RU2811477C2 - Multi-specific antibodies and methods of their production and use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a tetraspecific antibody monomer that binds to CD19, CD3, 4-1BB and PD-L1, as well as to an antibody containing it. Also an isolated nucleic acid encoding the above antibody is disclosed, as well as a vector and a cell containing it are disclosed. The invention also relates to a method of producing a tetraspecific antibody, including culturing the above host cell, as well as a composition containing the above antibody.

EFFECT: invention is effective for the treatment of cancer associated with CD19, CD3, 4-1BB and PD-L1.

21 cl, 8 dwg, 2 ex

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62524558, filed June 25, 2017, which is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The present invention generally relates to the field of biological drugs and, more particularly, relates to the production and use of multispecific antibodies.

State of the art

Tumor cells develop various mechanisms to evade immune system surveillance. One of the main mechanisms for evading immune system surveillance is decreased recognition of tumor cells by the immune system. Defective or absent presentation of tumor-specific antigens leads to immune tolerance and cancer progression. However, even in the presence of effective immunological recognition, tumors develop other mechanisms to avoid elimination by the immune system. Immunocompetent tumors create a suppressive microenvironment to suppress the immune response. Several players are involved in the formation of a tumor suppressive microenvironment, including tumor cells, regulatory T cells, myeloid suppressor cells, stromal cells, and other cell types. Suppression of the immune response can occur in a cell contact-dependent or cell-independent format through the secretion of immunosuppressive cytokines or the elimination of important survival factors from the local environment. Cell contact-dependent suppression relies on molecules expressed on the cell surface, such as programmed death protein ligand 1 (PD-L1), T-lymphocyte-associated protein 4 (CTLA-4), and others [Dunn et al., 2004, Immunity, 21 (2): 137-48; Adachi & Tamada, 2015, Cancer Sci., 106 (8): 945-50].

As the mechanisms by which tumors evade immune recognition continue to be better understood, new treatments targeting these mechanisms have recently emerged. On March 25, 2011, the US Food and Drug Administration (FDA) approved injectable ipilimumab (Yervoy, Bristol-Myers Squibb) for the treatment of unresectable or metastatic melanoma. Yervoy binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), expressed on activated T cells, and blocks the interaction of CTLA-4 with CD80/86 on antigen-presenting cells, thereby blocking the negative or inhibitory signal delivered to T cells. cell via CTLA-4, leading to reactivation of antigen-specific T cells, which in turn leads to tumor eradication in many patients. A few years later, in 2014, the FDA approved Keytruda (pembrolizumab, Merck) and Opdivo (nivolumab, Bristol-Myers Squibb) for the treatment of advanced melanoma. These monoclonal antibodies bind to PD-1, which is expressed on activated and/or exhausted T cells, and block the interaction of PD-1 with PD-L1, which is expressed on tumors, thereby eliminating the inhibitory signal through PD-1 to the T cell , which leads to reactivation of antigen-specific T cells, again leading to tumor eradication in many patients. Since then, further clinical trials have been conducted to compare the effectiveness of Yervoy's monoclonal antibody alone with the combination of Yervoy and Opdivo monoclonal antibodies in the treatment of advanced melanoma, which showed improved overall survival and progression-free survival in patients receiving the antibody combination (Hodi et al., 2016, Lancet Oncol. 17 (11): 1558-1568, Hellman et al., 2018, Cancer Cell 33 (5): 853-861). However, many clinical trials have shown that the significant benefit of treating cancer patients with monoclonal antibodies that are specific for one or more immune checkpoint molecules occurs only in patients with a high mutational load that generates new T cell epitope(s). , which is recognized by antigen-specific T cells, resulting in a clinical response (Snyder et al., 2014, NEJM, 371: 2189-2199). Those patients who have a low tumor mutational load generally do not demonstrate an objective clinical response (Snyder et al., 2014, NEJM, 371: 2189-2199, Hellman et al., 2018, Cancer Cell, 33 (5): 853- 861).

In recent years, other groups have developed an alternative approach that does not require neoepitope presentation by antigen-presenting cells to activate T cells. One example is the development of a bispecific antibody, in which an antibody binding domain that is specific for a tumor-associated antigen, such as CD19, is linked to an antibody binding domain specific for CD3 on T cells, thereby creating a bispecific T cell activator or BiTe molecule . In 2014, the FDA approved a bispecific antibody called blinatumomab for the treatment of B-cell precursor acute lymphoblastic leukemia. Blinatumomab binds the CD19-specific scFv expressed on leukemia cells to the CD3-specific scFv expressed on T cells (Bejnjamin and Stein 2016, Ther. Adv. Adv. Hematol., 7 (3): 142-146). However, despite an initial response rate of >50% of patients with relapsed or refractory ALL, many patients are refractory to blinatumomab therapy or develop relapses after successful treatment with blinatumumab. There is emerging evidence that resistance to blinatumomab or the development of relapses after treatment with blinatumomab is due to the expression of immune checkpoint inhibitory molecules expressed on tumor cells, such as PD-L1, which regulates the inhibitory signal through PD-1 expressed on activated T cells ( Feucht et al., 2016, Oncotarget, 7 (47): 76902-76919). In the case of a study patient who was resistant to blinatumomab therapy, a second round of blinatumomab therapy was performed, but with the addition of a monoclonal antibody, pembrolizumab (Keytruda, Merck), which is specific for PD-1 and blocks the interaction of PD-1 expressed by T -cells, with PD-L1 expressed by tumor cells, which resulted in a high response and a decrease in the number of tumor cells in the bone marrow from 45% to less than 5% in this one patient (Feucht et al., 2016, Oncotarget, 7 (47): 76902-76919). These results indicate that combining a bispecific BiTe molecule with one or more monoclonal antibodies can significantly improve clinical activity compared with either agent alone.

The essence of the invention

The present invention provides, among other things, tetraspecific antibody monomers, antibodies containing tetraspecific monomers, antigen binding fragments thereof, multispecific antibodies, immunoconjugates containing the disclosed antibodies, methods for preparing the disclosed monomers, antigen binding fragments and antibodies, and methods for using the disclosed molecules for the treatment of cancer .

In one aspect, the application provides tetraspecific antibody monomers. In one embodiment, the tetraspecific antibody monomer has an N terminus and a C terminus, and includes, sequentially from N terminus to C terminus, a first scFv domain at the N terminus, a second scFv domain, a Fab domain, an Fc domain, and a third scFv domain. at the C-terminus. The first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each have binding specificity for a different antigen.

In one embodiment, the antigen includes a tumor antigen, an immune signaling antigen, or a combination thereof. In one embodiment, the first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each have specificity for binding to a tumor antigen or immune signaling antigen. In one embodiment, the first domain of the scFv has specificity for binding to a tumor antigen. In one embodiment, the first domain of the scFv has specificity for binding to an immune signaling antigen. In one embodiment, the second domain of the scFv has specificity for binding to a tumor antigen. In one embodiment, the second scFv domain has binding specificity for an immune signaling antigen. In one embodiment, the Fab domain has tumor antigen binding specificity. In one embodiment, the Fab domain has binding specificity for an immune signaling antigen. In one embodiment, the third domain of the scFv has specificity for binding to a tumor antigen. In one embodiment, the third domain of the scFv has specificity for binding to a tumor antigen.

In one embodiment, the tetraspecific monomer includes a first scFv domain, a second scFv domain, a Fab domain, and a third scFv domain, each of which independently has binding specificity for an antigen selected from CD19, CD3, CD137, 4-1BB, PD-L1, ROR1, CD28, 41BB, CEA, HER2, EGFRvIII, EGFR, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glipimae-3, gpA33, GD2, TROP2, NKG2D, BCMA, CD20, CD33, CD123, CD22, CD30, PD1, OX40, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73 and CD39. In one embodiment, the scFv domain, the second scFv domain, the Fab domain, and the third scFv domain each independently have binding specificity for tumor-specific antigens, including, but not limited to, CD19, CD3, CD137, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD20, CD33, CD123, CD22, CD30, or immune checkpoint modulators including, but not limited to, PD-L1, PD1, OX40, 4 -1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, HVEM, CD73, CD39, etc. In one embodiment, one set of scFv domain may specifically bind to modulators immune checkpoints or tumor antigen. In one embodiment, the CD3-specific scFv may be located at the C-terminus or N-terminus of the heavy chain or light chains.

In one embodiment, the first scFv domain, the second scFv domain, the Fab domain, and the third scFv domain each independently have binding specificity for an antigen selected from CD19, CD3, CD137M, PD-L1, and 4-1BB. In one embodiment, the first domain of the scFv has CD19 binding specificity. In one embodiment, the second scFv domain has CD3 binding specificity. In one embodiment, the Fab domain has binding specificity for 4-1BB or CD137. In one embodiment, the third scFv domain has PD-L1 binding specificity.

In one embodiment, the first scFv domain has a CD19 binding specificity, the second scFv domain has a CD3 binding specificity, the Fab domain has a 4-1BB binding specificity, and the third scFv domain has a PD-L1 binding specificity. In one embodiment, the first scFv domain has a CD19 binding specificity, the second scFv domain has a CD3 binding specificity, the Fab domain has a CD137 binding specificity, and the third scFv domain has a PD-L1 binding specificity.

The scFv domain may include a linker linking the scFv domain to the heavy chain or light chain of the antibody. In one embodiment, the linker may include more than 10 amino acids. In one embodiment, the linker may include more than 15 amino acids. In one embodiment, the linker may comprise less than 20 amino acids.

In one embodiment, the linker may include a gly-gly-gly-ser(G4S)n linker, and n may be an integer from 1 to 20. For example, n may be 2, 4, or 6. In one embodiment, the first scFv domain , the second scFv domain or the third scFv domain may contain gly-gly-gly-ser(G4S)n, where n is 2 or 4.

The Fc domain may be humanized. In one embodiment, the Fc domain is a human IgG1 Fc.

In one embodiment, the application provides tetraspecific antibody monomers having an amino acid sequence having percent homology to SEQ ID NOs: 38 and 39. The percent homology is at least 70%, 80%, 90%, 95%, 98%, or 99%.

The application also provides antigen binding fragments. In one embodiment, the application provides scFv domains. In one embodiment, the scFv domain has an amino acid sequence having percent homology to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30 and 32, where the percent homology is at least 70%, 80%, 90%, 95%, 98% or 99%. In one embodiment, the application provides Fab domains. In one embodiment, the Fab domain includes an amino acid sequence having percent homology to SEQ ID NOs: 1-12 and 26-32, where the percent homology is at least 70%, 80%, 90%, 95%, 98%, or 99%. The antigen binding fragments disclosed herein can be used to construct tetraspecific antibody monomers or multispecific antibodies.

In one aspect, the application provides multispecific antibodies. In one embodiment, the multispecific antibody includes tetraspecific antibody monomers. In one embodiment, the multispecific antibody includes two tetraspecific antibody monomers disclosed herein. Since each tetraspecific antibody monomer has four antigen binding domains, the disclosed multispecific antibody may include 8 antigen binding domains. In one embodiment, the antigen binding domains in such a multispecific antibody each independently have binding specificity for a different antigen, resulting in an octaspecific antibody. In one embodiment, the multispecific antibody is a pentaspecific antibody. In one embodiment, the multispecific antibody is a pentaspecific antibody. In one embodiment, the multispecific antibody is a pentaspecific antibody and a hexaspecific antibody. In one embodiment, the multispecific antibody is a pentaspecific antibody and a heptaspecific antibody.

In one embodiment, the multispecific antibody includes a dimer of a tetraspecific antibody monomer to provide a tetraspecific antibody. In one embodiment, the application provides isolated, purified or non-naturally occurring multispecific antibodies. In one embodiment, the application provides a tetraspecific antibody having an amino acid sequence having percent homology to SEQ ID NO: 37-40. The percentage homology is at least 70%, 80%, 90%, 95%, 98% or 99%.

The application also provides an isolated nucleic acid sequence encoding tetraspecific antibody monomers, multispecific antibodies, or antigen binding fragments thereof. In one embodiment, the nucleic acid encodes an amino acid sequence having percent homology to a tetraspecific antibody monomer having SEQ ID NOs: 37 and 38. The percent homology is at least 70%, 80%, 90%, 95%, 98%, or 99%.

The application further provides expression vectors and host cells containing the nucleic acid sequences disclosed herein. In one embodiment, the host cell includes an expression vector. The host cell may be a prokaryotic cell or a eukaryotic cell.

The application further provides immunoconjugates. In one embodiment, the immunoconjugate includes a cytotoxic agent or imaging agent linked to a multispecific antibody disclosed herein via a linker.

The linker may be cleavable or non-cleavable. The linker may include a covalent bond such as an ester bond, an ether bond, an amide bond, a disulfide bond, an imide bond, a sulfone bond, a phosphate bond, a phosphodiester bond, a peptide bond, or a combination thereof. In one embodiment, the linker comprises a hydrophobic poly(ethylene glycol) linker.

The cytotoxic agent may include a chemotherapeutic agent, a growth inhibitory agent, a calicheamicin class cytotoxic agent, an antimitotic agent, a toxin, a radioactive isotope, a therapeutic agent, or a combination thereof. In one embodiment, the cytotoxic agent includes calicheamicin, ozogamicin, monomethyl auristatin E, emtansine, a derivative, or a combination thereof.

The imaging agent can be any compound suitable for imaging purposes. In one embodiment, the imaging agent may be a radionuclide, a fluorescent agent, quantum dots, or a combination thereof.

The application further provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier and a tetraspecific antibody monomer disclosed herein. In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier and a multispecific antibody disclosed herein. In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier and an antigen binding moiety disclosed herein. In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier and an immunoconjugate disclosed herein.

In one embodiment, the pharmaceutical composition further includes a therapeutic agent. Examples of therapeutic agents include, but are not limited to, a radioisotope, radionuclide, toxin, chemotherapeutic agent, or a combination thereof. In one embodiment, the therapeutic agent includes an antibody, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent includes an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof.

In one embodiment, the therapeutic agent includes an immune checkpoint inhibitor. In one embodiment, the therapeutic agent includes an inhibitor of PD1, PDL1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, their derivative or combination.

In one embodiment, the therapeutic agent includes an immune checkpoint inhibitor. In one embodiment, the therapeutic agent includes an inhibitor of PD1, PDL1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, their derivative or combination.

In an additional aspect, the application provides methods for producing tetraspecific antibody monomers, multi-specific antibodies, antigen binding fragments thereof, and immunoconjugates thereof.

In one embodiment, the method includes the steps of culturing a host cell containing the nucleic acid sequences disclosed herein such that the DNA sequence encoding the antibody is expressed, and isolating the antibody. In one embodiment, the antibody is a tetraspecific antibody.

In an additional aspect, the application provides methods of using tetraspecific antibody monomers, multi-specific antibodies, antigen binding fragments thereof, and immunoconjugates thereof for the treatment of cancer. In one embodiment, the method includes the step of administering tetraspecific antibody monomers, multispecific antibodies, antigen binding fragments thereof and immunoconjugates thereof, or a pharmaceutical composition thereof, to a subject in need of such treatment. In one embodiment, the method includes the step of administering to the subject an effective amount of a tetraspecific antibody.

In one embodiment, the method includes direct administration to the tumor site of an effective amount of multispecific monomers, multispecific antibodies, immunoconjugates, antigen-binding fragments thereof.

Various types of cancer can be prevented or treated. In one embodiment, the cancer may have cells expressing ROR1, CEA, HER2, EGFR, EGFR VIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D, BCMA, CD19, CD20, CD33, CD123, CD22 or CD30. Examples of malignant diseases include, but are not limited to, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, cancer urethra, lung cancer, non-small cell lung cancer, small cell lung cancer, brain tumor, glioma, neuroblastoma, esophageal cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer , sarcoma, bone cancer, leukemia, myeloma or lymphoma.

In one embodiment, the method may further include coadministration of an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent may include an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent may include an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof. In one embodiment, the therapeutic agent may include an immune checkpoint inhibitor. In one embodiment, the therapeutic agent may include an inhibitor of PD1, PD-L1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D , CD73, derivative or combination thereof.

In one embodiment, the therapeutic agent may include capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folinic acid, gemicitabine, oxaliplatin , irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopan ib, lapatinib , sunitinib, nilotinib, sorafenib, nab-palitaxel, everolimus, temsirolimus, dabrafenib, vemurafenib, trametinib, vintafolide, apatinib, crizotinib, periforsin, olaparib, bortezomib, tofacitinib, trastuzumab, a derivative or combination thereof.

The subject can represent a person. In one embodiment, the subject may have cancer. The application also provides solutions containing an effective concentration of the multispecific antibodies, monomers or immunoconjugates disclosed herein. In one embodiment, the solution is the subject's blood plasma.

The objects and advantages of the disclosure may become apparent from the following detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings. Other embodiments may become readily apparent to those skilled in the art from the following detailed description, which describes the embodiments by describing the best mode of implementation proposed. As will be understood, other and different embodiments are possible, and certain details of the embodiments may be changed in various obvious respects without departing from the spirit and scope of the invention. Accordingly, the figures and detailed description should be considered illustrative in nature and not limiting.

Brief description of the figures

The foregoing and other features of the present disclosure may become more apparent from the following description and the accompanying claims, when taken in conjunction with the accompanying drawings. It will be understood that these figures illustrate only a few embodiments arranged in accordance with the disclosure and, therefore, they are not to be construed as limiting the scope thereof; the disclosure may be described with further specificity and detail through the use of the accompanying figures, in which:

In fig. 1 is a diagram of the general format of a tetra-specific antibody guided by navigation control (GNC).

In fig. 2 shows experimental results showing a redirected T cell cytotoxicity (RTCC) assay with PBMC (peripheral blood mononuclear cells) as effectors and B-acute lymphoblastic leukemia (B-ALL) Kasumi-2 cells as targets.

In fig. 3 shows experimental results showing the proliferation of CD8+ T cells induced by GNC tetraspecific antibodies.

In fig. 4 shows experimental results showing the proliferation of CD4+ T cells induced by GNC tetraspecific antibodies.

In fig. 5 shows experimental results showing interferon-gamma secretion from PBMCs induced by GNC tetraspecific antibodies.

In fig. 6 shows experimental results showing granzyme B secretion from PBMCs induced by GNC tetraspecific antibodies.

In fig. 7 shows exemplary tetraspecific antibodies with a CD19 tumor antigen recognition domain.

In fig. 8 provides a list of exemplary tetraspecific antibodies disclosed herein.

Detailed Description of the Invention

In the following detailed description, reference is made to the accompanying figures, which form a part thereof. In the figures, like symbols typically identify like components unless the context requires otherwise. The exemplary embodiments described in the detailed description, figures and claims are not intended to be limiting. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It can be readily understood that aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, replaced, combined, separated, and configured in a wide variety of different configurations, all of which are expressly contemplated herein.

The disclosure provides, among other things, isolated antibodies, methods for producing such antibodies, tetraspecific or multispecific molecules, antibody-drug conjugates and/or immunoconjugates consisting of such antibodies or antigen-binding fragments, pharmaceutical compositions containing antibodies, tetraspecific or multispecific molecules, antibody conjugates -drug and/or immunoconjugates, method of their preparation and method of using the disclosed molecules or composition for the treatment of cancer.

The term "antibody" is used in the broadest sense and specifically covers individual monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitope specificity, as well as antibody fragments (e.g. Fab, F(ab') ₂ and Fv) , provided that they exhibit the desired biological activity. In some embodiments, the antibody may be a monoclonal, polyclonal, chimeric, single-chain, tetraspecific or bi-effective, simianized, human and humanized antibody, as well as active fragments thereof. Examples of active molecular fragments that bind known antigens include Fab, F(ab') ₂ , scFv and Fv fragments, including immunoglobulin Fab library expression products and fragments that bind epitopes of any of the antibodies and fragments mentioned above. In some embodiments, the antibody may include immunoglobulin molecules and immunologically active regions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds an antigen. Immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecules. In one embodiment, the antibody may be whole antibodies and any antigen binding fragment derived from whole antibodies. A typical antibody is a heterotetrameric protein typically containing two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, complementarity determining regions (CDRs), and more conserved regions called framework regions (FRs). Each variable region (VH or VL) usually consists of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. Within the variable regions of the light and heavy chains are binding regions that interact with the antigen.

As used herein, the term “monoclonal antibody” refers to an antibody derived from a population of essentially homogeneous antibodies, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific and directed against a single antigenic site. Additionally, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by a culture of hybridoma cells that does not contain impurities of other immunoglobulins. The term “monoclonal” refers to the nature of the antibody as being derived from an essentially homogeneous population of antibodies, and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described by Kohler & Milstein, Nature, 256: 495 (1975), or can be produced by recombinant DNA methods (see, for example, US Pat. No. 4816567).

Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which part of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US patent No. 4816567 and Morrison et al., Proc. Natl. Acad Sci USA 81: 6851-6855 [1984]).

Monoclonal antibodies can be produced using a variety of methods, including mouse hybridoma or phage display (reviewed in Siegel. Transfus. Clin. Biol., 9: 15-22 (2002)), or by molecular cloning of antibodies directly from primary B cells (See Tiller. New Biotechnol., 28: 453-7 (2011)). In the present disclosure, antibodies were generated by immunizing rabbits with human PD-L1 protein and cells transiently expressing human PD-L1 on the cell surface. Rabbits are known to produce antibodies of high affinity, diversity and specificity (Weber et al. Exp. Mol. Med., 49: e305). B cells from immunized animals were cultured in vitro and screened for the production of anti-PD-L1 antibodies. Antibody variable region genes were isolated using recombinant DNA techniques, and the resulting antibodies were recombinantly expressed and then screened for desired traits, such as the ability to inhibit PD-L1 binding to PD-1, the ability to bind to non-human primate PD-L1 , and the ability to enhance human T cell activation. This general method for generating antibodies is similar to that described in Seeber et al., PLOS One, 9: e86184 (2014).

The term “antigen- or epitope-binding site or fragment” refers to antibody fragments that are capable of binding to an antigen (in this case, PD-L1). These fragments may have antigen-binding function and additional functions of the intact antibody. Examples of binding fragments include, but are not limited to, a single chain Fv fragment (scFv), consisting of the VL and VH regions of one arm of an antibody linked into a single polypeptide chain by a synthetic linker, or a Fab fragment, which is a monovalent fragment consisting of the VL region constant light chain (CL), VH and heavy chain constant region 1 (CH1) regions. Antibody fragments may be even smaller subfragments and may consist of domains as small as a single CDR domain, in particular the CDR3 domain of the VL and/or VH regions (for example, see Beiboer et al., J. Mol. Biol ., 296: 833-49 (2000).Antibody fragments are prepared using conventional methods known to those skilled in the art.Antibody fragments can be screened for utility using the same techniques that are used for this purpose for intact antibodies.

"Antigen or epitope binding fragments" can be obtained from the antibody of the present invention using a number of methods known in the art. For example, purified monoclonal antibodies can be digested with an enzyme such as pepsin and subjected to HPLC-based gel filtration. The appropriate fraction containing Fab fragments is then collected and concentrated by membrane filtration and the like. For further description of general techniques for isolating active antibody fragments, see, for example, Khaw, B.A. et al. J. Nucl. Med., 23: 1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

When antibodies are digested by papain, two identical antigen-binding fragments are formed, called “Fab fragments,” each of which has one antigen-binding site, and a residual “Fc fragment,” the name of which reflects its ability to readily crystallize. Treatment with pepsin produces an F(ab') ₂ fragment that has two antigen-binding sites and is still capable of antigen cross-linking.

The Fab fragment may contain a light chain constant region and a first heavy chain constant region (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the CH1 region of the heavy chain, including one or more cysteines from the antibody hinge region. Fab'-SH herein refers to Fab' in which the constant region cysteine residue(s) bear a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments that have hinge region cysteines between them. Other chemical compounds of antibody fragments are also known.

"Fv" is the minimal antibody fragment that contains the complete antigen recognition and binding site. This region consists of a variable region dimer of one heavy and one light chain in close non-covalent association. It is in this configuration that the three CDRs of each variable region interact to form an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs give the antibody antigen binding specificity. However, even one variable region (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

Antibody "light chains" (immunoglobulins) from any vertebrate species can be classified into one of two clearly distinguishable types, called kappa and lambda (λ), based on the amino acid sequences of their constant regions.

Depending on the amino acid sequence of their heavy chain constant region, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chain constant regions that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

"Humanized antibody" refers to a type of engineered antibody whose CDRs are derived from a non-human donor immunoglobulin and the remaining portions, the immunoglobulin-derived molecules, are derived from one (or more) human immunoglobulin(s). In some embodiments, the framework supporting residues may be modified to maintain binding affinity. Methods for producing “humanized antibodies” are well known to those skilled in the art (see, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al., Bio/Technology, 9: 421 (1991)).

The terms "polypeptide", "peptide" and "protein" as used herein are used interchangeably and are defined to mean a biological molecule consisting of amino acids linked by a peptide bond.

As used herein, the terms "a", "an" and "the" mean "one or more" and include the plural unless the context otherwise requires.

The term “isolated” means a biological molecule that does not contain at least some of the components with which it occurs in nature. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or isolated from the cell or cell culture in which it was expressed. Typically, the isolated polypeptide can be obtained by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities.

“Recombinant” means that antibodies are produced using recombinant nucleic acid techniques in exogenous host cells.

The term "antigen" refers to a molecule or fragment thereof that can induce an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and their regions responsible for antigenicity or antigenic determinants.

Also as used herein, the term “immunogenic” refers to substances that induce or enhance the production of antibodies, T cells or other reactive immune cells directed against an immunogenic agent and promote an immune response in humans or animals. An immune response occurs when a subject produces a sufficient number of antibodies, T cells and other reactive immune cells against the administered immunogenic compositions of the present invention to modify or ameliorate the disorder to be treated.

“Specific binding” or “specifically binds to” or “specifically for” a particular antigen or epitope means binding that is distinctly different from a nonspecific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure but which does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Specific binding for a particular antigen or epitope may be exhibited, for example, by an antibody having a KD for the antigen or epitope of at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^{- 11} M, at least about 10 ^-12 M or more, where KD refers to the rate of dissociation of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen may have a KD that is 20, 50, 100, 500, 1000, 5000, 10,000 or more times higher for the control molecule relative to the antigen or epitope.

In addition, specific binding for a particular antigen or epitope may be exhibited, for example, by an antibody having a KA or Ka for the antigen or epitope at least 20, 50, 100, 500, 1000, 5000, 10,000 times higher or greater for the epitope compared to control, where KA or Ka refers to the rate of association of a particular antibody-antigen interaction.

"Homology" between two sequences is determined by sequence identity. If two sequences that are to be compared to each other differ in length, then sequence identity preferably refers to the percentage of nucleotide residues of the shorter sequence that are identical to nucleotide residues of the longer sequence. Sequence identity can be determined tentatively using computer programs. Deviations arising from comparisons between this sequence and the above-described sequences of the invention may be caused, for example, by addition, deletion, substitution, insertion or recombination.

In one aspect, the application provides tetraspecific antibody monomers, antigen binding fragments, and multispecific antibodies. In one embodiment, the application provides tetraspecific antibodies.

In one embodiment, the present invention provides tetraspecific antibodies with binding specificity for four different target antigens. In one embodiment, the target antigens are tumor-specific antigens, a T cell receptor component CD3, or immune checkpoint molecules. Tetraspecific antibodies can directly activate the body's endogenous T cells to kill tumor cells independent of MHC presentation of tumor antigen to antigen-specific T cell receptors. In some embodiments, the tetraspecific antibody immune checkpoint modulating component can overcome the immunosuppressive tumor microenvironment to fully activate exhausted T cells in the tumor microenvironment.

In one embodiment, tetraspecific antibodies have the unique properties of directly activating T cells while at the same time modulating immune checkpoints or inhibiting Tregs or other inhibitory immune cells or targeting tumor antigens. This may provide benefits for patients when BiTE or CAR-T treatments are not suitable. In one embodiment, tetraspecific antibodies may exhibit clinical benefit in solid tumors where BiTE-like technology or CAR-T treatments have not yet demonstrated clinical benefit due to limitations caused by the inhibitory tumor microenvironment.

In one embodiment, the application provides an engineered antibody with 4 different binding domains, or a “tetraspecific antibody.” One binding domain is specific for CD3 on T cells, the second binding domain is specific for tumor-associated antigen, including but not limited to ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypican-3 , gpA33, GD2, TROP2, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, and the third and fourth binding domains are specific for two different immune checkpoint modulators, such as PD-L1, PD-1, OX40, 4- 1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, HVEM, CD73, CD39, etc.

In one embodiment, the tetraspecific molecules (FIG. 1) target the human CD19 (SEQ ID NOs 25-32) tumor-associated antigen. In some embodiments, these targeted tetraspecific proteins carry anti-human PD-L1- (SEQ ID NO: 9-12), anti-human 4-1BB- (SEQ ID NO: 13-24), anti-human CD3 binding domain (SEQ ID NO: 1 -8). The binding domains of a tetraspecific molecule are arranged such that the placement of the binding domain is from the N-terminus to D1, scFv VLVH, followed by D2, scFv VLVH, D3, which is in the Fab position in this group of tetraspecific proteins, followed by Fc of human IgG1 and scFv , VHVL, in D4.

In one embodiment, the tetraspecific protein SI-38E34 (SEQ ID NO: 37-40) consists of anti-human scFv CD19 21D4, anti-human scFv CD3 284A10, anti-human CD137 Fab and anti-human scFv PD-L1 clone PL221G5 occupying positions D1, D2, D3 and D4 respectively. D1, D2, and D3 are genetically linked via a (G4S)×2 10 amino acid linker, as well as the C terminus of the human IgG1 Fc and D4, resulting in a continuous monomeric heavy chain peptide of ~150 kDa containing the binding specificities described above. All scFv molecules described here contain a 20 amino acid gly-gly-gly-gly-ser(G4S)×4 linker that functionally links VH and VL, regardless of the orientation of the V region (LH or HL). The remaining position in the tetraspecific protein, domain 3 (D3), consists of an IgG1 heavy chain, VH-CH1-hinge-CH2-CH3, and its corresponding light chain, VL-CL, which can be a kappa or lambda chain. D1 and D2 are genetically linked via a 10 amino acid (G4S)×2 linker, as are D2, D3, and D4, resulting in a continuous monomeric heavy chain peptide of ~150 kDa. When cotransfected with the appropriate light chain, the final symmetric tetraspecific peptide can be purified with IgG1 Fc (protein A/protein G) and analyzed to assess functional activity. Heavy and light chain gene "cassettes" have been constructed previously so that the V regions can be cloned using restriction sites (HindIII/NheI for the heavy chain and HindIII/BsiWI for the light chain) or "restriction free cloning" such as Gibson Assembly (SGI-DNA, La Jolla, CA), Infusion (Takara Bio USA), or NEBuilder (NEB, Ipswich, MA), the latter of which was used here.

Tetraspecific proteins are produced by a process that involves designing the intact molecule, synthesizing and cloning the nucleotide sequences for each domain, expressing it in mammalian cells, and purifying the final product. Nucleotide sequences were assembled using the Geneious 10.2.3 software package (Biomatters, Auckland, NZ) and separated into their component domains for gene synthesis (Genewiz, South Plainsfield, NJ).

In one embodiment, SI-35E18 (SEQ ID NOs: 65 and 67) is cleaved into its constituent domains, where anti-4-1BB scFv, VLVH, occupies D1, clone anti-human PD-L1 PL230C6 occupies D2 (Fab position), clone antispecific Ig 323H7 for the ROR1 domain of VHVL scFv occupies position D3, and the anti-human CD3 scFv, VHVL, occupies C-terminal D4. Using NEBuilder web tools, 5' and 3' nucleotides were added to each of the domains depending on their position in the larger protein, so that each domain overlaps its flanking domains by 20-30 nucleotides, which direct site-specific recombination, thereby most genetically merging each domain at one stage of gene assembly. Due to the large number of homologous regions in the tetraspecific nucleotide sequence, the N-terminal domains 1 and 2 are assembled separately from the C-terminal D3 and D4. The N- and C-terminal fragments are then assembled together in a second NEBuilder reaction. A small aliquot was transformed into E. coli DH10b (Invitrogen, Carlsbad, CA) and plated into 100 μg/ml TB+ carbenicillin media (Teknova, Hollister, CA) and incubated at 37°C overnight. The resulting colonies were selected and inoculated with 2 ml of overnight cultures in TB+ carbenicillin medium. DNA was prepared (Thermo-Fisher, Carlsbad, CA) from overnight cultures and then sequenced (Genewiz, South Plainsfield, NJ) using sequencing primers (Sigma, St. Louis, MO) flanking each domain. In some embodiments, DNA sequences were collected and analyzed in Geneious.

In yet another aspect, the application provides pharmaceutical compositions including multispecific antibody monomers, multispecific antibodies, antigen binding fragments and immunoconjugates thereof, and methods of using the disclosed antibodies or pharmaceutical compositions for the treatment of cancer.

Advantages of using the disclosed multispecific antibody monomers, multispecific antibodies or compositions for treatment purposes compared to any existing treatments include, but are not limited to: 1) inclusion of an IgG Fc domain may impart a longer serum half-life compared to the bispecific BiTe molecule ; 2) inclusion of two binding domains specific for immune checkpoint modulators, which can inhibit suppressor pathways and simultaneously activate costimulatory pathways; and 3) cross-linking CD3 on T cells to tumor-associated antigens, thereby “redirecting” the T cells to induce tumor death without the need to remove the T cells from the patient and genetically modify them to be tumor cell specific before rather than reintroducing them back into the patient, as is done for chimeric antigen receptor T (CAR-T) cells.

The formulation of the pharmaceutical composition can be accomplished in accordance with standard methodology known to those skilled in the art.

In one embodiment, the antibodies and monomers of the present invention can be formulated in a physiologically acceptable formulation and may contain a pharmaceutically acceptable carrier, diluent and/or excipient using known techniques. For example, an antibody of the invention and as described herein, including any functionally equivalent antibody or functional fragments thereof, in particular, a monoclonal antibody including any functionally equivalent antibody or functional fragments thereof, is combined with a pharmaceutically acceptable carrier, diluent and/or excipient to form a therapeutic compositions. The formulation of the pharmaceutical composition of the present invention can be accomplished in accordance with standard methodology known to those skilled in the art.

With respect to the formulation of suitable compositions for administration to a subject, such as a human patient in need of treatment, the antibodies disclosed herein can be mixed or combined with pharmaceutically acceptable carriers known in the art, depending on the route of administration chosen. There are no particular limitations on the methods of use of the antibodies disclosed herein, and the selection of suitable routes of administration and suitable compositions is known in the art without undue experimentation.

Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions such as oil/water emulsions.

"Pharmaceutically acceptable" refers to those compounds, materials, compositions and dosage forms which, within the bounds of sound medical judgment, are suitable for use in contact with tissues of humans or animals without exhibiting undue toxicity, irritation or other problems or complications commensurate with a reasonable balance of benefit. risk.

In one embodiment, the pharmaceutical composition may include protein carriers, such as, for example, serum albumin or immunoglobulin, in particular of human origin. Additional biologically active agents may be present in the pharmaceutical composition of the invention depending on the intended use. In one embodiment, the proteinaceous pharmaceutically active agent may be present in amounts from 1 ng to 10 mg per dose. Typically, the dosage regimen will be in the range of 0.1 μg to 10 mg of the antibody of the present invention, particularly in the range of 1.0 μg to 1.0 mg, and more particularly in the range of 1.0 μg to 100 μg , where all individual numbers falling within these ranges are also part of the expansion. If administration is by continuous infusion, a more suitable dosage may be in the range of 0.01 μg to 10 mg units per kilogram of body weight per hour, where all individual numbers falling within these ranges are also part of the disclosure.

The compositions of the present invention can be administered to a subject in the form of a solid, liquid or aerosol at a suitable pharmaceutically effective dose. Examples of solid compositions include pills, creams, and implantable dosage units. The tablets can be administered orally. Therapeutic creams can be applied topically. Implantable dosage units can be administered locally, for example, at the site of a tumor, or can be implanted for systemic release of a therapeutic composition, for example, subcutaneously. Examples of liquid compositions include those adapted for intramuscular, subcutaneous, intravenous, intra-arterial, and topical and intraocular injection. Examples of aerosol formulations include inhalation formulations for administration to the lungs.

It is well known to those skilled in the art that the dosage of the composition may depend on various factors, such as, for example, the pathological condition being treated, the particular composition used, and other clinical factors such as the body weight, size, gender, and general health of the patient. body surface area, the specific compound or composition to be administered, other drugs that are administered simultaneously, and the route of administration.

The term “therapeutically effective amount” or “effective amount” refers to an amount of antibody that, when administered to a human or animal, produces a response sufficient to result in a therapeutic effect in said human or animal, for example, to alleviate a disease. One of ordinary skill in the art will readily determine the effective amount after routine procedures. When the disease is cancer, an effective amount of the drug can inhibit (eg, slow to some extent, suppress or stop) one or more of the following exemplary features, including, without limitation, tumor cell growth, tumor cell proliferation, tumor cell motility, infiltration tumor cells to peripheral organs, tumor metastasis and tumor growth. When the disease is Mayer's disease, an effective amount of the drug may alternatively provide one or more of the following when administered to a subject: slow or stop tumor growth, reduce tumor size (e.g., volume or weight), relieve to some extent one or more symptoms, associated with cancer, increase progression-free survival, result in objective response (including, for example, partial response or complete response), and increase overall survival. To the extent that a drug can prevent the growth and/or cause the death of existing tumor cells, it is cytostatic and/or cytotoxic.

One of ordinary skill in the art will be able to determine an effective amount or concentration of the antibodies disclosed herein to effectively treat a pathological condition such as cancer. One skilled in the art can determine other parameters, such as the relative amounts of the various components in the pharmaceutical composition, routes and frequency of administration, without undue experimentation. For example, a suitable injection solution may contain, without limitation, about 1 mg to about 20 mg, about 1 mg to about 10 mg of antibodies per ml. An exemplary dose may be, without limitation, about 0.1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 5 mg/kg body weight. An approximate frequency of administration may be, without limitation, once a day or three times a week.

The compositions can be administered by conventional routes of administration. Typically, the composition may be administered by topical, oral, rectal, nasal, intradermal, intraperitoneal, or parenteral (eg, intravenous, subcutaneous, or intramuscular) routes. In some embodiments, the composition can be incorporated into sustained release matrices, such as biodegradable polymers, wherein the polymers are implanted adjacent to the site where delivery is desired, such as a tumor site. The method includes administering a single dose, administering repeated doses at predetermined intervals, and continuous administration over a predetermined period of time.

Although many forms of administration are possible, an exemplary form of administration may be an injection solution, particularly for intravenous or intra-arterial injection. Typically, a suitable pharmaceutical composition for injection may include pharmaceutically suitable carriers or excipients, such as, but not limited to, a buffer, surfactant or stabilizing agent. Examples of buffers may include, but are not limited to, acetate, phosphate, or citrate buffer. Examples of surfactants may include, but are not limited to, polysorbate. An example of a stabilizer may include, but is not limited to, human albumin.

In one embodiment, administration may be parenteral, such as intravenous. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solvents may be selected from the group consisting of water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers include fluid and nutrient replenishers, electrolyte replenishers (eg, Ringer's dextrose based), and others. Preservatives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, etc.

Antibody monomers, antibodies, antigen binding fragments and immunoconjugates thereof can be used in combination with a therapeutic agent or a composition containing a therapeutic agent for treatment purposes.

In some embodiments, the multispecific antibody molecule is used in combination with one or more additional therapeutic agents in an effective amount. The additional therapeutic agent includes an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In some embodiments, the additional therapeutic agent may be an anti-estrogenic agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof. In some embodiments, the additional therapeutic agent may be an immune checkpoint inhibitor. In some embodiments, the therapeutic agent includes inhibitors of PD1, PDL1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73. their derivative or combination.

In one embodiment, the therapeutic agent may include capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopanib, lapatinib sunitinib , nilotinib, sorafenib, nab-palitaxel, their derivative or combination. In one embodiment, the therapeutic agent may include capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, mustine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folinic acid, gemicitabine, oxaliplatin , irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, vandetanib, afatinib, imatinib, pazopan ib, lapatinib , sunitinib, nilotinib, sorafenib, nab-palitaxel, everolimus, temsirolimus, dabrafenib, vemurafenib, trametinib, vintafolide, apatinib, crizotinib, periforsin, olaparib, bortezomib, tofacitinib, trastuzumab, a derivative or combination thereof.

Malignant tumors, including breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer , stomach cancer, bladder cancer, cervical cancer or brain cancer, may express tumor-associated genes. Inhibition of tumor-associated activity using specific monoclonal antibodies or antigen-binding fragment may have a therapeutic effect on cancer. In addition, administration of a therapeutically effective amount of a composition containing a monoclonal antibody or antigen-binding fragment specific for a tumor-associated protein can cure, prevent, attenuate, and delay the development or metastasis of malignant tumors through the action of a cytotoxic agent.

The present disclosure may be more readily understood by reference to the following detailed description of specific embodiments and examples included herein. Although the present disclosure has been described with reference to specific details of certain embodiments thereof, such details are not intended to be construed as limiting the scope of the disclosure.

Examples

Example 1: Redirected T cell cytotoxicity (RTCC) assay with PBMC (peripheral blood mononuclear cells) as effectors and B-acute lymphoblastic leukemia (B-ALL) cell lines Kasumi-2 and NALM-6 as targets

Tetraspecific antibodies shown in FIG. 7 and 8 were tested for RTCC activity against the B-ALL cell lines Kasumi-2 and Nalm-6 using human PBMC as effectors. Kasumi-2 and Nalm-6 target cells were previously transfected with green fluorescent protein (GFP) and FACS sorted to obtain a population of cells in which GFP expression exceeded 99%. GFP+ Kasumi-2 and GFP+ Nalm-6 cells were counted and set at a density of 100,000 cells/ml in assay media. Human PBMCs were counted and set at a density of 100,000 cells/ml. Antibodies were prepared at 2× final concentration and titrated 1:10 in 6 wells of a 96-well plate in assay medium. In a final 96-well plate, target cells, PBMCs, and serial antibody dilutions were combined by adding 50 μl of target cells (5000), 50 μl of PBMCs (5000), and 100 μl of each antibody dilution to each well of the assay plate. The assay plate was incubated at 37°C for 8 days, and then 100 μl of the supernatant was transferred to a new 96-well plate and frozen at -80°C for subsequent analysis. Cells were resuspended by pipetting and transferred to a 384-well plate. Cells were stained with an antibody mixture that included commercial antibodies directly conjugated to anti-CD4 and anti-CD8. Cells remaining in the wells were washed and resuspended with assay buffer containing commercial directly conjugated anti-CD4 (Biolegend Cat #317436) and anti-CD8 (Biolegend Cat #557746) antibodies plus 7AAD live/dead spot and bead count and then assayed on BD LSRIII Fortessa. The number of CD4+, CD8+, and GFP+ target cells per well was determined. As shown in Figure 2, tetraspecific antibodies SI-38X34, SI-38X35 and SI-38X36 cause T cell killing of most NALM-6 target cells at a concentration of 0.05 pM, which is approximately 10 times stronger, than for bispecific antibodies SI-38X19 and HD37ХI2C. Because bispecific antibody SI-38X19 has binding domains 21D4 (CD19) and 284A10 (CD3), as in tetraspecific antibodies SI-38E34, SI-38E35 and SI-38E36, but tetraspecific antibodies have additional domains 420H5, 466F6, 460C3 (41BB) and PL221 (PDL1), suggesting that additional 41BB and/or PDL1 binding domains in tetraspecific antibodies have an enhancing effect on T cell killing of target cells. In addition, as shown in Fig. 3, the 21D4X284A10 bispecific antibody strongly induced CD8+ T cell proliferation, as did the HD37XI2C bispecific at a 10-fold higher concentration than the 21D4X284A10 bispecific antibody. However, the tetraspecific antibodies SI-38E34, SI-38E35, and SI-38E36 induced much lower levels of CD8+ T cell proliferation at similar concentrations. This data combined with the RTCC data in FIG. 2 suggest that tetraspecific antibodies induce differentiation of CD8+ T cells into final cytotoxic T cells more effectively than bispecific antibodies. As shown in FIG. 4, the bispecific antibodies tested also induced greater CD4+ T cell proliferation, similar to the effect on CD8+ T cell proliferation, and tetraspecific antibodies induced much lower levels of CD4+ T cell proliferation.

Example 2: ELISA analysis of gamma interferon and granzyme B in culture supernatants on day 8 RTCC with CD19-specific GNC antibodies

Supernatants in wells that were stored at -80°C were thawed and assayed for gamma interferon and granzyme B using g-IFN and GrB assay kits from R&D systems (No. DY285B and No. DY2906-05) according to the protocol recommended by the manufacturer. QuantaRed ^™ Enhanced Chemifluorescent HRP Substrate (ThermoFisher Scientific #15159) was added to each well of the ELISA plates and used according to the manufacturer's instructions. As shown in FIG. 6, the bispecific antibody 21D4X284A10 induced a high level of gamma interferon secretion from PBMC at a concentration of 50 pM antibody, almost identical to the tetraspecific antibody SI-34E34, while other tetraspecific antibodies SI-34E35 and SI-34E36, as well as the bispecific antibody HD37XI2C induced secretion tion gamma -interferon from PBMC, but at a much lower level. As shown in Fig. 6, the bispecific antibody 21D4X284A10 induced a high level of granzyme B secretion from PBMCs at 50 pM antibody concentration, almost identical to the tetraspecific antibody SI-34E34, while the other tetraspecific antibodies SI-34E35 and SI-34E36, as well as the bispecific antibody HD37XI2C induced the secretion of granzyme B from PBMCs, but at slightly lower levels. Although tumor cell death mediated by the tetraspecific antibodies SI-38E34, SI-38E35, and SI-38E36 was quite similar, as shown in Fig. 3, the amount of granzyme B secreted from PBMCs was highest with the tetraspecific antibody SI -38E34 at approximately 2-fold higher levels compared to the other 2 tetraspecific antibodies SI-38E35 and SI-38E36.

Although the present disclosure has been described with reference to specific embodiments or examples, it will be understood that the embodiments are illustrative and that the scope of the disclosure is not so limited. Alternative embodiments of the present disclosure may become apparent to those skilled in the art to which the present disclosure pertains. Such alternative embodiments are included within the scope of this disclosure. Accordingly, the scope of the present disclosure is defined by the appended claims and is supported by the foregoing description. All references cited or referred to in this disclosure are incorporated herein by reference in their entirety.

List of sequences

List of tetraspecific antibody sequences

CDRs are underlined in amino acid sequences

>SEQ ID:01 anti-CD3 284A10 VHv1 nucleotide sequence

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATG AACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGCGACGGTGGATCATCTGCTATTACTAGTAACAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCA

>SEQ ID:02 anti-CD3 284A10 VHv1 amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNNI WGQGTLVTVSS

>SEQ ID:03 anti-CD3 284A10 VLv1 nucleotide sequence

GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATG ATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:04 anti-CD3 284A10 VLv1 amino acid sequence

DVVMTQSPSTLSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIK

>SEQ ID:05 anti-CD3 I2C VH nucleotide sequence

CAGGTGCAATTGGTGGAAAGCGGAGGGGGACTGGTGCAGCCCGGGGGAAGTCTGAAGCTGTCCTGTGCCGCCAGCGGCTTTACCTTCAACAAGTACGCCATGAATTGGGTCCGACAGGCCCCAGGGAAAGGCCTGGAATGGGTGGCACGGATTCGGTCCAAGTACAACAACTACGCCACCTACTACGCTGACTCCGTGAAGGACAGATTCACCATCAGCCGGGACGACTCTAAGAACACCGCCTATC TGCAGATGAACAACCTGAAACCGAGGATACAGCTGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACTCCTACATCTCCTACTGGGCCTATTGGGGACAGGGAACACTGGTCACCGTGTCTAGC

>SEQ ID:06 anti-CD3 I2C VH amino acid sequence

QVQLVESGGGLVQPGGSLKLSCAASGFTFN KYAMN WVRQAPGKGLEWVA RIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAY WGQGTLVTVSS

>SEQ ID:07 anti-CD3 I2C VL nucleotide sequence

CAGACCGTGGTCACCCAGGAACCTTCCCTGACCGTCTCCCCAGGCGGCACCGTGACCCTGACCTGTGGCTCCTCTACCGGCGCTGTGACCTCCGGCAACTACCCTAACTGGGTGCAGCAGAAACCCGGACAGGCTCCTAGAGGCCTGATCGGCGGCACCAAGTTTCTGGCCCCTGGCACCCCTGCCAGATTCTCCGGCTCCCTGCTGGGAGGCAAGGCCGCTCTGACCCTGTCTGGCGTGCAGC CTGAGGACGAGGCCGAGTACTACTGTGTGCTGTGGTACTCCAACAGATGGGTGTTCGGAGGCGGCACAAAGCTGACCGTGCTGTCCTCG

>SEQ ID:08 anti-CD3 I2C VL amino acid sequence

QTVVTQEPSLTVSPGGTVTLTC GSSTGAVTSGNYPN WVQQKPGQAPRGLIG GTKFLAP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC VLWYSNRWV FGGGTKLTVLSS

>SEQ ID:09 anti-PD-L1 PL221G5 VHv1 nucleotide sequence

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTACGACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:10 anti-PD-L1 PL221G5 VHv1 amino acid sequence

EVQLLESGGGLVQPGGSLRLSCAASGFSFSS GYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDL WGQGTLVTVSS

>SEQ ID:11 anti-PD-L1 PL221G5 VLv1 nucleotide sequence

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGAT TTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:12 anti-PD-L1 PL221G5 VLv1 amino acid sequence

DIQMTQSPSTLSASVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIK

>SEQ ID:13 anti-4-1BB 420H5 VHv3 nucleotide sequence

CAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCAACTACTGGATATGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATGTTGGTAGTAGTGGTGACACTTACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATC TGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGATAGTAGTAGTTATTATATGTTTAACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:14 anti-4-1BB 420H5 VHv3 amino acid sequence

QSLVESGGGLVQPGGSLRLSCAASGFSFSS NYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSS

>SEQ ID:15 anti-4-1BB 420H5 VLv3 nucleotide sequence

GCCCTTGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTGAGGACATTGATACCTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTTTATGCATCCGATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGAT TTTGCAACTTATTACTGCCAAGGCGGTTACTATACTAGTAGTGCTGATACGAGGGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:16 anti-4-1BB 420H5 VLv3 amino acid sequence

ALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK

>SEQ ID:17 anti-4-1BB 466F6 VHv2 nucleotide sequence

CGGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCATCAGTAGCTACCACATGCAGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAACCATTAGTAGTGGTGGTAATGTATACTACGCGAGCTCCGCGAGAGGCAGATTCACCATCTCCAGACCCTCGTCCAAGAACACGGTGGATCTTCAAA TGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACTCTGGTTATAGTGATCCTATGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:18 anti-4-1BB 466F6 VHv2 amino acid sequence

RSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCARDSGYSDPMWGQGTLVTVSS

>SEQ ID:19 anti-4-1BB 466F6 VLv5 nucleotide sequence

GACGTTGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACCTGTCAGGCCAGTCAGAACATTAGGACTTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAGCCAATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGACCTGGAGCCTGGCGAT GCTGCAACTTACTATTGTCAGTCTACCTATCTTGGTACTGATTATGTTGGCGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:20 anti-4-1BB 466F6 VLv5 amino acid sequence

DVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK

>SEQ ID:21 anti-4-1BB 460C3 VHv1 nucleotide sequence

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAATCGACTTCAGTAGGAGATACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATATATACTGGTAGCCGCGATACTCCTCACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGAAGGTAGCCTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:22 anti-4-1BB 460C3 VHv1 amino acid sequence

EVQLLESGGGLVQPGGSLRLSCAASGIDFSR RYYMC WVRQAPGKGLEWIA CIYTGSRDTPHYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGSL WGQGTLVTVSS

>SEQ ID:23 anti-4-1BB 460C3 VLv1 nucleotide sequence

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAGTAACTGGTTCTCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGA TGATTTTGCAACTTATTACTGCGCAGGCGGTTACAATACTGTTATTGATACTTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:24 anti-4-1BB 460C3 VLv1 amino acid sequence

DIQMTQSPSTLSASVGDRVTITC QSSQSVYSNWFS WYQQKPGKAPKLLIY SASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC AGGYNTVIDTFA FGGGTKVEIK

>SEQ ID:25 anti-CD19 21D4 VH nucleotide sequence

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAACCAGGAGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAGCAGTTCATGGATCGGCTGGGTGCGCCAGGCACCTGGGAAAGGCCTGGAATGGATGGGGATCATCTATCCTGATGACTCTGATACCAGATACAGTCCATCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGGACTGCCTACCTGCAG TGGAGTAGCCTGAAGGCCTCGGACACCGCTATGTATTACTGTGCGAGACATGTTACTATGATTTGGGGAGTTATTATTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

>SEQ ID:26 anti-CD19 21D4 VH amino acid sequence

EVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSS

>SEQ ID:27 anti-CD19 21D4 VL nucleotide sequence

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAG ATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

>SEQ ID:28 anti-CD19 21D4 VL amino acid sequence

AIQLTQSPSSLSSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPFT FGPGTKVDIK

>SEQ ID:29 anti-CD19 HD37 VH nucleotide sequence

CAGGTCCAACTCCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATGCCTTCAGTAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTGGCCTGGAGATGGTGATACTAACTACAATGGAAAGTTCAAGGGGAAAGCCACTCTGACTGCAGACGAATCCTCCAGCACAGCCTACATG CAACTCAGCAGCCTAGCATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGACGGGAGACTACGACGGTAGGCCGTTATTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCC

>SEQ ID:30 anti-CD19 HD37 VH amino acid sequence

QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAMDY WGQGTTVTVSS

>SEQ ID:31 anti-CD19 HD37 VL nucleotide sequence

GATATCCAGCTGACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGTGAGTTACTTGAACTGGTATCAACAGATTCCAGGACAGCCACCCAAACTCCTCATCTATGATGCTTCCAATCTAGTTTCTGGGATCCCACCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGA GAAGGTGGATGCTGCAACCTATCACTGTCAGCAAAGTACTGAGGATCCGTGGACGTTCGGTGGAGGGACCAAGCTCGAGATTAAA

>SEQ ID:32 anti-CD19 HD37 VL nucleotide sequence

DIQLTQSPASLAVSLGQRATISC KASQSVDYDGVSYLN WYQQIPGQPPKLLIY DASNLVS GIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIK

>SEQ ID:33 human IgG1 null mutation (G1m-fa null mutation ADCC/CDC) nucleotide sequence

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAC CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT

>SEQ ID:34 human IgG1 null mutation (G1m-fa null mutation ADCC/CDC) amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLS LSPG

>SEQ ID:35 kappa chain human Ig nucleotide sequence

CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

>SEQ ID:36 kappa chain human Ig amino acid sequence

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

>SEQ ID:37 SI-38E34 (21D4-LH-scFv x 284A10-L1H1-scFv x 420H5-Fab x PL221G5-H1L1-scFv) heavy chain nucleotide sequence

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAG ATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCCGGCGGTGGAGGATCAGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAACCAGGAGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAGCAGTTCATGGATCGGCTGGG TGCGCCAGGCACCTGGGAAAGGCCTGGAATGGATGGGGATCATCTATCCTGATGACTCTGATACCAGATACAGTCCATCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGGACTGCCTACCTGCAGTGGAGTAGCCTGAAGGCCTCGGACACCGCTATGTATTACTGTGCGAGACATGTTACTATGATTTGGGGAGTTATTATTGACTTCTGGGGCCAGGGAACCCTGTCACCGTCTC CTCAGGCGGTGGAGGGTCCGGCGGTGGTGGATCCGACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAG TTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCCGGCGGTGGAGGATCAGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCT CCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGCGACGGTGGATCATCTGCTATT ACTAGTAACAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCAGGGCGGTGGAGGGTCCGGAGGTGGTGGCTCCCAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCAACTACTGGATATGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATGTTGGTAGTAG TGGTGACACTTACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGATAGTAGTAGTTATTATATGTTTAACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTT GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTATACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCTTAAGCCTGTCTCCGGGTGGCGGTGGAGGGTCCGGCGGTGGTGGATCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACA GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTACGACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTA TATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGATCTGGCGGAGGTGGTTCCGGCGGTGGCGGCTCCGGTGGAGGCGGCTCTGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACT TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:38 SI-38E34 (21D4-LH-scFv x 284A10-L1H1-scFv x 420H5-Fab x PL221G5-H1L1-scFv) heavy chain amino acid sequence

AIQLTQSPSSLSSASVGDRVTITTCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGPGTKVDIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFSSSWIGWVRQAPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISADKSIRTAYLQWSSLKASDTAMYYCARHVTMIWGVIIDFWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISTNAMSWVRQAPGKGLEWIGVITGRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSGGGGSGGGGSQSLVESGGGLVQPGGSLRLSCAASGFSFSSNYWICWVRQAPGKGLEWIACIYVGSSGDTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSSYYMFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYSWGNVDNVFGGGTKVEIK

>SEQ ID:39 SI-38E34 (21D4-LH-scFv x 284A10-L1H1-scFv x 420H5-Fab x PL221G5-H1L1-scFv) light chain nucleotide sequence

GCCCTTGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTGAGGACATTGATACCTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTTTATGCATCCGATCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGAT TTTGCAACTTATTACTGCCAAGGCGGTTACTATACTAGTAGTGCTGATACGAGGGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

>SEQ ID:40 SI-38E34 (21D4-LH-scFv x 284A10-L1H1-scFv x 420H5-Fab x PL221G5-H1L1-scFv) light chain amino acid sequence

ALVMTQSPSTLSASVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

--->

List of sequences

<110> SYSTIMMUNE, INC.

SICHUAN BAILI PHARMACEUTICAL CO. LTD.

<120> MULTI-SPECIFIC ANTIBODIES AND METHODS FOR THEIR OBTAINING AND

APPLICATIONS

<130>SIBA036PCT

<140> PCT/US2018/039157

<141> 2018-06-22

<150> US62524558

<151> 2017-06-25

<160> 40

<170> Patent In version 3.5

<210> 1

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 1

gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt caccatcagt accaatgcaa tgagctgggt ccgccaggct

120

ccagggaagg ggctggagtg gatcggagtc attactggtc gtgatatcac atactacgcg

180

agctgggcga aaggcagatt caccatctcc agagacaatt ccaagaacac gctgtatctt

240

caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgcg cgacggtgga

300

tcatctgcta ttactagtaa caacatttgg ggccaaggaa ctctggtcac cgtttcttca

360

<210> 2

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 2

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 Thr Ile Ser Thr Asn

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Thr Gly Arg Asp Ile Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Gly Gly Ser Ser Ala Ile Thr Ser Asn Asn Ile Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 3

gacgtcgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc

60

atcaattgcc aagccagtga gagcattagc agttggttag cctggtatca gcagaaacca

120

gggaaagccc ctaagctcct gatctatgaa gcatccaaac tggcatctgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagag ttcactctca ccatcagcag cctgcagcct

240

gatgattttg caacttatta ctgccaaggc tatttttatt ttattagtcg tacttatgta

300

aattctttcg gcggagggac caaggtggag atcaaa

336

<210> 4

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 4

Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Tyr Phe Tyr Phe Ile Ser

85 90 95

Arg Thr Tyr Val Asn Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 5

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 5

caggtgcaat tggtggaaag cggaggggga ctggtgcagc ccgggggaag tctgaagctg

60

tcctgtgccg ccagcggctt taccttcaac aagtacgcca tgaattgggt ccgacaggcc

120

ccagggaaag gcctggaatg ggtggcacgg attcggtcca agtacaacaa ctacgccacc

180

tactacgctg actccgtgaa ggacagattc accatcagcc ggacgactc taagaacacc

240

gcctatctgc agatgaacaa cctgaaaacc gaggatacag ctgtgtacta ttgtgtgcgg

300

cacggcaact tcggcaactc ctacatctcc tactgggcct attggggaca gggaacactg

360

gtcaccgtgt ctagc

375

<210> 6

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 6

Gln 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 Lys 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 Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 7

cagaccgtgg tcacccagga accttccctg accgtctccc caggcggcac cgtgaccctg

60

acctgtggct cctctaccgg cgctgtgacc tccggcaact accctaactg ggtgcagcag

120

aaacccggac aggctcctag aggcctgatc ggcggcacca agtttctggc ccctggcacc

180

cctgccagat tctccggctc cctgctggga ggcaaggccg ctctgaccct gtctggcgtg

240

cagcctgagg acgaggccga gtactactgt gtgctgtggt actccaacag atgggtgttc

300

ggaggcggca caaagctgac cgtgctgtcc tcg

333

<210> 8

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 8

Gln Thr 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

Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn

85 90 95

Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser

100 105 110

<210> 9

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 9

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt ctccttcagt agcgggtacg acatgtgctg ggtccgccag

120

gctccaggga aggggctgga gtggatcgca tgcattgctg ctggtagtgc tggtatcact

180

tacgacgcga actgggcgaa aggccggttc accatctcca gagacaattc caagaacacg

240

ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaga

300

tcggcgtttt cgttcgacta cgccatggac ctctggggcc agggaaccct ggtcaccgtc

360

tcgagc

366

<210> 10

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 10

Glu Val Gln Leu Leu 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 Ser Phe Ser Ser Gly

20 25 30

Tyr Asp Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Ala Cys Ile Ala Ala Gly Ser Ala Gly Ile Thr Tyr Asp Ala Asn

50 55 60

Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Ala Phe Ser Phe Asp Tyr Ala Met Asp Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 11

gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc

60

atcacttgcc aggccagtca gagcattagt tcccacttaa actggtatca gcagaaacca

120

gggaaagccc ctaagctcct gatctataag gcatccactc tggcatctgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagaa tttactctca ccatcagcag cctgcagcct

240

gatgattttg caacttatta ctgccaacag ggttatagtt ggggtaatgt tgataatgtt

300

ttcggcggag ggaccaaggt ggagatcaaa

330

<210> 12

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Trp Gly Asn

85 90 95

Val Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 13

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 13

cagtcgctgg tggagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc

60

tgtgcagcct ctggattctc cttcagtagc aactactgga tatgctgggt ccgccaggct

120

ccagggaagg ggctggagtg gatcgcatgc atttatgttg gtagtagtgg tgacacttac

180

tacgcgagct ccgcgaaagg ccggttcacc atctccagag acaattccaa gaacacgctg

240

tatctgcaaa tgaacagcct gagagccgag gacacggccg tatattactg tgcgagagat

300

agtagtagtt attatatgtt taacttgtgg ggccagggaa ccctggtcac cgtctcgagc

360

<210> 14

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 14

Gln Ser Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

1 5 10 15

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Asn Tyr

20 25 30

Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Cys Ile Tyr Val Gly Ser Ser Gly Asp Thr Tyr Tyr Ala Ser Ser

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Ser Ser Ser Tyr Tyr Met Phe Asn Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 15

gcccttgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc

60

atcaattgcc aggccagtga ggacattgat acctatttag cctggtatca gcagaaacca

120

gggaaagccc ctaagctcct gatcttttat gcatccgatc tggcatctgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct

240

gatgattttg caacttatta ctgccaaggc ggttactata ctagtagtgc tgatacgagg

300

ggtgctttcg gcggagggac caaggtggag atcaaa

336

<210> 16

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 16

Ala Leu Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Glu Asp Ile Asp Thr Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Tyr Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Gly Tyr Tyr Thr Ser Ser

85 90 95

Ala Asp Thr Arg Gly Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 17

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 17

cggtcgctgg tggagtctgg gggaggcttg gtccagcctg gggggtccct gagactctcc

60

tgtacagcct ctggattcac catcagtagc taccacatgc agtgggtccg ccaggctcca

120

gggaaggggc tggagtacat cggaaccatt agtagtggtg gtaatgtata ctacgcgagc

180

tccgcgagag gcagattcac catctccaga ccctcgtcca agaacacggt ggatcttcaa

240

atgaacagcc tgagagccga ggacacggct gtgtattact gtgcgagaga ctctggttat

300

agtgatccta tgtggggcca gggaaccctg gtcaccgtct cgagc

345

<210> 18

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 18

Arg Ser Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

1 5 10 15

Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Ile Ser Ser Tyr His

20 25 30

Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Thr Ile Ser Ser Gly Gly Asn Val Tyr Tyr Ala Ser Ser Ala Arg Gly

50 55 60

Arg Phe Thr Ile Ser Arg Pro Ser Ser Lys Asn Thr Val Asp Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Asp Ser Gly Tyr Ser Asp Pro Met Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 19

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 19

gacgttgtga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc

60

atcacctgtc aggccagtca gaacattagg acttacttat cctggtatca gcagaaacca

120

gggaaagccc ctaagctcct gatctatgct gcagccaatc tggcatctgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcga cctggagcct

240

ggcgatgctg caacttacta ttgtcagtct acctatcttg gtactgatta tgttggcggt

300

gctttcggcg gagggaccaa ggtggagatc aaa

333

<210> 20

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 20

Asp Val Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Arg Thr Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ala Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asp Leu Glu Pro

65 70 75 80

Gly Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Thr Tyr Leu Gly Thr Asp

85 90 95

Tyr Val Gly Gly Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 21

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 21

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggaat cgacttcagt aggagatact acatgtgctg ggtccgccag

120

gctccaggga aggggctgga gtggatcgca tgcatatata ctggtagccg cgatactcct

180

cactacgcga gctccgcgaa aggccggttc accatctcca gagacaattc caagaacacg

240

ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgaga

300

gaaggtagcc tgtggggcca gggaaccctg gtcaccgtct cgagc

345

<210> 22

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 22

Glu Val Gln Leu Leu 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 Ile Asp Phe Ser Arg Arg

20 25 30

Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Ala Cys Ile Tyr Thr Gly Ser Arg Asp Thr Pro His Tyr Ala Ser

50 55 60

Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Gly Ser Leu Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 23

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 23

gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc

60

atcacttgcc agtccagtca gagtgtttat agtaactggt tctcctggta tcagcagaaa

120

ccagggaaag cccctaagct cctgatctat tctgcatcca ctctggcatc tggggtccca

180

tcaaggttca gcggcagtgg atctgggaca gaattcactc tcaccatcag cagcctgcag

240

cctgatgatt ttgcaactta ttactgcgca ggcggttaca atactgttat tgatactttt

300

gctttcggcg gagggaccaa ggtggagatc aaa

333

<210> 24

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Ser Val Tyr Ser Asn

20 25 30

Trp Phe Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Asn Thr Val

85 90 95

Ile Asp Thr Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 25

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 25

gaggtgcagc tggtgcagtc tggagcagag gtgaagaaac caggagagtc tctgaagatc

60

tcctgtaagg gttctggata cagctttagc agttcatgga tcggctgggt gcgccaggca

120

cctgggaaag gcctggaatg gatggggatc atctatcctg atgactctga taccagatac

180

agtccatcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag gactgcctac

240

ctgcagtgga gtagcctgaa ggcctcggac accgctatgt attactgtgc gagacatgtt

300

actatgattt ggggagttat tattgacttc tggggccagg gaaccctggt caccgtctcc

360

tca

363

<210> 26

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 26

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Ser Ser

20 25 30

Trp Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Arg Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Val Thr Met Ile Trp Gly Val Ile Ile Asp Phe Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 27

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 27

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc

60

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaacca

120

gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct

240

gaagatttg caacttatta ctgtcaacag tttaatagtt acccattcac tttcggccct

300

gggaccaaag tggatatcaa a

321

<210> 28

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 28

Ala Ile Gln Leu 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 Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 29

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 29

caggtccaac tccagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt

60

tcctgcaagg cttctggcta tgccttcagt agctactgga tgaactgggt gaagcagagg

120

cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga tactaactac

180

aatggaaagt tcaaggggaa agccactctg actgcagacg aatcctccag cacagcctac

240

atgcaactca gcagcctagc atctgaggac tctgcggtct atttctgtgc aagacgggag

300

actacgacgg taggccgtta ttactatgct atggactact ggggccaagg gaccacggtc

360

accgtctcctcc

372

<210> 30

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 30

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 31

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 31

gatatccagc tgacccagtc tccagcttct ttggctgtgt ctctagggca gagggccacc

60

atctcctgca aggccagcca aagtgttgat tatgatggtg tgagttactt gaactggtat

120

caacagattc caggacagcc acccaaactc ctcatctatg atgcttccaa tctagtttct

180

gggatcccac ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat

240

cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga ggatccgtgg

300

acgttcggtg gagggaccaa gctcgagatt aaa

333

<210> 32

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 32

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Val Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 33

<211> 987

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 33

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg

60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg

120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca

180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc

240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc

300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgcgggggca

360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

600

gagtacaagt gcgcggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc

660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag

720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc

780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg

840

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg

900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

960

cagaagagcc tctccctgtc tccggggt

987

<210> 34

<211> 329

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 34

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

Arg 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 Ala 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 Ala 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 Thr 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

325

<210> 35

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 35

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct

60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag

120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac

180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag

240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag

300

agcttcaaca ggggagagtg t

321

<210> 36

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 36

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> 37

<211> 3693

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 37

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc

60

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaacca

120

gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct

240

gaagatttg caacttatta ctgtcaacag tttaatagtt acccattcac tttcggccct

300

gggaccaaag tggatatcaa aggcggtggc ggtagtgggg gaggcggttc tggcggcgga

360

gggtccggcg gtggaggatc agaggtgcag ctggtgcagt ctggagcaga ggtgaagaaa

420

ccaggagagt ctctgaagat ctcctgtaag ggttctggat acagctttag cagttcatgg

480

atcggctggg tgcgccaggc acctgggaaa ggcctggaat ggatggggat catctatcct

540

gatgactctg ataccagata cagtccatcc ttccaaggcc aggtcaccat ctcagccgac

600

aagtccatca ggactgccta cctgcagtgg agtagcctga aggcctcgga caccgctatg

660

tattactgtg cgagacatgt tactatgatt tggggagtta ttattgactt ctggggccag

720

ggaaccctgg tcaccgtctc ctcaggcggt ggagggtccg gcggtggtgg atccgacgtc

780

gtgatgaccc agtctccttc caccctgtct gcatctgtag gagacagagt caccatcaat

840

tgccaagcca gtgagagcat tagcagttgg ttagcctggt atcagcagaa accagggaaa

900

gcccctaagc tcctgatcta tgaagcatcc aaactggcat ctggggtccc atcaaggttc

960

agcggcagtg gatctgggac agagttcact ctcaccatca gcagcctgca gcctgatgat

1020

tttgcaactt attactgcca aggctatttt tattttatta gtcgtactta tgtaaattct

1080

ttcggcggag ggaccaaggt ggagatcaaa ggcggtggcg gtagtggggg aggcggttct

1140

ggcggcggag ggtccggcgg tggaggatca gaggtgcagc tggtggagtc tgggggaggc

1200

ttggtccagc ctggggggtc cctgagactc tcctgtgcag cctctggatt caccatcagt

1260

accaatgcaa tgagctgggt ccgccaggct ccagggaagg ggctggagtg gatcggagtc

1320

attactggtc gtgatatcac atactacgcg agctgggcga aaggcagatt caccatctcc

1380

agagacaatt ccaagaacac gctgtatctt caaatgaaca gcctgagagc cgaggacacg

1440

gctgtgtatt actgtgcgcg cgacggtgga tcatctgcta ttactagtaa caacatttgg

1500

ggccaaggaa ctctggtcac cgtttcttca ggcggtggag ggtccggagg tggtggctcc

1560

cagtcgctgg tggagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc

1620

tgtgcagcct ctggattctc cttcagtagc aactactgga tatgctgggt ccgccaggct

1680

ccagggaagg ggctggagtg gatcgcatgc atttatgttg gtagtagtgg tgacacttac

1740

tacgcgagct ccgcgaaagg ccggttcacc atctccagag acaattccaa gaacacgctg

1800

tatctgcaaa tgaacagcct gagagccgag gacacggccg tatattactg tgcgagagat

1860

agtagtagtt attatatgtt taacttgtgg ggccagggaa ccctggtcac cgtctcgagc

1920

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg

1980

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg

2040

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca

2100

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc

2160

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc

2220

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgcgggggca

2280

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

2340

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

2400

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

2460

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

2520

gagtacaagt gcgcggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc

2580

aaagccaaag ggcagccccg agaaccacag gtgtataccc tgcccccatc ccgggatgag

2640

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc

2700

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg

2760

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg

2820

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

2880

cagaagagct taagcctgtc tccgggtggc ggtggagggt ccggcggtgg tggatccgag

2940

gtgcagctgt tggagtctgg gggaggcttg gtacagcctg gggggtccct gagactctcc

3000

tgtgcagcct ctggattctc cttcagtagc gggtacgaca tgtgctgggt ccgccaggct

3060

ccagggaagg ggctggagtg gatcgcatgc attgctgctg gtagtgctgg tatcacttac

3120

gacgcgaact gggcgaaagg ccggttcacc atctccagag acaattccaa gaacacgctg

3180

tatctgcaaa tgaacagcct gagagccgag gacacggccg tatattactg tgcgagatcg

3240

gcgttttcgt tcgactacgc catggacctc tggggccagg gaaccctggt caccgtctcg

3300

agcggtggag gcggatctgg cggaggtggt tccggcggtg gcggctccgg tggaggcggc

3360

tctgacatcc agatgaccca gtctccttcc accctgtctg catctgtagg agacagagtc

3420

accatcactt gccaggccag tcagagcatt agttcccact taaactggta tcagcagaaa

3480

ccagggaaag cccctaagct cctgatctat aaggcatcca ctctggcatc tggggtccca

3540

tcaaggttca gcggcagtgg atctgggaca gaatttactc tcaccatcag cagcctgcag

3600

cctgatgatt ttgcaactta ttactgccaa cagggttata gttggggtaa tgttgataat

3660

gttttcggcg gagggaccaa ggtggagatc aaa

3693

<210> 38

<211> 1231

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 38

Ala Ile Gln Leu 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 Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser

130 135 140

Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Ser Ser Trp

145 150 155 160

Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly

165 170 175

Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

180 185 190

Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Arg Thr Ala Tyr Leu

195 200 205

Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala

210 215 220

Arg His Val Thr Met Ile Trp Gly Val Ile Ile Asp Phe Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser

260 265 270

Val Gly Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Ile Ser

275 280 285

Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

290 295 300

Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe

305 310 315 320

Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu

325 330 335

Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Tyr Phe Tyr Phe

340 345 350

Ile Ser Arg Thr Tyr Val Asn Ser Phe Gly Gly Gly Thr Lys Val Glu

355 360 365

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

385 390 395 400

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

405 410 415

Phe Thr Ile Ser Thr Asn Ala Met Ser Trp Val Arg Gln Ala Pro Gly

420 425 430

Lys Gly Leu Glu Trp Ile Gly Val Ile Thr Gly Arg Asp Ile Thr Tyr

435 440 445

Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

450 455 460

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

465 470 475 480

Ala Val Tyr Tyr Cys Ala Arg Asp Gly Gly Ser Ser Ala Ile Thr Ser

485 490 495

Asn Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

500 505 510

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Leu Val Glu Ser Gly Gly

515 520 525

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Ser Phe Ser Ser Asn Tyr Trp Ile Cys Trp Val Arg Gln Ala

545 550 555 560

Pro Gly Lys Gly Leu Glu Trp Ile Ala Cys Ile Tyr Val Gly Ser Ser

565 570 575

Gly Asp Thr Tyr Tyr Ala Ser Ser Ala Lys Gly Arg Phe Thr Ile Ser

580 585 590

Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

595 600 605

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Ser Ser Ser Tyr

610 615 620

Tyr Met Phe Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

625 630 635 640

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

645 650 655

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

660 665 670

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

675 680 685

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

690 695 700

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

705 710 715 720

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

725 730 735

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

740 745 750

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

755 760 765

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

770 775 780

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

785 790 795 800

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

805 810 815

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

820 825 830

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn

835 840 845

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

850 855 860

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

865 870 875 880

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

885 890 895

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

900 905 910

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

915 920 925

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

930 935 940

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

945 950 955 960

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly

965 970 975

Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

980 985 990

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe

995 1000 1005

Ser Ser Gly Tyr Asp Met Cys Trp Val Arg Gln Ala Pro Gly Lys

1010 1015 1020

Gly Leu Glu Trp Ile Ala Cys Ile Ala Ala Gly Ser Ala Gly Ile

1025 1030 1035

Thr Tyr Asp Ala Asn Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg

1040 1045 1050

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

1055 1060 1065

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ala Phe Ser

1070 1075 1080

Phe Asp Tyr Ala Met Asp Leu Trp Gly Gln Gly Thr Leu Val Thr

1085 1090 1095

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1100 1105 1110

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

1115 1120 1125

Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

1130 1135 1140

Cys Gln Ala Ser Gln Ser Ile Ser Ser His Leu Asn Trp Tyr Gln

1145 1150 1155

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser

1160 1165 1170

Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

1175 1180 1185

Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp

1190 1195 1200

Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Trp Gly Asn Val

1205 1210 1215

Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1220 1225 1230

<210> 39

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> synthesized

<400> 39

gcccttgtga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc

60

atcaattgcc aggccagtga ggacattgat acctatttag cctggtatca gcagaaacca

120

gggaaagccc ctaagctcct gatcttttat gcatccgatc tggcatctgg ggtcccatca

180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct

240

gatgattttg caacttatta ctgccaaggc ggttactata ctagtagtgc tgatacgagg

300

ggtgctttcg gcggagggac caaggtggag atcaaacgta cggtggctgc accatctgtc

360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg

420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa

480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc

540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa

600

gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt

657

<210> 40

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized

<400> 40

Ala Leu Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Glu Asp Ile Asp Thr Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Tyr Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Gly Tyr Tyr Thr Ser Ser

85 90 95

Ala Asp Thr Arg Gly Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (27)

1. Tetraspecific antibody monomer binding to CD19, CD3, 4-1BB and PD-L1, having an N-terminus and a C-terminus, including sequentially located from the N-terminus to the C-terminus: first domain of scFv at the N-terminus, second scFv domain, Fab domain, Fc domain and third scFv domain at the C-terminus, wherein the first scFv domain, the second scFv domain, the Fab domain and the third scFv domain each have specificity for binding to a different antigen, and where the first scFv domain, the second scFv domain, the Fab domain and the third scFv domain each independently have the specificity for binding to an antigen, selected from CD19, CD3, 4-1BB and PD-L1. 2. The tetraspecific antibody monomer of claim 1, wherein the first scFv domain has CD19 binding specificity. 3. The tetraspecific antibody monomer of claim 1, wherein the second scFv domain has CD3 binding specificity. 4. The tetraspecific antibody monomer of claim 1, wherein the Fab domain has binding specificity for 4-1BB. 5. The tetraspecific antibody monomer of claim 1, wherein the third scFv domain has PD-L1 binding specificity. 6. The tetraspecific antibody monomer of claim 1, wherein the first scFv domain has a CD19 binding specificity, wherein the second scFv domain has a CD3 binding specificity, wherein the Fab domain has a 4-1BB binding specificity, and wherein the third scFv domain has a PD binding specificity. -L1. 7. The tetraspecific antibody monomer of claim 1, wherein the first scFv domain, second scFv domain, or third scFv domain comprises a gly-gly-gly-gly-ser(G4S)n linker, where n is 2, 3, or 4. 8. The tetraspecific antibody monomer of claim 1, comprising an amino acid sequence having percent homology to SEQ ID NO: 38 or 40, where the percent homology is at least 98%. 9. Tetraspecific antibody monomer according to claim 1, characterized in that the tetraspecific antibody monomer contains an scFv domain containing an amino acid sequence having a percentage of homology with SEQ ID NO: 2, 4, 6, 8, 10, 12, 26, 28, 30 or 32, where the percentage of homology is at least 98%. 10. Tetraspecific antibody monomer according to claim 1, characterized in that the tetraspecific antibody monomer contains a Fab domain containing an amino acid sequence having a percentage of homology with SEQ ID NO: 2, 4, 6, 8, 10, 12, 26, 28, 30 or 32, where the percentage of homology is at least 90%. 11. The tetraspecific antibody monomer of claim 1, wherein the monomer contains: (i) 3 complementarity determining regions (CDRs) SEQ ID NO: 26 and 3 CDRs SEQ ID NO: 28; (ii) 3 CDR SEQ ID NO: 2 and 3 CDR SEQ ID NO: 4; (iii) 3 CDR SEQ ID NO: 14 and 3 CDR SEQ ID NO: 16 and/or (iv) 3 CDR SEQ ID NO: 10 and 3 CDR SEQ ID NO: 12. 12. The tetraspecific antibody monomer of claim 11, wherein the monomer comprises: (i) 3 complementarity determining regions (CDRs) SEQ ID NO: 26 and 3 CDRs SEQ ID NO: 28; (ii) 3 CDR SEQ ID NO: 2 and 3 CDR SEQ ID NO: 4; (iii) 3 CDR SEQ ID NO: 14 and 3 CDR SEQ ID NO: 16 and (iv) 3 CDR SEQ ID NO: 10 and 3 CDR SEQ ID NO: 12. 13. A tetraspecific antibody that binds to CD19, CD3, 4-1BB and PD-L1, comprising a tetraspecific antibody monomer according to claim 11 or 12. 14. A tetraspecific antibody that binds to CD19, CD3, 4-1BB and PD-L1, comprising a tetraspecific antibody monomer according to claim 1, wherein the tetraspecific antibody contains an amino acid sequence having 98% homology to SEQ ID NO: 38, and an amino acid sequence, having 98% homology with SEQ ID NO: 40. 15. Isolated nucleic acid encoding the tetraspecific antibody according to claim 14. 16. An expression vector containing the isolated nucleic acid sequence according to claim 15. 17. A host cell expressing the tetraspecific antibody of claim 14, comprising the isolated nucleic acid sequence of claim 15, wherein the host cell is a prokaryotic cell or a eukaryotic cell. 18. A method for producing a tetraspecific antibody, comprising culturing the host cell of claim 17 such that the tetraspecific antibody of claim 14 is expressed, and purifying said tetraspecific antibody. 19. A pharmaceutical composition for the treatment of CD19, CD3, 4-1BB and PD-L1 related cancer in humans, comprising a pharmaceutically acceptable carrier and a tetraspecific antibody according to claim 13 or 14 in an effective amount. 20. Use of the tetraspecific antibody of claim 13 or 14 in the manufacture of a medicament for the treatment of CD19, CD3, 4-1BB and PD-L1 related cancer in humans, wherein an effective amount of said tetraspecific antibody is administered to the subject. 21. The use of a tetraspecific antibody according to claim 13 or 14 and an effective amount of a therapeutic agent for combination therapy for the treatment of CD19, CD3, 4-1BB and PD-L1 related cancer in humans, wherein an effective amount of the therapeutic agent is to be co-administered and wherein the therapeutic agent comprises an antibody, chemotherapeutic agent, enzyme, anti-estrogenic agent, receptor tyrosine kinase inhibitor, kinase inhibitor, cell cycle inhibitor, checkpoint inhibitor, DNA, RNA or protein synthesis inhibitor, RAS inhibitor, PD1, PD-L1, CTLA4, 4 inhibitor -1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, CD3, CD19 or a combination thereof.

Images