containing a pharmaceutical composition comprising an anti-CD3 antibody or fragment thereof;

[0304] - a second container containing a pharmaceutical composition comprising the other therapeutic agent.

[0305] X. Uses and Methods

[0306] In one aspect, the present application provides a method of preventing or treating a tumor (e.g., a cancer) in a subject, comprising administering to the subject an effective amount of an anti-CD3 antibody or fragment thereof, immunoconjugate, pharmaceutical composition, pharmaceutical combination, or kit of the present application.

[0307] In some embodiments, the tumor, e.g., cancer, comprises solid tumors and blood tumors, as well as metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in an early, intermediate, or advanced stage or be metastatic. In some embodiments, the tumor is a tumor immune escape.

[0308] In a particular embodiment, the anti-CD3 antibody of the present application is capable of activating T cells. In a particular embodiment, the antibody of the present application is capable of killing tumor cells, and / or inhibiting tumor cell proliferation.

[0309] Accordingly, the CD3 antibody of the present application is suitable for use in the prevention or treatment of any tumor or cancer in which the effector mechanisms of cytotoxic T cells are required, or any tumor or cancer in which T cell recruitment is required. In some embodiments, the tumor or cancer treatment would benefit from T cell activation, or the effector mechanisms of cytotoxic T cells, or T cell recruitment.

[0310] In a particular aspect, the anti-CD3 antibody of the present application is a multispecific antibody (e.g., a bispecific antibody) that specifically binds CD3, and one or more cancer-associated antigen (e.g., a cancer-specific antigen / target or an antigen / target that is overexpressed in or associated with cancer), e.g., the antigen is HER2 or CD70 or CLAUDIN18.2. In some embodiments, the tumor (e.g., cancer) patient has (altered, e.g., elevated levels of, e.g., nucleic acid or protein levels of) a cancer-associated antigen. In some embodiments, the tumor treatment would benefit from increasing or inhibiting the cancer-associated antigen at the nucleic acid or protein level.

[0311] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual having or at risk of having a disease described herein). In one embodiment, the subject has a disease described herein (e.g., a cancer) or is at risk of having a disease described herein. In certain embodiments, the subject receives or has received other treatment, e.g., chemotherapy treatment and / or radiation therapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0312] In other aspects, the present application provides the use of an antibody molecule or fragment thereof or an immunoconjugate thereof or a pharmaceutical composition or a pharmaceutical combination or a kit of the present application in the manufacture or preparation of a medicament for the uses described herein, e.g., for the prevention or treatment of a relevant disease or condition mentioned herein.

[0313] In some embodiments, the antibody molecule of the application, or a fragment thereof, or an immunoconjugate or pharmaceutical composition or drug combination or kit thereof, will delay the onset of a disorder and / or symptoms associated with a disorder.

[0314] In some embodiments, the antibody molecule of the application, or a fragment thereof, or an immunoconjugate or pharmaceutical composition thereof, can also be administered in combination with one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for the prophylaxis and / or treatment of a relevant disease or disorder mentioned herein. In some embodiments, the treatment modalities include surgery; radiation therapy, local or focused irradiation, etc. In some embodiments, the therapeutic agents are selected from chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulatory agents. Exemplary immunomodulatory agents include immunosuppressants or anti-inflammatory agents.

[0315] In some embodiments, the antibody combinations described herein can be administered separately, e.g., as separate antibodies.

[0316] Such combination therapy encompasses combined administration (e.g., two or more therapeutic agents are included in the same formulation or separate formulations), and separate administration, in which case, administration of the antibody of the application can occur prior to, simultaneously with, and / or following, administration of the other therapeutic agent and / or agents.

[0317] The route of administration of the pharmaceutical compositions is in accordance with known methods, e.g., orally, by intravenous injection, intraperitoneally, intracerebrally (intraparenchymal), intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraportally, or intralesionally; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or by continuous infusion or by implantation devices.

[0318] The compositions can also be administered topically via an implanted membrane, sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In certain embodiments, when implantation devices are used, the devices can be implanted into any suitable tissue or organ, and can deliver the desired molecules via diffusion, timed release bolus, or continuous administration.

[0319] X. Methods and compositions for diagnosis and detection

[0320] In certain embodiments, the anti-CD3 antibodies or fragments thereof, preferably antigen binding fragments, provided herein can be used to detect the presence of CD3 or cancer-specific targets in a biological sample.

[0321] The term "detecting" as used herein includes quantitative or qualitative detection. Exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion associated lesion.

[0322] In one embodiment, the antibodies of the application can be used to diagnose a tumor, e.g., a cancer, e.g., to evaluate (e.g., monitor) the treatment or progression of a disease described herein in an individual, its diagnosis and / or staging. In certain embodiments, a labeled anti-CD3 antibody or fragment thereof is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophoric labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by enzymatic reaction or molecular interaction.

[0323] In some embodiments, the sample is formalin-fixed, paraffin-embedded (FFPE). In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

[0324] These and other aspects and embodiments of the application are described in and illustrated by the accompanying drawings (a brief description of which follows) and the detailed description of the application below. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the application. The following examples further illustrate the application, however, it is understood that the examples are described in illustrative, and not restrictive, manner, and that modifications can be made by those skilled in the art.

[0325] Examples

[0326] Example 1 Humanization sequence design of murine CD3 antibody sp34

[0327] Murine CD3 antibody sp34 (U.S. Pat. No. 8,236,308; J. Immunol. Methods., 1994, 178: 195) has the function of activating T cells, and can form a CD3 adapter with antibodies to tumor cell specific antigen molecules, thereby enabling T cells to target and kill tumor cells. To reduce its immunogenicity, the sequence of the antibody was first humanized.

[0328] The CDR regions of the sp34 antibody were defined, wherein the heavy chain CDR1 was integrated using the AbM naming scheme, and the remaining CDRs used the Kabat naming scheme. By sequence similarity comparison, the antibody germline with the highest similarity to sp34 was selected as the antibody template, and the CDRs of the light chain and the heavy chain were replaced with the CDR regions of the template, and then key amino acids were back-mutated according to the simulated three-dimensional structure. The specific humanization process is as follows:

[0329] (1) IGHV3-73*01 and IGHJ6*01 (sequences see the following table) were selected as the antibody template of the heavy chain variable region of sp34, and IGKV3-7*02 and IGKJ1*01 (sequences see the following table) of Kappa and IGLV7-46*02 and IGLJ3*02 (sequences see the following table) of Lambda were selected as the antibody template of the light chain variable region of sp34, respectively. The CDR regions of the heavy chain or light chain of sp34 were replaced with the CDR regions of the antibody template, and the variable region sequences husp34h.g0 (SEQ ID NO: 48), husp34k.g0 (SEQ ID NO: 81) and husp34l.g0 (SEQ ID NO: 77) were obtained.

[0330]

[0331]

[0332] (2) The variable region of the sp34 antibody was homologous modeling using Discovery Studio software, and the three-dimensional structure model of the variable region of sp34 was obtained.

[0333] (3) According to the variable region structure of the sp34 antibody, the key amino acids affecting the interaction between the heavy chain and the light chain and the interaction with the CDR were judged, and the amino acid sites for back-mutation were determined, and three heavy chain variable region sequences husp34h.g1 (SEQ ID NO: 49), husp34h.g2 (SEQ ID NO: 50) and husp34h.g3 (SEQ ID NO: 51) were obtained, and six light chain variable region sequences husp34k.g1 (SEQ ID NO: 82), husp34k.g2 (SEQ ID NO: 83), husp34k.g3 (SEQ ID NO: 84), husp34l.g1 (SEQ ID NO: 78), husp34l.g2 (SEQ ID NO: 79) and husp34l.g3 (SEQ ID NO: 80) were obtained.

[0334] Example 2 Preparation of humanized antibody

[0335] The antibody variable region sequences in Example 1 were combined with light and heavy chains, and the specific combinations are shown in Table 1. The heavy chain constant region was selected as human IgG1 L234AL235A sequence (SEQ ID NO: 100), and the light chain constant region was selected as CL-Kappa (SEQ ID NO: 102) and CL-Lambda (SEQ ID NO: 101) according to whether the variable region was Kappa or Lambda. Then the heavy chain sequence and the light chain sequence of the antibody were respectively constructed into the expression vector pcDNA3.1 (Invitrogen, V790-20) to obtain each plasmid, and Expi293 cells (Invitrogen, A14527) were used for transient transfection to obtain the corresponding humanized antibodies. The specific transfection and purification process is as follows:

[0336] According to the required transfection volume, the Expi293 cells were passaged, and the cell density was adjusted to 1.5x10 6 cells / ml the day before transfection. The cell density on the transfection day was about 3x10 6 cells / ml. Take 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as the transfection buffer, add appropriate plasmids to the transfection cells at 1.0 μg / ml, and mix well. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix well, and incubate at room temperature for 20 min to obtain the DNA / PEI mixture. Slowly add the DNA / PEI mixture to the cells, shake the flask gently while adding, then place it in a 36.5°C, 8% CO2 incubator for culture. After seven days, the cell broth was obtained, and the cell supernatant was collected for purification.

[0337] The Protein A column (Hitrap Mabselect Sure, GE, 11-0034-95) used for purification was treated with 0.1 M NaOH for 2 h and the glass bottle etc. were washed with distilled water and baked at 180°C for 4 h. The cell broth collected was centrifuged at 4500 rpm for 30 min and the cells were discarded. The supernatant was filtered using a 0.22 μm filter. The Protein A column was equilibrated with 10 column volumes of binding buffer (Na phosphate 20 mM, NaCl 150 mM, pH 7.0). The filtered supernatant was loaded into the purification column and equilibrated with 10 column volumes of binding buffer. 5 ml of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) was added and the eluate was collected. 80 μl of 2 M Tris-HCl was added per 1 ml of eluate. The collected antibody was ultrafiltered and concentrated and exchanged into PBS (Gibco, 70011-044) and the concentration was checked.

[0338] Table 1. Control table of light and heavy chain combinations of humanized sp34 antibodies

[0339]

[0340]

[0341] wherein the sequence of the heavy chain HCDR1 of the antibody listed in Table 1 having a VH of husp34h.g0 or husp34h.g1 is set forth in SEQ ID NO: 1, the sequence of the HCDR2 is set forth in SEQ ID NO: 2, the sequence of the HCDR3 is set forth in SEQ ID NO: 3, the sequence of the light chain LCDR1 is set forth in SEQ ID NO: 29, the sequence of the LCDR2 is set forth in SEQ ID NO: 30, and the sequence of the light chain LCDR3 is set forth in SEQ ID NO: 31; the sequence of the heavy chain HCDR1 of the antibody listed in Table 1 having a VH of husp34h.g2 or husp34h.g3 is set forth in SEQ ID NO: 1, the sequence of the HCDR2 is set forth in SEQ ID NO: 2, the sequence of the HCDR3 is set forth in SEQ ID NO: 8, the sequence of the light chain LCDR1 is set forth in SEQ ID NO: 29, the sequence of the LCDR2 is set forth in SEQ ID NO: 30, and the sequence of the light chain LCDR3 is set forth in SEQ ID NO: 31

[0342] Example 3. Affinity determination of humanized antibodies

[0343] 3.1 Determination of the binding kinetics of the antibodies of the application to human CD3 protein and cynomolgus CD3 protein using the Bio-Layer Interferometry technique

[0344] The equilibrium dissociation constant (KD) of the antibodies of the present application binding to human CD3 protein and cynomolgus CD3 protein was determined using the Bio-Layer Interferometry assay (BLI). The BLI method affinity assay was performed according to the existing method (Estep, P, et al. High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0345] Half an hour before the experiment, the appropriate number of AHC (18-5060, Fortebio) sensors were immersed in SD buffer (1x PBS, BSA 0.1%, Tween-200.05%) according to the number of samples.

[0346] 100 μl of SD buffer, each antibody, human CD3 E&D complex protein (Beijing Yiqioshen Zhou, CT026H0323H), cynomolgus CD3 E&D complex protein (Beijing Yiqioshen Zhou, CT032-C0323H) were added to a 96-well black polystyrene half-volume microplate (Greiner, 675076). The detection was performed using Fortebio Octet Red 96, and the sensor position was selected according to the sample position layout. The instrument setting parameters were as follows: running steps: Baseline, Loading ~ 1 nm, Baseline, Association and Dissociation; the running time of each step depends on the binding and dissociation speed of the sample, the rotation speed is 1000 rpm, and the temperature is 30°C. The ForteBio Octet analysis software was used to analyze the K D values.

[0347] In the experiments described in the above assay, the affinity of the antibodies is shown in Table 2, where N.B represents no binding, the affinities of hzsp34.12-hzsp34.15, hzsp34.17-hzsp34.20, hzsp34.22-hzsp34.25 are equivalent to that of the murine sp34 (10 -10 ~ 10 -9 M), while the affinities of hzsp34.37-hzsp34.40, hzsp34.42-hzsp34.45 are slightly reduced (10 -9 10 -8 M).

[0348] Table 2 Binding kinetics constants of sp34 humanized antibodies

[0349]

[0350]

[0351] 3.2 Using Jurkat cell line to detect the binding of the antibody of the present invention to human CD3

[0352] Jurkat cells are immortalized human T lymphocytes that express human CD3 complexes. This cell line was used to detect the binding of the antibodies of the present invention to these cells. The detailed procedure was as follows: Jurkat cells (Promega, J1621) were seeded in a U-shaped 96-well plate, with 2×10 5 The antibodies to be tested were added to the corresponding cell wells according to a series of concentration gradients (the starting concentration of the antibody molecule was 500nM and diluted 3 times), incubated at 4°C for 30 minutes, and then the unbound parts were washed with PBS. The PE fluorescent secondary antibody of goat anti-human Fc (Southern Biotech, J2815-5H87B) was added and incubated at 4°C for 15 minutes before detection on a flow cytometer (FACS CELESTA, BD). The results showed that the humanized antibodies hzsp34.17~hzsp34.20, hzsp34.22~hzsp34.25 and the chimeric antibody sp34 showed comparable affinity at the cellular level, while the affinity of hzsp34.37~hzsp34.40 and hzsp34.42~hzsp34.45 was reduced, which had a one-to-one correspondence with the protein level ( Figure 1 A, B and C). We selected some of the antibodies to conduct further cell-level binding curves. The results are shown in Figure 2. Figure 1 D, better shows the cellular binding of humanized antibodies with different CD3 affinities, among which hzsp34.24 has the closest T cell binding to the chimeric antibody sp34.

[0353] Example 4 Detection of T cell activation function of humanized antibodies

[0354] The present invention uses Jurkat NFAT (Nuclear Factor of Activated T) reporter cells (Promega, J1621) to detect the T cell activation function of the sp34 humanized antibody. The cells are engineered Jurkat T cells. When the cells are activated through the TCR-CD3 pathway, they release luciferase substrate into the experimental system through the downstream signal NFAT, thereby detecting the degree of T cell activation.

[0355] The detailed operation of this example is as follows: a white 96-well flat-bottom plate was used, and 4×10 Jurkat NFAT cells were plated in each well. 4The antibody molecules were mixed with the corresponding concentrations, and the initial concentration of the antibody molecules was 500 nM, and the dilution was performed in 3-fold gradient. Then, the mixture was incubated in a 37 °C incubator for 6-8 hours, and then 100 μL of Bio-Glo (Promega, G7940) was added to each well. The wavelength was detected by using an enzyme-labeled instrument (Spectra, Molecular Devices). The results are shown in Table 2. Figure 2 As shown in Table 2, the T cell activation ability of the humanized sp34 antibody was related to its affinity.

[0356] Example 5 CDR mutants of the humanized antibody

[0357] 5.1 Design and preparation of the mutants

[0358] The CDR regions of the humanized sp34 antibody were also subjected to amino acid mutation, aiming to reduce the affinity to CD3, so as to obtain molecules with different T cell activation abilities, and to meet the needs of CD3 adapters of different tumor-specific antigens.

[0359] The specific operation is as follows: taking hzsp34.24 as the initial sequence, the amino acids at positions H31, H32, H33, H52, H52A, H52C, H53, H54, H95, H96, H97, H98, H99, H100, H100A, H100B, H100C (Kabat number) and L24, L28, L29, L30, L31, L53, L91, L92, L93, L94 (Kabat number) of the heavy chain and the light chain were selected as target amino acids, and then point mutation and combination mutation were performed, respectively, to obtain the heavy chain variable region sequences husp34h.g2.1-husp34h.g2.24 (the sequences are shown in the sequence listing) and the light chain variable region sequences husp341g3.1-husp341g3.15 (the sequences are shown in the sequence listing). The basic principle of the point mutation is as follows (Table 3): the aromatic amino acids Y, W and F are mutated to amino acids G, A and S with smaller side chains; the positively charged amino acids R, K and H are mutated to amino acids G, A and S with smaller side chains; the amino acid G with hydrogen atom in the side chain is mutated to an aromatic amino acid Y; the amino acids N and Q with amide group in the side chain are mutated to amino acids G, S, D and E; the non-aromatic amino acids T and S with hydroxyl group in the side chain are mutated to G, L and R. Among them, the aromatic amino acids Y, W and F are mutated to amino acids G, A and S with smaller side chains because the aromatic amino acids often participate in the hydrophobic interaction between molecules and between molecules through the structure of the side chain hydrophobic benzene ring, and the side chain occupies a larger space, and therefore, the mutation to amino acids with smaller side chains can potentially change the interaction between the antibody and the antigen;

[0360] The positive charge amino acids R, K, H are mutated to the amino acids G, A, S with smaller side chains because the positive charge amino acids often participate in the charge interaction between molecules and molecules through the positive charge of the side chain, and the side chain of R\K occupies a larger space, so mutating to the amino acids with smaller side chains can potentially change the interaction between the antibody and the antigen;

[0361] The amino acid G with a hydrogen atom in the side chain is mutated to the aromatic amino acid Y because the side chain of the amino acid G occupies the smallest space among all amino acids, and mutating to the amino acid Y with a larger side chain can potentially change the interaction between the antibody and the antigen due to the increase in steric hindrance;

[0362] The amino acids N, Q with amide groups in the side chain are mutated to the amino acids G, S, D, E because the amino acids N\Q with amide groups in the side chain are structurally similar to D\E, mutating them to D\E can slightly weaken the interaction between the antibody and the antigen without completely destroying it, and mutating to G, S is to directly reduce the side chain, which can potentially change the interaction between the antibody and the antigen;

[0363] The non-aromatic amino acids T, S with hydroxyl groups in the side chain are mutated to G, L, R because the side chain of the amino acid T\S contains a hydroxyl group, and mutating them to G with the smallest side chain, L with a medium hydrophobic amino acid in the side chain, and R with a larger side chain and a positive charge, respectively, is expected to change the interaction between the antibody and the antigen to different degrees.

[0364] Table 3 Amino acid mutation control table

[0365]

[0366] The antibody variable region sequences obtained above are combined with light and heavy chains, and the specific combination is shown in Table 4. The heavy chain constant region is selected as the human IgG1L234AL235A sequence (SEQ ID NO: 100), and the light chain constant region is selected as the corresponding constant region CL-Kappa (SEQ ID NO: 102) and CL-Lambda (SEQ ID NO: 101) according to whether the variable region is Kappa or Lambda. Then the heavy chain sequence and the light chain sequence of the antibody are constructed into the expression vector pcDNA3.1 (Invitrogen, V790-20), and Expi293 cells (Invitrogen, A14527) are used for transient transfection to obtain the corresponding humanized antibody. The specific transfection process is the same as that of Example 2.

[0367] Table 4 sp34 humanized CDR mutation antibody combination light and heavy chain combination control table

[0368]

[0369]

[0370]

[0371] 5.2 Affinity testing of mutants and T cell activation function testing

[0372] The affinity of the humanized CDR mutant of Sp34 to human CD3 protein was determined using biofilm thin layer interferometry (BLI) technology. The specific method was the same as in Example 3.1. The results are shown in Table 5. The affinity level of the antibody to the human CD3E&G complex (Beijing Sino Biological, CT041-H0305H) was 1.11×10 -8 M~9.50×10 -9 M, and the affinity level for human CD3E&D complex (Beijing Sino Biological, CT026H0323H) was 1.03×10 -8 M~9.77×10 -10 Between M.

[0373] Table 5 Affinity of sp34 humanized CDR mutant antibodies to human CD3

[0374]

[0375]

[0376] The affinity test method of Sp34 humanized CDR mutants to CD3 at the cellular level was the same as that in Example 3.2. The results are shown in FIG. Figure 3 A and Figure 3 As shown in B, compared with the sp34 antibody, except for hzsp34.40, hzsp34.42, and hzsp34.45, the affinities of hzsp34.80, hzsp34.81, hzsp34.82, hzsp34.87, hzsp34.88, hzsp34.89, hzsp34.91, hzsp34.97, hzsp34.99, hzsp34.101, hzsp34.107, and hzsp34.116 decreased to varying degrees. Some clones were selected for binding to Jurkat cells at a gradient of antibody concentrations, and the results are shown in Figure 2. Figure 3 C shows the difference in affinity of different CDR mutants for CD3.

[0377] S p The T cell activation function of the humanized CDR mutant of 34 was detected using Jurkat NFAT cells. The detection method was the same as that in Example 4. The results are shown in FIG. Figure 4As shown, the CDR mutants of sp34 humanized antibody can all activate the downstream signaling pathway of T cells, but the degree of activation is different.

[0378] Example 6 Her2xCD3 bispecific antibody in vitro activity experiment

[0379] In order to detect the monovalent affinity of sp34 humanized antibody and the activation ability of tumor associated antigen dependent T cells, a "1+1" form of bispecific molecule targeting Her2xCD3 was constructed, in which the variable region sequence of anti-Her2 was from the marketed drug trastuzumab, the heavy chain variable region sequence was EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTV (SEQ ID NO: 114); the light chain variable region sequence was DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 115), the variable region sequence of anti-CD3 was the variable region sequence of sp34 humanized antibody (hzsp34.24, hzsp34.80, hzsp34.87, hzsp34.97, hzsp34.99, hzsp34.101), the Fc segment of the antibody was selected as the IgG1 LALA sequence of Knob-into-hole (A. Margaret Merchant et al., Nature Biotechnology, 1998) (the sequence of each domain is as follows), and the schematic diagram of the antibody is as follows Figure 5 A.

[0380] CL:

[0381]

[0382] CH1+CH2+CH3(knob):

[0383]

[0384] CH1+CH2+CH3(hole):

[0385]

[0386] The CD3 equilibrium dissociation constant (KD) of Her2xCD3 bispecific antibodies was determined using Biacore (GE Healthcare, T200) as follows:

[0387] After human CD3E&G antigen (Beijing Yiqioshen Zhou, CT041-H0305H) and cynomolgus CD3E&D antigen protein (Beijing Yiqioshen Zhou, CT032-C0323H) were coupled to the chip surface respectively, the affinity and kinetic constants were obtained by detecting the binding and dissociation between the chip surface antigen and the antibody in the mobile phase. The method includes chip preparation and affinity detection. The determination process uses 10x HBS-EP+ (BR-1006-69, GE Healthcare) diluted by 10 times as the experimental buffer. The chip preparation process uses an amino coupling kit (BR-1006-33, GE Healthcare) to couple human CD3E&G antigen and cynomolgus CD3E&D antigen to the surface of a CM5 chip (29-1496-03, GE Healthcare), and the coupling level is not more than 100 RU to avoid high coupling density causing strong affinity. After coupling, 1M ethanolamine is injected to block the remaining activated sites. Affinity detection cycles are performed for each concentration of antibody, and each cycle includes binding the antibody at that concentration and chip regeneration. Gradient-diluted antibodies (Her2-sp34.24: starting concentration 32nM, 2-fold concentration dilution; Her2-sp34.87: starting concentration 200nM, 2-fold concentration dilution; Her2-sp34.80, Her2-sp34.97, Her2-sp34.99, Her2-sp34.101: starting concentration 800nM, 2-fold concentration dilution; 5 concentration points are diluted) are sequentially flowed through the chip surface at a flow rate of 30μl / min from low concentration to high concentration, with a binding time of 180s and a dissociation time of 600s. Finally, 10mM Glycine pH1.5 (BR-1003-54, GE Healthcare) is used to regenerate the chip. The data results are analyzed using Biacore T200 analysis software (version number 3.1), and the analysis model used is a 1:1 binding model for kinetic analysis. The detection results are shown in Table 6, and the affinities of the monovalent sp34 humanized antibodies and their CDR mutants in Her2xCD3 bispecific antibodies to human and cynomolgus CD3 show different gradients.

[0388] Table 6 Affinity of sp34 humanized antibodies in Her2xCD3 bispecific antibodies to CD3

[0389]

[0390] To verify the T cell activation ability of Her2xCD3 bispecific antibody, luciferase reporter system of Jurkat NFAT (Nuclear Factor of Activated T) cell (J1621, Promega) was used to detect the activation ability of bispecific antibody molecule to T cell. The cell line activates the expression of luciferase through the NFAT signal pathway of TCR / CD3 intracellular downstream signal, thereby detecting the activation of T cell.

[0391] The specific method is as follows: 96-well white flat-bottom cell culture plates are used, 8x10 5 cells of target cell SK-BR-3 (JCRB0834, JCRB cell bank) and 4x10 6 cells of effector cell Jurkat NFAT are added to each well, then Her2xCD3 bispecific antibody molecules (the initial concentration of Her2-sp34.24, Her2-sp34.87 and Her2-sp34.101 is 10 nM, 4-fold dilution; the initial concentration of Her2-sp34.80 is 200 nM, 4-fold dilution; the initial concentration of Her2-sp34.97 and Her2-sp34.99 is 100 nM, 4-fold dilution) corresponding to the concentration are added, and the plates are incubated in a 37°C incubator for 16 hours. Then the culture plates are taken out, Bio-Glo (G7940, Promega) is added, and the wavelength is detected by using an enzyme-labeled instrument (Spectra, Molecular Devices). The results are shown in Figure 5 Figure B. The T cell activation ability from high to low is Her2-sp34.24, Her2-sp34.87, Her2-sp34.101, Her2-sp34.97, Her2-sp34.99, Her2-sp34.80.

[0392] Example 7 In vitro activity experiment of CD70 / CD3 bispecific antibody

[0393] The present application also constructs a bispecific antibody expressing a CD70xCD3 "1+1" form, in which the variable region sequence of anti-CD70 is from SGN70 (SEQ ID NO: 14 and SEQ ID NO: 24 in WO2004073656), and the variable region sequence of anti-CD3 is from the antibody variable region sequence of sp34 humanized, and adopts a scFv form Figure 6A), each sp34 sequence uses the order of "heavy chain-light chain (HL)" and "light chain-heavy chain (LH)" respectively (where sp34.24LH represents the light chain-heavy chain order, sp34.24HL represents the heavy chain-light chain order, and other molecules are the same). They are connected using (GGGGS)4, and the Fc segment uses the IgG1LALA sequence of knob-into-hole (A. Margaret Merchant et al., Nature Biotechnology, 1998). The schematic diagram of the antibody structure is shown in the figure. Figure 6 As shown in A, the CH3 of the Knob uses the point mutation Y349C (EU numbering system), and the CH3 of the Hole uses the point mutation S354C ((EU numbering system)). The sequences are as follows:

[0394] CL: SEQ ID NO: 116

[0395] CH1+CH2+CH3 (pestle):

[0396]

[0397] CH1+CH2+CH3(mortar):

[0398]

[0399] The CD3 equilibrium dissociation constant (KD) of each bispecific antibody was determined using Biacore (GE Healthcare, T200) using the same method as in Example 6. The results are shown in Table 7. Some bispecific antibodies SGN70-sp34.80HL and SGN70-sp34.99HL have lower affinity due to structural changes.

[0400] Table 7 Affinity of the sp34 humanized antibody in scFv format in the SGN70×CD3 bispecific antibody to CD3

[0401]

[0402] The Jurkat NFAT cell reporter system was used for detection. The specific method is as follows: a 96-well white flat-bottom cell culture plate was used, and 8×10 target cells NOMO-1 (CBP60515, Nanjing Kebai Biotechnology) were added to each well. 5 and effector cells Jurkat NFAT cells 4×10 6Then, add the corresponding concentration of CD70×CD3 bispecific antibody molecules, with the starting concentration of 100mM and 5-fold dilution. Incubate at 37℃ for 16 hours. Then remove the culture plate, add Bio-Glo (G7940, Promega), and use a microplate reader (Spectra, Molecular Devices) for wavelength detection. The results are as follows Figure 6 As shown in Figure 2B, some bispecific antibodies SGN70-sp34.24LH, SGN70-sp34.24HL, SGN70-sp34.87LH, SGN70-sp34.87HL, SGN70-sp34.101HL and SGN70-sp34.101LH have strong T cell activation ability, while some bispecific antibodies such as SGN70-sp34.80HL, SGN70-sp34.80LH, SGN70-sp34.97HL, SGN70-sp34.97LH, SGN70-sp34.99HL and SGN70-sp34.99LH have relatively weak T cell activation ability, which may be related to the low affinity of the SGN70 antibody at the CD70 end and the low abundance of CD70 on the surface of NOMO-1 cells.

[0403] Example 8 Construction and Preparation of CD3 / Claudin18.2 Bispecific Antibody

[0404] According to Table 8, the anti-Claudin18.2 (CLDN18.2) monoclonal antibody HB37A6 (see CN202010570517.X, sequence information is shown in Table 9) was combined with the sequences of the antigen-binding regions of the above-mentioned three different anti-human CD3 monoclonal antibodies HzSP34.24, HzSP34.87 and HzSP34.97 to construct the "1+1" bispecific antibodies 030, 032 and 033 targeting CLDN18.2×CD3, respectively. The schematic diagram of the antibody structure is shown in Figure 7 Specifically, the Fc region of the antibody is selected from the IgG1 LALA sequence with a knob-in-hole structure (A. Margaret Merchant et al., Nature Biotechnology, 1998). Therefore, the heavy chain of the CLDN18.2-terminal portion of the bispecific antibody is SEQ ID NO: 121, and the light chain is SEQ ID NO: 122. The heavy chains of the CD3-terminal portion of the bispecific antibody are SEQ ID NOs: 123, 125, and 126, respectively, and the light chain is SEQ ID NO: 124.

[0405] The plasmid construction process of the bispecific antibody is as follows: the heavy chain sequence of CLDN18.2 (SEQ ID NO: 121), the light chain sequence of CLDN18.2 (SEQ ID NO: 122), the heavy chain sequence of CD3 (SEQ ID NO: 123, 125 and 126), and the light chain sequence of CD3 (SEQ ID NO: 124) are inserted into the vector pcDNA3.1 (Invitrogen, V790-20) respectively to obtain the heavy chain plasmid and the light chain plasmid of the CLDN18.2 end, and the heavy chain plasmid and the light chain plasmid of the CD3 end. Then, PEI (Polysciences, 23966) is used to transiently transfect the heavy chain plasmid and the light chain plasmid of the CLDN18.2 end and the heavy chain plasmid and the light chain plasmid of the CD3 end into Expi293 cells (Invitrogen, A14527) to express the half-antibody of the CLDN18.2 end and the three half-antibodies of the CD3 end. After 7 days, the cell fermentation broth is obtained, filtered and clarified, and then captured by a Hitrap Mabselect Sure Protein A column (GE Healthcare, 11-0034-95) to obtain the half-antibodies of the CLDN18.2 end and the CD3 end. After detecting the half-antibody concentration by the A280 method, the half-antibodies of the two ends are mixed in a molar ratio of 1:1. An appropriate amount of reducing agent GSH is added, and the reaction is carried out at room temperature overnight. The reducing agent is removed by ultrafiltration, and the reaction is terminated. Then, fine purification is carried out by MonoS cation exchange chromatography (GE Healthcare, 17-5168-01), the A liquid is 20 mM sodium phosphate buffer (pH 6.6), the B liquid is 20 mM sodium phosphate buffer containing 1 M sodium chloride (pH 6.6), and the elution gradient is 0-50% (30 column volumes). The eluted protein solution is ultrafiltered and exchanged into PBS (Gibco, 70011-044), and the molecular weight is determined by mass spectrometry, and the purity is identified by SEC-HPLC. The obtained 030, 032 and 033 bispecific antibodies are used in the following examples.

[0406] Table 8. List of CD3 / CLDN18.2 bispecific antibodies

[0407] Anti-CLDN18.2 C-terminus Anti-CD3 C-terminus 030 HB37A6 HzSP34.24 032 HB37A6 HzSP34.87 033 HB37A6 HzSP34.97

[0408] Table 9: Bispecific antibody information

[0409]

[0410]

[0411]

[0412] Example 9. Affinity determination of bispecific antibodies

[0413] The equilibrium dissociation constant (KD) of the bispecific antibodies of the present application binding to human CD3 protein was determined by using the Bio-Layer Interferometry (BLI) assay. The BLI method for affinity determination was performed according to the existing method (Estep, P, et al. High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0414] Half an hour before the experiment, according to the number of samples, the appropriate number of AHC (18-5060, Fortebio) sensors were soaked in SD buffer (1x PBS, BSA 0.1%, Tween-200.05%). 100 μl of SD buffer, each bispecific antibody, human CD3 protein (CT026H0323H, Beijing Yiqiao God) were added to a 96-well black polystyrene half-volume microplate (Greiner, 675076). The detection was performed using Fortebio Octet Red96, and the sensor position was selected according to the sample position layout. The instrument setting parameters were as follows: running steps: Baseline, Loading ~ 1 nm, Baseline, Association and Dissociation; the running time of each step depends on the binding and dissociation speed of the sample, the rotation speed is 1000 rpm, and the temperature is 30℃. The KD value was analyzed using ForteBio Octet analysis software.

[0415] The affinity of the bispecific antibodies is shown in Table 10. Among them, the affinity of 030 molecule CD3 end is the highest, which is 7.4 nM. The affinity of 032 and 033 molecule CD3 end decreases in turn, which is 89 nM and 440 nM, respectively.

[0416] Table 10. Affinity of bispecific antibody CD3 end

[0417] Ka (1 / Ms) Kd (1 / s) KD (M) 030 6.927E+5 0.005164 7.455E-9 032 8.066E+5 0.07183 8.906E-8 033 3.788E+5 0.1689 4.459E-7

[0418] The equilibrium dissociation constant (KD) of the binding to human CLDN18.2 was determined by surface plasmon resonance (SPR). According to the manufacturer's instructions, the antigen human Claudin 18.2 (GenScrip, P50251802) was coupled to the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino coupling kit (GE Healthcare, BR-1006-33). After coupling, 1M ethanolamine was injected to block the remaining activated sites. According to the manufacturer's instructions, the Biacore (GE Healthcare, T200) was used to detect the binding and dissociation between the antigen on the chip surface and each bispecific antibody in the mobile phase to obtain the affinity and kinetic constants. The gradient diluted antibody (0-100 nM, 2-fold dilution) was sequentially flowed through the chip surface from low concentration to high concentration, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). The data results were analyzed using the Biacore T200 analysis software with a 1:1 binding model. The results are shown in Table 11. The same clone HB37A6 was used for the CLDN18.2 end of the 030, 032 and 033 bispecific antibodies, and the affinity of the CLDN18.2 end was consistent, with very strong affinity of 0.57 nM.

[0419] Table 11. Affinity of CLDN18.2 end of bispecific antibody

[0420]

[0421] Example 10. In vitro T cell killing experiments

[0422] The 030, 032 and 033 bispecific antibodies obtained in Example 9 were subjected to in vitro T cell killing experiments. Human peripheral blood mononuclear cells (PBMC, Allcells or Saily) were resuspended with complete culture medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), and the PBMC were adjusted to 2x10 6

[0423] ​NUGC-4 (JCRB Cell Bank, JCRB0834) or DAN-G tumor target cells DAN-G-hCLDN18.2 overexpressing Claudin18.2 prepared as follows, non-target cells L363 (DSMZ, ACC49) were labeled with Far-Red (Invitrogen) for 10 min, washed twice and resuspended in complete medium, cell concentration was adjusted to 2x10 5 cells / ml.

[0424] Wherein DAN-G-Hcldn18.2 was constructed as follows:

[0425] Full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into vector pWPT-GFP (Addgene, 12255) to replace the GFP sequence therein, co-transfected into HEK293T (ATCC, CRL-3216) cells with packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) for virus packaging. Culture supernatants after 48 and 72 hours of culture were collected and concentrated using PEG8000. The concentrated virus was used to transfect pancreatic cancer DAN-G cells, and then cells expressing CLDN18.2 were sorted out using a flow cytometry sorter (MoFlo XDP, Beckman Coulter) to obtain tumor cell line DAN-G-hCLDN18.2 stably transfected with CLDN18.2.

[0426] PBMCs were mixed with bispecific antibodies 030, 032 and 033 respectively (030, 032 were used at an initial concentration of 1 nM, 033 was used at an initial concentration of 400 nM, all antibodies were 5-fold diluted, a total of 10 concentration points), incubated at 37°C for 30 min, then 50 μl of tumor target cells (1x10 4 cells) were added to 50 μl of PBMC effector cells at an effector to target ratio of 10:1. Incubate at 37°C for 24 hours, centrifuge, resuspend the cells with propidium iodide (PI, Invitrogen) at a final concentration of 10 μg / ml, detect Far-Red and PI double positive cells using a flow cytometer (BD, FACSCELESTA). Calculate the tumor target cell killing ratio by FACSDiva software (BD, Celestsa).

[0427] From Figure 8 The results show that 030 and 032 molecules have very strong killing activity on gastric cancer cells NUGC-4, with EC50 values less than 1 pM. From Figure 9Results show that the EC50 values of both are even less than 0.1 pM on the pancreatic cancer cell DAN-G-hCLDN18.2 which has high expression of CLDN18.2. The killing activity of 033 molecule on the two tumor cell lines is weaker than 030 and 032, about 1000 times lower, but still can reach the maximum killing (close to 100% lysis of cells). But in the case of negative expression of CLDN18.2, 030, 032 and 033 molecules have no non-specific killing Figure 10 ). This shows that 030, 032 and 033 molecules all exhibit tumor cell specific killing dependent on the expression of CLDN18.2. Moreover, the killing effect of the bispecific antibody of the application is related to the abundance of CLDN18.2 on the cell surface, within a certain expression abundance range, the higher the expression of CLDN18.2 on the cell surface, the better the killing effect.

[0428] Example 11. In vitro cytokine release experiment

[0429] Resuspend human peripheral blood mononuclear cells (PBMC, Allcells or Saily) with complete culture medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), and adjust the PBMC to 2 x 10 6 cells / ml. Adjust the NUGC-4 or Claudin18.2 overexpressing DAN-G tumor target cells DAN-G-hCLDN18.2 to 2 x 10 5 cells / ml.

[0430] Mix PBMC with bispecific antibodies 030, 032 and 033 respectively, incubate at 37°C for 30 min, then add 50 μl of tumor target cells (1 x 10 4 cells) to 50 μl of PBMC effector cells at an effector to target ratio of 10:1. Incubate at 37°C for 24 hours, centrifuge, and take the cell supernatant. Detect the cytokines using the Human Th1 / Th2 / Th17 Kit kit (BD, catalog number 560484), incubate at room temperature for 3 hours, detect using a flow cytometer (BD, FACSCELESTA), and analyze the release of cytokines in the supernatant by FCAPArray software (BD).

[0431] From the results of Figure 11 and Figure 12 , it can be seen that 030, 032 and 033 molecules can all mediate the release of large amounts of IL-2, TNFα and IFNgamma cytokines on gastric cancer cells NUGC-4 and pancreatic cancer cells DAN-G-hCLDN18.2, and are positively correlated with the affinity of the CD3 end.

[0432] Example 12. In vitro T cell activation experiment

[0433] Human peripheral blood mononuclear cells (PBMC, Allcells or Saily) were resuspended in complete medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03) and the PBMC were adjusted to 2×10 6 pcs / ml.

[0434] The concentration of DAN-G-CLDN18.2 in NUGC-4 or Claudin18.2-overexpressing DAN-G tumor target cells was adjusted to 2×10 5 PBMCs were mixed with bispecific antibodies 030, 032, and 033, respectively, and incubated at 37°C for 30 min. Then, 50 μl of tumor target cells (1×10 4 ) were added to 50 μl PBMC effector cells. The cells were incubated at 37°C for 24 hours, centrifuged, and the supernatant was removed. The cells were incubated with BV421 anti-human CD3 (Biolegend, 317344), PerCP / Cy5.5 mouse anti-human CD4 (BD, 552838), APC / Cy7 anti-human CD8a (Biolegend, 300926), PE anti-human CD25 (Biolegend, 302606), and FITC anti-human CD69 (Biolegend, 310904) at 4°C for one hour, and then washed three times with 1× PBS. The proportion of CD25 and CD69 double-positive cells in CD4+ and CD8+ T cells was detected by flow cytometry (BD, FACSCELESTA), respectively. The proportion of CD25 and CD69 double-positive cells in CD4+ and CD8+ T cells was calculated using FACSDiva software (BD, Celestsa), which is the proportion of activated CD4 and CD8+ T cells.

[0435] according to Figure 13 As shown, 30, 032 and 033 molecules can specifically activate T cells when co-cultured with gastric cancer cell NUGC-4, and the activation ability is positively correlated with the affinity of the CD3 end.

[0436] Example 13. In vivo efficacy experiment - gastric cancer model

[0437] This study investigated the anti-tumor effect of bispecific antibodies against NUGC-4 tumors in NOG female mice. 49 NOG female mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected.

[0438] PBMC cells (Allcells) were resuscitated, and the cells were centrifuged to disperse the PBMC cells with PBS (1x) to obtain a cell suspension with a cell density of 2x10 7 / ml. 200 μl of the cell suspension was used to perform an orbital venous injection of the PBMC cells into the mice, 4x10 6 / mouse.

[0439] NUGC-4 cells were resuscitated and subcultured for subsequent in vivo experiments. The cells were collected by centrifugation and dispersed with PBS (1x) to obtain a cell suspension with a cell density of 6x10 7 / ml. The NUGC-4 cells were mixed with matrigel 1:1 to prepare a cell suspension with a cell density of 3x10 7 / ml. On day 3, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of the NOG humanized mice to establish a NUGC-4 tumor-bearing mouse model.

[0440] On day 7 after cell inoculation, the maximum width and maximum length of the tumor of the mice were measured with a vernier caliper, and the tumor volume was calculated. Mice with a tumor volume of 53.35 mm 3 ~ 168.07 mm 3 were selected, and the mice were grouped in a serpentine manner according to the tumor volume (6 mice per group). Each mouse was intravenously injected with the bispecific antibodies 030, 032 and 033 of the present application, and a negative control h-IgG (Equitech-Bio, batch number 160308-02) at a dose of 0.3 mg / kg and 1 mg / kg, once a week for a total of 4 times. The tumor volume of the mice was measured twice a week. The tumor inhibition rate (TGI%) was calculated according to the following formula:

[0441] TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration).

[0442] According to the results shown in Table 1, in the human gastric cancer NUGC-4 tumor-bearing mouse model, 030 and 032 achieved a TGI of 100% at a low dose of 0.3 mg / kg, and even achieved a 50% CR (complete remission) at a high dose of 1 mg / kg (3 out of 6 mice achieved complete tumor regression). The 033 molecule had almost no efficacy at a low dose, and the TGI reached 20% at a dose of 1 mg / kg. During the entire experiment, the body weight of the mice in the experimental and control groups did not decrease. Figure 14 Example 14. In vivo efficacy experiment - pancreatic cancer model

[0443]

[0444] ​This study investigated the antitumor effect of bispecific antibodies against DAN-G-Claudin18.2 tumors in NOG female mice. PBMC cells were injected into the orbital vein of 49 NOG mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) at 4×10 6 / mouse, inoculated with 200ul / mouse (as in Example 13). This time is recorded as day 0.

[0445] The human pancreatic cancer cell line DAN-G-CLDN18.2 constructed in Example 10 was routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation and DAN-G-CLDN18.2 was dispersed in PBS (1×) to obtain a cell density of 10×10 6 The cell suspension was mixed with matrigel gel at a ratio of 1:1 to prepare a cell concentration of 5×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOD-SCID mice to establish a DNA-G pancreatic cancer humanized model with overexpression of CLDN18.2.

[0446] Seven days after tumor cell inoculation, the maximum width and maximum length of the mouse tumor were measured with a vernier caliper to calculate the tumor volume. The mice were divided into snake groups according to the tumor volume. 3 ~120.64mm 3 Mice within the range were divided into groups according to tumor size (6 mice per group).

[0447] Each mouse was intravenously injected with the bispecific antibodies 030, 032, and 033 of the present invention, as well as a negative control h-IgG (Equitech-Bio, batch number 160308-02) at intraperitoneal doses of 0.3 mg / kg and 1 mg / kg once weekly for a total of four doses. Tumor volume was measured twice weekly. Tumor inhibition rate (TGI%) was calculated using the following formula:

[0448] TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before administration).

[0449] like Figure 15 As shown, in a DNA-G humanized pancreatic cancer model overexpressing CLDN18.2, both 030 and 032 achieved a TGI of 100% at both doses. The 033 molecule also achieved TGIs of 42% (0.3 mg / kg) and 76% (1 mg / kg), respectively. This may be related to the high expression of CLDN18.2 in DNA-G-CLDN18.2 pancreatic cancer cells. Throughout the experiment, mice in both the experimental and control groups did not lose weight.

[0450] Example 15. Mouse PK experiment

[0451] The pharmacokinetic properties of 030, 032 and 033 in mice were investigated by injecting 10 mg / kg of 030, 032 and 033 into female Balb / C mice (Vital River Laboratory Animal Technology Co., Ltd.) via tail vein. The mice were taken blood from the eyeball at 0.086 hr, 0.5 hr, 2 hr, 6 hr, 24 hr, 48 hr, 4 day, 7 day, 14 day and 21 day after administration, respectively. The blood was centrifuged at 4°C at 3000 rpm for 10 min, and the serum was collected. The antibody content in the serum was determined by ELISA, and the half-life of 030, 032 and 033 in mice was calculated.

[0452] The experimental results are shown in Table 1. Figure 16 As shown in Table 1, the half-lives of 030, 032 and 033 in mice all reached the PK similar to that of normal monoclonal antibodies, which further indicated that the bispecific antibody constructed in the application did not affect the half-life of the antibody.

[0453] Sequence information:

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467] SEQUENCE LISTING <110> Sinobiological Pharmaceuticals (Suzhou) Co., Ltd. <120> Anti-CD3 antibodies and uses thereof <130> PF 210773 PCT <160> 126 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic <400> 1 Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn 1 5 10 <210> 2 <211> 19 <212> PRT <213> Artificial <220> <223> Synthetic <400> 2 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 3 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 3 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic <400> 4 Gly Phe Thr Phe Gly Thr Tyr Ala Met Asn 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic <400> 5 Gly Phe Thr Phe Asn Gly Tyr Ala Met Asn 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic <400> 6 Gly Phe Thr Phe Asn Thr Ala Ala Met Asn 1 5 10 <210> 7 <211> 19 <212> PRT <213> Artificial <220> <223> Synthetic <400> 7 Arg Ile Ser Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 8 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 8 His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 9 <211> 19 <212> PRT <213> Artificial <220> <223> synthetic <400> 9 Arg lie Arg Leu Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 10 <211> 19 <212> PRT <213> Artificial <220> <223> synthetic <400> 10 Arg lie Arg Ser Lys Ala Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 11 <211> 19 <212> PRT <213> Artificial <220> <223> synthetic <400> 11 Arg lie Arg Ser Lys Tyr Gly Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 12 <211> 19 <212> PRT <213> Artificial <220> <223> synthetic <400> 12 Arg Ile Arg Ser Lys Tyr Asn Gly Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 13 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 13 Ala Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 14 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 14 His Tyr Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 15 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 15 His Gly Gly Phe Gly Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 16 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 16 His Gly Asn Ala Gly Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 17 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 17 His Gly Asn Phe Tyr Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 18 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 18 His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 19 His Gly Asn Phe Gly Gin Arg Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 20 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 20 His Gly Asn Phe Gly Gin Ser Ala Val Ser Trp Phe Ala Tyr 1 5 10 <210> 21 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 21 His Gly Asn Phe Gly Gin Ser Tyr Ala Ser Trp Phe Ala Tyr 1 5 10 <210> 22 <211> 10 <212> PRT <213> Artificial <220> <223> synthetic <400> 22 Gly Phe Thr Phe Ser Gly Ser Ala Met Asn 1 5 10 <210> 23 <211> 19 <212> PRT <213> Artificial <220> <223> synthetic <400> 23 Arg Ile Ser Leu Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 24 <211> 19 <212> PRT <213> Artificial <220> <223> synthetic <400> 24 Arg Ile Arg Ser Lys Ala Gly Gly Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 25 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 25 His Tyr Gly Phe Gly Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 26 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 26 His Gly Asn Ala Tyr Gin Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 27 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 27 His Gly Asn Phe Gly Gly Arg Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 28 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 28 His Gly Asn Phe Gly Gin Ser Ala Ala Ser Trp Phe Ala Tyr 1 5 10 <210> 29 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 29 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 30 <211> 7 <212> PRT <213> artificial <220> <223> synthetic <400> 30 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 31 <211> 9 <212> PRT <213> artificial <220> <223> synthetic <400> 31 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 32 <211> 14 <212> PRT <213> artificial <220> <223> synthetic <400> 32 Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 33 <211> 14 <212> PRT <213> artificial <220> <223> synthetic <400> 33 Arg Ser Ser Thr Gly Ala Val Gly Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 34 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 34 Arg Ser Ser Thr Gly Ala Val Thr Gly Ser Asn Tyr Ala Asn 1 5 10 <210> 35 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 35 Arg Ser Ser Thr Gly Ala Val Thr Thr Arg Asn Tyr Ala Asn 1 5 10 <210> 36 <211> 14 <212> PRT <213> Artificial <220> <223> synthetic <400> 36 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Gly Tyr Ala Asn 1 5 10 <210> 37 <211> 9 <212> PRT <213> Artificial <220> <223> synthetic <400> 37 Ala Leu Ala Tyr Ser Asn Leu Trp Val 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <400> 38 Ala Leu Trp Ala Ser Asn Leu Trp Val 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <400> 39 Ala Leu Trp Tyr Arg Asn Leu Trp Val 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <400> 40 Ala Leu Trp Tyr Ser Gly Leu Trp Val 1 5 <210> 41 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 41 Arg Ser Ser Thr Gly Ala Val Gly Gly Ser Asn Tyr Ala Asn 1 5 10 <210> 42 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <400> 42 Arg Ser Ser Thr Gly Ala Val Thr Thr Arg Gly Tyr Ala Asn 1 5 10 <210> 43 <211> 9 <212> PRT <213> Artificial <220> <223> synthetic <400> 43 Ala Leu Ala Ala Ser Asn Leu Trp Val 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial <220> <223> synthetic <400> 44 Ala Leu Trp Tyr Arg Gly Leu Trp Val 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial <220> <223> synthetic <400> 45 Gly Thr Asn Ser Arg Ala Pro 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial <220> <223> synthetic <400> 46 Ala Leu Trp Tyr Ser Asp Leu Trp Val 1 5 <210> 47 <211> 125 <212> PRT <213> Artificial <220> <223> synthetic <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 125 <212> PRT <213> 人工 <220> <223> 合成 <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 49 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 49 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 50 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 51 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 52 <211> 125 <212> PRT <213> 人工 <220> <223> 合成 <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 53 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Gly Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 54 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Ala 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 55 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Ser Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 56 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Leu Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 57 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 57 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Ala Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 58 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 58 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Gly Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 59 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 59 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Gly Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 60 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 60 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ala Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe ​​​​​​​​100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 61 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 61 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Tyr Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 62 <211> 125 <212> PRT <213> 人工 <220> <223> 合成 <400> 62 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Gly Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 63 <211> 125 <212> PRT <213> Artificial <220> <223> synthetic <400> 63 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Ala Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 64 <211> 125 <212> PRT <213> Artificial <220> <223> synthetic <400> 64 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Tyr Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 65 <211> 125 <212> PRT <213> 人工 <220> <223> 合成 <400> 65 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gly Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 66 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 66 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Arg Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 67 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Ala Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 68 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 68 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Ala Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 69 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 69 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 70 <211> 125 <212> PRT <213> 人工 <220> <223> 合成 <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Ser Leu Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 71 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ala Gly Gly Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 72 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Tyr Gly Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 73 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Ala Tyr Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 74 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gly Arg Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 75 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 75 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Ala Ala Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 76 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 76 Gln Ala Val Val Thr Gin Gin Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu He Gly Asp Lys Ala Ala Leu Thr He Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala He Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 77 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 77 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Thr 35 40 45 Leu Ile Tyr Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 78 <211> 109 <212> PRT <213> Artificial <220> <223> synthetic <400> 78 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Thr 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 79 <211> 109 <212> PRT <213> Artificial <220> <223> synthetic <400> 79 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 80 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 80 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 81 <211> 110 <212> PRT <213> 人工 <220> <223> 合成 <400> 81 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 Ser Ser Thr Gly Ala Val Thr Thr 20 25 30 Ser Asn Tyr Ala Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 35 40 45 Leu Leu Ile Tyr Gly Thr Asn Lys Arg Ala Pro Gly Ile Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ala Leu Trp Tyr Ser 85 90 95 Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 82 <211> 110 <212> PRT <213> Artificial <220> <223> Synthetic <400> 82 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 Ser Ser Thr Gly Ala Val Thr Thr 20 25 30 Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg 35 40 45 Leu Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ala Leu Trp Tyr Ser 85 90 95 Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 83 <211> 110 <212> PRT <213> Artificial <220> <223> synthetic <400> 83 Glu Ile Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Thr Gly Ala Val Thr Thr 20 25 30 Ser Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg 35 40 45 Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gin Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ala Leu Trp Tyr Ser 85 90 95 Asn Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 84 <211> 110 <212> PRT <213> Artificial <220> <223> synthetic <400> 84 Glu Ile Val Met Thr Gin Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Thr Gly Ala Val Thr Thr 20 25 30 Ser Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg 35 40 45 Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Asp Glu Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gin Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ala Leu Trp Tyr Ser 85 90 95 Asn Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 85 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 85 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 86 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 86 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Gly Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 87 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 87 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Gly Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 88 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 88 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Arg 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 89 <211> 109 <212> PRT <213> Artificial <220> <223> synthetic <400> 89 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Gly Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 90 <211> 109 <212> PRT <213> Artificial <220> <223> synthetic <400> 90 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Ala Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 91 <211> 109 <212> PRT <213> 人工 <220> <223> 合成 <400> 91 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Ala Ser Asn 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 92 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 92 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Arg Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 93 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 93 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Gly 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 94 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 94 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Gly Gly Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 95 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 95 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Arg 20 25 30 Gly Tyr Ala Asn Trp Val Gin Gin Lys Pro Gly Gin Ala Pro Arg Gly 35 40 45 Leu He Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gin Gly Thr Lys Leu Thr Val Leu 100 105 <210> 96 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 96 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Ala Ala Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 97 <211> 109 <212> PRT <213> 人工 <220> <223> 合成 <400> 97 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Arg Gly 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 98 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 98 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Ser Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 99 <211> 109 <212> PRT <213> Artificial <220> <223> Synthetic <400> 99 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Ser Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asp 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 <210> 100 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 100 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 101 <211> 106 <212> PRT <213> Artificial <220> <223> Synthetic <400> 101 Gly Gin Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gin Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gin Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gin Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gin Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 102 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic <400> 102 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 103 <211> 10 <212> PRT <213> Artificial <220> <223> synthetic <220> <221> misc_feature <222> (5) <223> Xaa is selected from N, G, S, D or E,preferably N, G or S <220> <221> misc_feature <222> (6) <223> Xaa is selected from T, G, L or R,preferably T or G <220> <221> misc_feature <222> (7) <223> Xaa is selected from Y, G, A or S,preferably Y, A or S <400> 103 Gly Phe Thr Phe Xaa Xaa Xaa Ala Met Asn 1 5 10 <210> 104 <211> 19 <212> PRT <213> artificial <220> <223> synthetic <220> <221> misc_feature <222> (3) <223> Xaa is selected from R, G, A or S,preferably R or S <220> <221> misc_feature <222> (4) <223> Xaa is selected from S, G, L or R,preferably S or L <220> <221> misc_feature <222> (6) <223> Xaa is selected from Y, G, A or S,preferably Y or A <220> <221> misc_feature <222> (7) <223> Xaa is selected from N, G, S, D or E,preferably N or G <220> <221> misc_feature <222> (8) <223> Xaa is selected from N, G, S, D or E,preferably N or G <400> 104 Arg Ile Xaa Xaa Lys Xaa Xaa Xaa Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 105 <211> 14 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1) <223> Xaa is selected from H, G, A or S,preferably H or A <220> <221> misc_feature <222> (2) <223> Xaa is G or Y <220> <221> misc_feature <222> (3) <223> Xaa is selected from N, G, S, D or E,preferably N or G <220> <221> misc_feature <222> (4) <223> Xaa is selected from F, G, A or S,preferably F or A <220> <221> misc_feature <222> (5) <223> Xaa is G or Y <220> <221> misc_feature <222> (6) <223> Xaa is selected from N, G, S, D or E,preferably N, Q or G <220> <221> misc_feature <222> (7) <223> Xaa is selected from S, G, L or R,preferably S or R <220> <221> misc_feature <222> (8) <223> Xaa is selected from Y, G, A or S,preferably Y or A <220> <221> misc_feature <222> (9) <223> Xaa is selected from V or A <400> 105 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Trp Phe Ala Tyr 1 5 10 <210> 106 <211> 13 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(1) <223> Xaa is selected from R, G, A or S, preferably R or G <220> <221> misc_feature <222> (8) <223> Xaa is selected from T, G, L or R, preferably T or G <220> <221> misc_feature <222> (9) <223> Xaa is selected from T, G, L or R, preferably T or G <220> <221> misc_feature <222> (10) <223> Xaa is selected from S, G, L or R, preferably S or R <400> 106 Xaa Ser Ser Thr Gly Ala Val Xaa Xaa Xaa Tyr Ala Asn 1 5 10 <210> 107 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (3) <223> Xaa is selected from W, G, A or S,preferably W or A <220> <221> misc_feature <222> (4) <223> Xaa is selected from Y, G, A or S,preferably Y or A <220> <221> misc_feature <222> (5) <223> Xaa is selected from S, G, L or R,preferably S or R <220> <221> misc_feature <222> (6) <223> Xaa is selected from N, G, S, D or E,preferably N, G or D <400> 107 Ala Leu Xaa Xaa Xaa Xaa Leu Trp Val 1 5 <210> 108 <211> 100 <212> PRT <213> Artificial <220> <223> Synthetic <400> 108 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg 100 <210> 109 <211> 96 <212> PRT <213> Artificial <220> <223> Synthetic <400> 109 Glu Ile Val Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Leu Pro 85 90 95 <210> 110 <211> 98 <212> PRT <213> Artificial <220> <223> synthetic <400> 110 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 His Tyr Pro Tyr Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Thr 35 40 45 Leu Ile Tyr Asp Thr Ser Asn Lys His Ser Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Leu Leu Ser Tyr Ser Gly 85 90 95 Ala Arg <210> 111 <211> 11 <212> PRT <213> Artificial <220> <223> synthetic <400> 111 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 112 <211> 10 <212> PRT <213> 人工 <220> <223> 合成 <400> 112 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 113 <211> 10 <212> PRT <213> 人工 <220> <223> 合成 <400> 113 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 114 <211> 118 <212> PRT <213> 人工 <220> <223> 合成 <400> 114 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val 115 <210> 115 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic <400> 115 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 Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 <210> 116 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic <400> 116 Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 35 40 45 Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 117 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 117 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 118 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 118 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80 Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 119 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 119 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 120 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 120 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 121 <211> 452 <212> PRT <213> Artificial <220> <223> Synthetic <400> 121 Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Leu 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 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asn Trp Val 35 40 45 Ser Thr Ile Ser His Ser Gly Gly Ser Thr 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 Ile Asp Ala Pro Tyr Tyr Asp Ile Leu Thr Gly Tyr Arg Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 122 <211> 214 <212> PRT <213> Artificial <220> <223> Synthetic <400> 122 Asp Ile Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Gin Ser Gly Val Pro Gin Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gin Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Asp Asp Phe Gin Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Tyr Ser Tyr 85 90 95 Thr Phe Gly Gin Gly Thr Lys Leu Gin Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 123 <211> 455 <212> PRT <213> Artificial <220> <223> Synthetic <400> 123 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg 340 345 350 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 124 <211> 215 <212> PRT <213> Artificial <220> <223> Synthetic <400> 124 Gln Ala Val Val Thr Gin Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Leu Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 125 <211> 455 <212> PRT <213> Artificial <220> <223> Synthetic <400> 125 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Tyr Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 405 410 415 ​​​​​​​​​​​​​​​​​​​Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 126 <211> 455 <212> PRT <213> Artificial <220> <223> Synthetic <400> 126 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg He Arg Ser Lys Ala Gly Gly Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Gin Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Gin 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Gin 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455

Claims

1. A humanized anti-CD3 antibody or an antigen-binding fragment thereof, comprising: HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 14; LCDR1 as shown in SEQ ID NO: 29, LCDR2 as shown in SEQ ID NO: 30 and LCDR3 as shown in SEQ ID NO:

31.

2. The humanized antibody or antigen-binding fragment thereof of claim 1, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 61, and the VL comprises the amino acid sequence shown in SEQ ID NO:

80.

3. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain constant region and / or a light chain constant region.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein The heavy chain constant region HC comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 100; and / or The light chain constant region LC comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 101 or 102.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant region HC comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 100; and / or the light chain constant region LC comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 101 or 102.

6. The antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant region HC comprises an amino acid sequence having 1-20 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 100; and / or the light chain constant region LC comprises an amino acid sequence having 1-20 amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 101 or 102.

7. The antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant region HC comprises the amino acid sequence of SEQ ID NO: 100, and the LC comprises the amino acid sequence of SEQ ID NO: 101 or 102.

8. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain constant region HC consists of the amino acid sequence of SEQ ID NO: 100, and the LC consists of the amino acid sequence of SEQ ID NO: 101 or 102.

9. A humanized anti-CD3 antibody or antigen-binding fragment thereof, comprising VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO: 61, and the VL consists of the amino acid sequence shown in SEQ ID NO:

80.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment is in the IgG1 form, or the IgG2 form, or the IgG3 form, or the IgG4 form. The antibody or antigen-binding fragment thereof according to claim 10 , wherein the antibody or antigen-binding fragment is in the IgG1 format.

12. The CD3-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antibody is a monoclonal antibody.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody or (Fab')2.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a multispecific antibody.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody is a bispecific antibody.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the antibody is a bispecific antibody and comprises a first antigen-binding region that specifically binds to CD3 and a second antigen-binding region that binds to a tumor-associated antigen.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the tumor-associated antigen is HER2 or CD70 or CLAUDIN18.

2.

18. An isolated nucleic acid encoding the CD3-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 17.

19. A vector comprising the isolated nucleic acid of claim 18, wherein the vector is an expression vector.

20. A host cell comprising the nucleic acid of claim 18 or the vector of claim 19.

21. The host cell of claim 20, wherein the host cell is prokaryotic or eukaryotic.

22. The host cell of claim 20, wherein the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antibodies or antigen-binding fragments thereof.

23. The host cell of claim 20, wherein the host cell is a 293 cell or a CHO cell.

24. The host cell of claim 23, wherein the host cell is a CHO-S cell or a HEK293 cell or a 293F cell.

25. A method for producing an antibody or antigen-binding fragment thereof that binds to CD3, the method comprising culturing the host cell of any one of claims 20 to 24 under conditions suitable for expression of a nucleic acid encoding the antibody or antigen-binding fragment thereof that binds to CD3 of any one of claims 1 to 17.

26. The method of claim 25, wherein the method further comprises isolating the antibody or antigen-binding fragment thereof.

27. The method of claim 25 or 26, wherein the method further comprises recovering the CD3-binding antibody or antigen-binding fragment thereof from the host cell.

28. An immunoconjugate comprising the CD3-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 and other substances.

29. The immunoconjugate of claim 28, wherein the other substance is a marker.

30. A pharmaceutical composition comprising the CD3-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 17 or the immunoconjugate of claim 28 or 29.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

32. The pharmaceutical composition of claim 30 or 31 further comprising one or more additional therapeutic agents.

33. The pharmaceutical composition of claim 32, wherein the additional therapeutic agent is a chemotherapeutic agent.

34. The pharmaceutical composition of claim 32, wherein the additional therapeutic agent is a cytokine.

35. The pharmaceutical composition of claim 32, wherein the additional therapeutic agent is a cytotoxic agent.

36. The pharmaceutical composition of claim 32, wherein the additional therapeutic agent is an additional antibody.

37. The pharmaceutical composition of claim 32, wherein the additional therapeutic agent is a small molecule drug.

38. The pharmaceutical composition of claim 32, wherein the additional therapeutic agent is an immunomodulatory agent.

39. A pharmaceutical combination comprising the CD3-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 17 or the immunoconjugate of claim 28 or 29, and one or more other therapeutic agents.

40. The pharmaceutical combination of claim 39, wherein the additional therapeutic agent is a chemotherapeutic agent.

41. The pharmaceutical combination of claim 39, wherein the additional therapeutic agent is a cytokine.

42. The pharmaceutical combination of claim 39, wherein the additional therapeutic agent is a cytotoxic agent.

43. The pharmaceutical combination of claim 39, wherein the additional therapeutic agent is an additional antibody.

44. The pharmaceutical combination of claim 39, wherein the additional therapeutic agent is a small molecule drug.

45. The pharmaceutical combination of claim 39, wherein the additional therapeutic agent is an immunomodulator.

46. ​​Use of the CD3-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 17, or the immunoconjugate of claim 28 or 29, or the pharmaceutical composition of any one of claims 30-38, or the pharmaceutical combination of any one of claims 39-45 in the preparation of a medicament for preventing or treating a tumor in a subject.

47. The use of claim 46, wherein the medicament is administered in combination with one or more therapies.

48. The use according to claim 47, wherein the therapy is a treatment modality and / or another therapeutic agent.

49. The use according to claim 48, wherein the treatment modality comprises surgery and / or radiotherapy.

50. The use of claim 48 or 49, wherein the additional therapeutic agent is a chemotherapeutic agent.

51. The use of claim 48 or 49, wherein the other therapeutic agent is a cytokine.

52. The use of claim 48 or 49, wherein the additional therapeutic agent is a cytotoxic agent.

53. The use of claim 48 or 49, wherein the other therapeutic agent is another antibody.

54. The use of claim 48 or 49, wherein the other therapeutic agent is a small molecule drug.

55. The use of claim 48 or 49, wherein the additional therapeutic agent is an immunomodulator.

56. A method for detecting CD3 in a sample, the method comprising (a) contacting a sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or the immunoconjugate of claim 28 or 29; and (b) detecting formation of a complex between the antibody or antigen-binding fragment thereof and CD3; optionally, the antibody is detectably labeled.

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