KR20220116141A - Monoclonal Antibodies Targeting Unique Cancer-Associated Epitopes of CD43 - Google Patents

Abstract

The present invention relates to monoclonal mouse antibodies produced by hybridoma cells deposited under ICLC Accession No. ICLC PD n° 16001. The present invention also relates to an antibody comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, comprising: CDRH1, CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 are amino acid sequences GFTFSSFGMH (SEQ ID NO: 1), YISSGSGNFYYVDTVKG (SEQ ID NO: 43), STYYHGSRGAMDY (SEQ ID NO: 3), SASSSVSSMYWY (SEQ ID NO: 4), DTSKMAS (SEQ ID NO: 4), respectively. : 5), and QQWSSYPPIT (SEQ ID NO: 6). Furthermore, the present invention relates to antibodies that recognize the same epitope.

Description

Monoclonal Antibodies Targeting Unique Cancer-Associated Epitopes of CD43

1. Cross-Reference to Related Applications

This application claims priority to U.S. Application Serial No. 16/449,255, filed on June 21, 2019, the entire contents of which are incorporated herein by reference.

2. Biological Deposit

Hybridomas secreting mAb UMG1 were deposited on August 4, 2016 by the Budapest Treaty with the Centro di Biotecnologie Avanzate (CBA) Interlab Cell Line Collection (ICLC) under ICLC Accession No. ICLC PD n° 16001 .

CD43 is a leukocyte marker that is usually restricted to cells of the hematopoietic lineage. CD43 is broadly expressed on most peripheral and bone marrow-derived cell components. The precursor form of CD43 is migratory with an apparent molecular weight of 54 kD. In its mature form, CD43 is highly glycosylated, giving it a molecular weight of 115-200 kD. CD4 ⁺ thymocytes and monocytes express the 115 kD morphology, while activated CD4 ⁺ and CD8 ⁺ T cells, B cells, neutrophils and platelets express the 130 kD morphology. CD43 is involved in multiple functions such as cell adhesion, apoptosis and migration (Ostberg et al. , Immunology Today 19:546-50, 1998).

UN1, a murine anti-human CD43 monoclonal antibody, was first described 25 years ago. Although originally selected for its high reactivity to human immature thymocytes (Tassone et al. , Tissue Antigens 44:73-82, 1994), the UN1 mAb was subsequently used in immature thymocytes as well as various fetal tissues. (Cecco et al. , Tissue Antigens 51:528-535, 1998; Tassone et al. , Int. J. Oncology 20:707-711, 2002) and breast, rectum, stomach and squamous cell lung Binds to a variety of solid tumors, including carcinomas, but not normal tissues and benign lesions (Tassone et al. , Int. J. Oncol. 20:707-11, 2002; Tassone et al., Anticancer Res . 22:2333-40, 2002]). In addition, the expression level of the UN1 epitope in breast cancer cells has been shown to correlate with the stage of disease (Tassone et al., Anticancer Res . 22:2333-40, 2002). Evidence that an epitope recognized by UN1 is a tumor-fetal antigen that is expressed in cancer tissues but not in most non-neoplastic adult tissues has made UN1 mAbs an attractive tool for tumor detection and immunotherapy (Tuccillo et al . al ., Mol. Cancer Ther . 13(3), 2014).

Using immunoprecipitation and tandem mass spectrometry, the UN1 antibody was found to recognize an epitope on CD43 comprising GalNAc, a monosaccharide O-linked to the polypeptide chain of CD43 (de Laurentiis et al ., Int. J. Biological Macromol. 39:122-126, 2006; de Laurentiis et al ., Molecular & Cellular Proteomics 10:1-12, 2011).

However, despite extensive functional characterization of the UN1 antibody, its CDR sequence has never been determined. Hybridomas secreting UN1 antibody have never been deposited in biological repositories and no UN1 hybridoma master cell bank or working cell bank has been created. There is a need for an antibody that binds to the same or similar epitope as the UN1 antibody for the treatment of cancer, particularly for the treatment of T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma, and for the diagnosis of cancer.

For the past 25 years, we have ultimately propagated cells derived from the original UN1 hybridoma. A recent subclonal secretes a monoclonal antibody termed UMG1, which retains, but not all, of the specific binding properties of the original UN1 antibody, with distinct binding specificities that provide certain advantages.

Briefly, the UMG1 antibody binds to a small subset of lymphocytes in peripheral blood mononuclear cells (PBMCs) of healthy human donors (Example 1). UMG1 positive lymphocytes are mostly CD45 ⁺ CD3 ⁺ CD4 ⁺ CD8 ^- CD127 ⁺ CCR7 ⁺ T lymphocytes (Example 2).

Like UN1, the UMG1 antibody mostly binds to the T-ALL cell line belonging to the EGIL T3 class (Example 3). However, unlike the UN1 antibody, the UMG1 antibody does not bind to breast cancer cells (Example 3). In a first trial, the UMG1 antibody inhibited cancer cells in lung, colorectal, and breast cancer tumors, in contrast to previous observations of UN1 (Laurentiis et al ., Molecular & Cellular Proteomics 10:1-12, 2011, FIG. 9). did not show any binding to (Example 5). However, UMG1 binds to cellular immune infiltrates in a variety of tumors, including lung, colorectal, and breast cancer tumors (Example 5). Although UMG1 does not bind to bone marrow-derived cells in PBMCs of healthy donors (Example 1), UMG1 epitope is expressed in tumor-associated macrophages, and expression of the UMG1 epitope results when macrophages co-culture and interact with cancer cells. raised (Example 6).

UMG1 also binds to some B-cell derived malignancies, including Waldenstrom's macroglobulinemia cell line (Example 3).

UMG1 binds to neutrophils in a small population of healthy donors (Example 9). UMG1 does not bind to activated T lymphocytes of healthy donors (Example 10).

Tissue microarrays of healthy human tissues show a unique distribution of UMG1 mAb binding, limited primarily to the thymus (mostly in cortical thymocytes) and immune infiltrates rarely interspersed in organs such as lymph nodes, intestines, and lungs (Example). 11).

Tissue microarray data show that in addition to lymphoma, the UMG1 epitope is also expressed in melanoma and testicular cancer of various origins (Example 11). In addition, expanded screening of multi-tissue microarrays shows that binding of UMG1 antibodies to immune infiltrates and neoplastic cells is a pediatric tumor of different origin (Example 11).

The chimeric antibody constructed by fusing the variable region of the UMG1 murine antibody to the human IgG Fc region (ch-UMG1) is an antibody-dependent cell against the T-ALL cell line HPB-ALL and the T lymphoma cell line H9 in the presence of effector cells of human PBMC. - were able to induce cytotoxicity (ADCC) (Example 17). The ch-UMG1 antibody was also able to induce ADCC against Waldenstrom's macroglobulinemia cells (Example 18). Humanized antibodies constructed by grafting UGM1 heavy and light chains into a human framework (h-UMG1) were able to reduce the growth of HPB-ALL xenografts in the NSG mouse model (Example 21). Finally, third generation chimeric antigen receptor (CAR) T cells, in which the CAR targeting moiety is an scFv having all six CDRs of the UMG1 antibody, were activated in the presence of H9 T lymphocyte cells (Example 22), and UMG1-derived CAR- It is predicted that T therapy will be effective in treating T cell lymphoma.

UMG1-CD3 bispecific antibodies were able to bind to UMG1 ⁺ or CD3 ⁺ positive cells and redirect T-cell cytotoxicity to target cancer cells (Examples 23-30).

The specificity of the UMG1 antibody is specific to lymphoma (such as non-Hodgkin's lymphoma derived from peripheral B cells or peripheral T cells, but not limited to diffuse large B cell lymphoma, MALT lymphoma, T-lymphoma, anaplastic large including anaplastic large cell lymphoma, follicular lymphoma, and mantle cell lymphoma), testicular cancer (such as seminoma, embryonal carcinoma), yolk sac tumor ), and its targets, including teratoma), multiple myeloma, melanoma, and solid tumors that aberrantly express epitopes such as pediatric cancer or tumors in which the depletion of tumor-associated macrophages has been shown to be therapeutically beneficial. It makes them particularly useful for the treatment of a subset of tumors that express the epitope.

Accordingly, in a first aspect, an anti-CD43 antibody or antigen-binding fragment thereof for use in a method of treating a CD43 positive cancer comprising administering to a patient having a CD43 positive cancer a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment thereof. Provided herein is an antigen-binding fragment, wherein the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 61 to 91 of wild-type CD43, wherein the CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinocytoma (retinoblastoma), hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyolysis It is selected from the group consisting of alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds amino acids 71 to 78 of wild-type CD43. In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds amino acids 73 to 78 of wild-type CD43.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the VH CDR1 sequence of SEQ ID NO: 1; VH CDR2 sequence of SEQ ID NO: 43; and the VH CDR3 sequence of SEQ ID NO: 3; The VL domain comprises the VL CDR1 sequence of SEQ ID NO: 4; VL CDR2 sequence of SEQ ID NO: 5; and the VL CDR3 sequence of SEQ ID NO: 6.

In some embodiments, the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 12. In some embodiments, the anti-CD43 antibody is a murine antibody produced by a hybridoma cell line deposited under ICLC Accession No. ICLC PD No. 16001 (UMG1). In some embodiments, the anti-CD43 antibody is a chimeric antibody further comprising a human constant region domain. In some embodiments, the human constant region domain is an IgG domain. In some embodiments, the antibody heavy chain sequence is SEQ ID NO: 34 and the antibody light chain sequence is SEQ ID NO: 35.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment comprises a human variable domain framework region. In some embodiments, the VH domain has a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; The VL domain has a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

In some embodiments, the anti-CD43 antibody is a monoclonal antibody. In some embodiments, the anti-CD43 antibody or antigen-binding fragment is a F(ab), F(ab)'2, scFv, diabody, single domain antibody, tandab, or flexibody. )to be.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment is capable of inducing antibody dependent cellular cytotoxicity (ADCC) in the presence of effector cells. In some embodiments, the anti-CD43 antibody or antigen-binding fragment is capable of depleting tumor-associated macrophages (TAMs).

In some embodiments, the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug.

In some embodiments, the patient has diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, or mantle cell lymphoma.

In some embodiments, the patient has multiple myeloma.

In some embodiments, the patient has melanoma.

In some embodiments, the patient has testicular cancer. In some embodiments, the testicular cancer is selected from the group consisting of seminal carcinoma, embryonic carcinoma, yolk tumor, and teratoma.

In some embodiments, the patient has nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroidal plexiform papilloma ( choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, or leiomyosarcoma has

In another aspect, provided herein is a bispecific antibody for use in a method of treating a CD43 positive cancer comprising administering to a patient having a therapeutically effective amount of the bispecific antibody, said dual The specific antibody has a first binding specificity for an epitope within amino acids 71 to 78 of wild-type CD43, wherein said CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle Cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, Choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and smooth muscle is selected from the group consisting of leiomyosarcoma. In some embodiments, the bispecific antibody has a second binding specificity for CD3.

In another aspect, provided herein is a CAR-T cell for use in a method of treating a CD43 positive cancer comprising administering to a patient having a therapeutically effective amount of the CAR-T cell, the CAR-T cell comprising: -T cells bind to an epitope within amino acids 71 to 78 of wild-type CD43, wherein said CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma , melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma), glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma selected from the group consisting of

In another aspect, an anti-CD43 antibody or an anti-CD43 antibody or its for use in a method of identifying CD43 positive cancers comprising detectably contacting a sample comprising CD43 positive cancer cells with an anti-CD43 antibody or antigen-binding fragment. Provided herein is an antigen-binding fragment, wherein the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43, and wherein the CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma. , anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, liver Hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma sarcoma , immaturity teratoma, and leiomyosarcoma.

In another embodiment, detectably contacting a sample of the patient with an anti-CD43 antibody or antigen-binding fragment, diagnosing the patient as a CD43 positive cancer if binding to the anti-CD43 antibody or antigen-binding fragment is detected. An anti-CD43 antibody or antigen-binding fragment thereof for use in a method of diagnosing and treating a CD43 positive cancer comprising the steps of, and administering to the patient a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment is provided herein. wherein said anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43, and wherein said CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma. , follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, male medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity immature teratoma), and leiomyosarcoma.

In another aspect, provided herein is a method of treating a CD43 positive cancer. The method comprises administering to a patient having a CD43 positive cancer a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment thereof, wherein the anti-CD43 antibody or antigen-binding fragment is within amino acids 61-91 of wild-type CD43. binds to an epitope, and wherein said CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuronal Neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoblastoma ( ependymoma), primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds amino acids 71 to 78 of wild-type CD43. In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds amino acids 73 to 78 of wild-type CD43.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the VH CDR1 sequence of SEQ ID NO: 1; VH CDR2 sequence of SEQ ID NO: 43; and the VH CDR3 sequence of SEQ ID NO: 3; The VL domain comprises the VL CDR1 sequence of SEQ ID NO: 4; VL CDR2 sequence of SEQ ID NO: 5; and the VL CDR3 sequence of SEQ ID NO: 6.

In some embodiments, the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 12. In some embodiments, the anti-CD43 antibody is a murine antibody produced by a hybridoma cell line deposited under ICLC Accession No. ICLC PD No. 16001 (UMG1). In some embodiments, the anti-CD43 antibody is a chimeric antibody further comprising a human constant region domain. In some embodiments, the human constant region domain is an IgG domain. In some embodiments, the antibody heavy chain sequence is SEQ ID NO: 34 and the antibody light chain sequence is SEQ ID NO: 35.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment comprises a human variable domain framework region. In some embodiments, the VH domain has a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; The VL domain has a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

In some embodiments, the anti-CD43 antibody is a monoclonal antibody. In some embodiments, the anti-CD43 antibody or antigen-binding fragment is a F(ab), F(ab)'2, scFv, diabody, single domain antibody, tandab, or flexibody. )to be.

In some embodiments, the anti-CD43 antibody or antigen-binding fragment is capable of inducing antibody dependent cellular cytotoxicity (ADCC) in the presence of effector cells. In some embodiments, the anti-CD43 antibody or antigen-binding fragment is capable of depleting tumor-associated macrophages (TAMs).

In some embodiments, the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug.

In some embodiments, the patient has diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, or mantle cell lymphoma.

In some embodiments, the patient has multiple myeloma.

In some embodiments, the patient has melanoma.

In some embodiments, the patient has testicular cancer. In some embodiments, the testicular cancer is selected from the group consisting of seminal carcinoma, embryonic carcinoma, yolk tumor, and teratoma.

In some embodiments, the patient has nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroidal plexiform papilloma ( choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, or leiomyosarcoma has

In another aspect, provided herein is a method of treating a CD43 positive cancer comprising administering to a patient having a therapeutically effective amount of a bispecific antibody, wherein the bispecific antibody comprises amino acid 71 of wild-type CD43. to 78, wherein the CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, Testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, Selected from the group consisting of glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma do. In some embodiments, the bispecific antibody has a second binding specificity for CD3.

In another aspect, provided herein is a method of treating a CD43 positive cancer comprising administering to a patient having a therapeutically effective amount of CAR-T cells, wherein the CAR-T cell comprises amino acid 71 of wild-type CD43. to 78, wherein the CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma. ), neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.

In another aspect, provided herein is a method of identifying a CD43 positive cancer comprising detectably contacting a sample comprising CD43 positive cancer cells with an anti-CD43 antibody or antigen-binding fragment thereof, said anti-CD43 The antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43, wherein said CD43 positive cancer is characterized by diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, Multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroidal plexiform papilloma (choroid plexus papilloma), glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma ) is selected from the group consisting of

In another aspect, provided herein is a method of diagnosing and treating a CD43 positive cancer, the method comprising the steps of detectably contacting a sample of a patient with an anti-CD43 antibody or antigen-binding fragment, the anti-CD43 antibody or antigen- diagnosing the patient as a CD43 positive cancer if binding to the binding fragment is detected, and administering to the patient a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment, the anti-CD43 antibody or antigen the binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43, wherein said CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, Melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma), glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma consisting of selected from the group.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, including color drawings, will be provided by the Patent Office upon request and payment of the necessary fees.
A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth exemplary embodiments in which the principles of the present invention are practiced, and the accompanying drawings:
1A and 1B demonstrate expression of an epitope recognized by UMG1 antibody on peripheral blood mononuclear cells from a panel of healthy donors and comparison with commercial CD43 antibody. The scatter plot in FIG. 1A represents data obtained by flow cytometry. The x-axis shows the detected forward scatter (FSC), and the y-axis shows side scatter (SSC). Each dot corresponds to one cell. The histogram of FIG. 1B plots the phycoerythrin signal intensity on the x-axis. The y-axis relates to the signal intensity for the unstained sample at maximum signal intensity (ie 100%). Red curve represents unstained control, blue curve represents scrambled IgG1 stained cells (i.e. negative control), orange curve represents mAb UMG1 stained cells and green curve represents commercial anti-CD43 antibody stained cells .
2A-2D depict four representative scatter plots of cell populations recognized by UMG1 antibodies produced by hybridoma cells deposited in accordance with the present invention. Figures 2a and 2c show two scatter plots belonging to lymphocytes. 2b and 2d are from lymphocytes detected by the UMG1 antibody produced by the hybridoma cells deposited according to the present invention. 2A and 2D , the x-axis shows the CD4 signal intensity, while the y-axis shows the CD8 signal intensity. 2c and 2d , the x-axis represents the CD45ro signal intensity and the y-axis represents the CCR7 signal intensity.
3A to 3B show two histograms. 3A shows UMG1 expression detected by UMG1 antibody on BCWM.1 cell line. 3B shows UMG1 expression on MWCL.1 cell line. The unfilled curve represents unstained control, the curve filled with horizontal lines represents cells stained with secondary mAb, the curve filled with vertical line represents cells stained with scrambled IgG and secondary mAb and the curve filled with diagonal lines represents cells stained with mAb UMG1. Stained cells are shown.
4 depicts tumor associated macrophages (TAMs) recognized by UMG1 antibody. Arrows indicate TAM infiltrating samples of colorectal carcinoma.
5A and 5B show THP1-derived macrophages. Figure 5a shows THP1-derived macrophages stained with: control IgG1 in the absence of tumor cells (first row), ch-UMG1 in the absence of tumor cells (chimeric antibody according to embodiment 2 of the present invention, wherein the native murine The Fc region was replaced with a fully human IgG1 Fc region (second line) and ch-UMG1 in the presence of the PANC1 pancreatic cancer cell line (shown in more detail in the third line and Figure 5b ). The first column shows DAPI staining, the second column shows the antibody + Alexa-fluor 488 labeled secondary antibody, and the third column shows the superimposed image.
Figures 6a and 6b are degranulation assays to evaluate antibody-dependent cell mediated cytotoxicity ( ADCC : Antibody -Dependent Cell mediated Cytotoxicity ) in HPB-ALL ( Figure 6a ) and H9 cell lines ( Figure 6b ) . A bar graph showing the results. Numbers on the x-axis represent the different samples tested: no target is indicated by (1), effector + target cells (E+T) (2), negative control (NC) 200 μg/ml (3), ch- UMG1 10 μg/ml (4), ch-UMG1 50 μg/ml (5), ch-UMG1 100 μg/ml (6), ch-UMG1 200 μg/ml (7), positive control (PC) 200 μg/ ml (8). The y-axis represents the percentage of CD107a ⁺ NK cells affected by ADCC in relation to the total number of CD107a ⁺ NK cells tested per sample.
7 is a bar graph depicting the results of a degranulation assay to evaluate antibody-dependent cell mediated cytotoxicity ( ADCC ) in BCWM.1 cell line. Numbers on the x-axis represent different samples. Numbers on the x-axis represent the different samples tested: no target is indicated by (1), effector + target cells (E+T) (2), negative control (NC) 200 μg/ml (3), ch- UMG1 10 μg/ml (4), ch-UMG1 50 μg/ml (5), ch-UMG1 100 μg/ml (6), ch-UMG1 200 μg/ml (7), positive control (PC) 200 μg/ ml (8). The y-axis represents the percentage of CD107a ⁺ NK cells affected by ADCC in relation to the total number of CD107a ⁺ NK cells tested per sample.
8 is a bar graph illustrating that CD3 ⁺ expressing lymphocytes (CAR-T) were able to release significantly higher amounts of interferon gamma (IFNγ) in the presence of H9 cells. The y-axis represents the concentration of IFNγ expressed in ng/ml. Numbers on the x-axis represent different cells tested: (1) represents non-transduced T cells (negative control); (2) shows T cells transduced with control CAR (vehicle control); and (3) shows T cells transduced with CAR-UMG1.
9 is a bar graph illustrating the ability to release significantly higher amounts of interleukin 2 (IL-2) in the presence of H9 cells. The y-axis represents the concentration of IL2 expressed in ng/ml. Numbers on the x-axis represent different cells tested: (1) represents non-transduced T cells (negative control); (2) shows T cells transduced with control CAR (vehicle control); and (3) shows T cells transduced with CAR-UMG1.
10 is a bar graph showing that CAR-T was able to induce selective apoptosis of H9 cells. The y-axis reports the ratio of dead/viable cells. The x-axis reports: H9 alone (1), also referred to as H9(2) in the presence of non-transduced T cells, H9(3) and UMG1-CAR in the presence of T cells transduced with control CAR. H9(4) in the presence of CAR-UMG1 transduced T cells.
Figure 11 shows tumor volume curves of in vivo experiments comparing control IgG1 (rituximab) versus humanized version of UMG1-mAb (h-UMG1) and afucosylated version of UMG1-mAb (ah-UMG1). It is a linear graph. In the graph, h-UMG1 is indicated by a line with a square, ah-UMG1 is indicated by a (line with a triangle), and the control IgG1 is indicated by a line with a circle.
12A and 12B show representative flow cytometry results of direct staining of h-UMG1-PE and three commercially available CD43 antibodies. 12A shows staining in ALL-SIL human cell line. 12B shows staining in the KE-37 cell line.
13A and 13B show competitive binding assays. 13A shows representative results of a competitive binding assay between h-UMG1, h-UMG1-PE, and three commercially available CD43 antibodies on CEM cell lines. 13B shows representative results of a competitive binding assay between h-UMG1, h-UMG1-PE, and three commercially available CD43 antibodies on HPB-ALL cell line.
14A-14C show representative images of m-UMG1 staining of inflammatory infiltrates in three different human tumors. 14A shows m-UMG1 staining in colorectal adenocarcinoma. 14B shows m-UMG1 staining in lung adenocarcinoma. 14C shows m-UMG1 staining in breast cancer.
15A to 15F show representative results of Example 12 . 15A shows the amino acid sequence of full-length CD43 (SEQ ID NO: 17). 15B is an illustration depicting the CD43 protein variant used to transfect HEK293T cells. 15C and 15E show Western blot results for protein lysates of transfected HEK293T cells. 15D and 15F are bar graphs showing FACS results for transfected HEK293T cells.
16 shows screening of h-UMG1 antibodies for their affinity to HPB-ALL and H9 cell lines, known to be positive for the UMG1 epitope.
17A and 17B show comparative flow cytometry profiles of h-UMG1 and UN1 in four different cell lines of hematopoietic lineage. 17A shows the reported UN1 flow cytometry profile in a cell line of hematopoietic lineage as provided by (Tassone et al. , Tissue Antigens 44:73-82, 1994). 17B shows the UMG1 flow cytometry profile in a cell line of hematopoietic lineage as provided by Example 8 .
18A and 18B show T-cell cytotoxicity assays on cell lines ALL-SIL ( FIG. 18B ) and KE-37 ( FIG. 18A ) as provided by Example 23 with UMG1-CD3 bispecific antibodies. Representative FACS images processed are shown.
19 shows an evaluation of the binding kinetics of h-UMG1 mAb to a recombinant human CD43 analyte (aa 20-253, SEQ ID NO: 42 ) expressed in an E. coli vector, an aglycosylated-CD43 protein. . See Example 15a .
20 depicts a plasmid map for the constructs used to prepare various embodiments of CAR-T provided herein.
Figures 21a and 21b show the efficacy of ah-UMG1 mAb (defucosylated h-UMG1) and UMG1-CD3 bispecific antibodies against T-ALL cells, Figure 21a shows the efficacy of ah-UMG1 mAb against CEM cell lines and Efficacy of UMG1-CD3 bispecific antibody and FIG. 21B shows efficacy of ah-UMG1 mAb and UMG1-CD3 bispecific antibody against T-ALL primary blast cells.
22A-22C show the efficacy of UMG1-CD3 bispecific antibody against T-ALL cell line at different concentrations, FIG. 22A shows the efficacy of UMG1-CD3 bispecific antibody against CEM cell line, and FIG. 22B shows KE37 The potency of the UMG1-CD3 bispecific antibody against the cell line is shown, and FIG. 22C shows the potency of the UMG1-CD3 bispecific antibody against the ALL-SIL cell line.
23 shows induction of apoptosis by different doses of UMG1-CD3 bispecific compared to non-treated control (NC).
24 shows the role of CD8 ⁺ and CD4 ⁺ T-cells in inducing a response to UMG1-CD3 bispecific treatment.
25 shows proliferation of PBMCs in the absence or presence of increasing concentrations of UMG1-CD3 bispecific antibody.
26A and 26B show proliferation of PBMCs in the absence or presence of UMG1-CD3 bispecific antibody, FIG. 26A shows proliferation of PBMCs in the absence of UMG1-CD3 bispecific antibody and FIG. 26B shows UMG1-CD3 bispecific antibody. Proliferation of PBMCs is indicated in the presence of an enemy antibody.
27A and 27B show expression of T cell activation markers in the absence or presence of increasing concentrations of UMG1-CD3-bispecific antibody, FIG. 27A shows the percentage of CD69 positive cells and FIG. 27B shows the percentage of CD25 positive cells. indicates
28A-28D show the induction of IFNγ and TNFα in CD4 ⁺ and CD8 ⁺ T cells, FIG. 28A shows the induction of IFNγ in CD4 ⁺ T cells, and FIG. 28B shows the induction of IFNγ in CD8 ⁺ T cells, Fig. 28c shows the induction of TNFα in CD4 ⁺ T cells, and Fig. 28d shows the induction of TNFα in CD8 ⁺ T cells.
29 shows the effect of UMG1-CD3 bispecific treatment on NFκB protein expression in PBMC and CCRF-CEM cell lines, respectively.
30A -30F show the in vitro efficacy of UMG1-CD3 bispecificity against multiple myeloma cell lines, FIG. 30A shows the expression of the UMG1 epitope on the Delta 47 cell line, and FIG. 30B shows UMG1 against the Delta 47 cell line. - shows the in vitro efficacy of CD3 bispecific, Figure 30c shows the expression of the UMG1 epitope on the H929 cell line, Figure 30d shows the in vitro efficacy of UMG1-CD3 bispecific on the H929 cell line, and Figure 30e shows the in vitro efficacy of the UMG1-CD3 bispecific on the H929 cell line. Expression of the UMG1 epitope for , and FIG. 30c shows the in vitro efficacy of UMG1-CD3 bispecificity against the KMS26 cell line.
Figures 31A-31C show the in vitro efficacy of UMG1-CD3 bispecific against TCAM2, a testicular cancer (spermatoma) cell line, Figure 31A shows the expression of the UMG1 epitope on the TCAM2 cell line, and Figure 31B shows the negative control (NC). shows the in vitro efficacy of UMG1-CD3 bispecificity for TCAM2 cell line compared to , Figure 31c shows the in vitro efficacy of UMG1 -CD3 bispecificity for TCAM2 cell line compared to negative control (NC) and ah-UMG1 monoclonal antibody shows efficacy.
Figure 32 shows the binding activity of different concentrations of UMG1-CD3 bispecific to CEM, Jurkat, and KE37 cell lines.
33 shows binding of h-UMG1 mAb on inactivated and activated neutrophils.
Figures 34a -d show the FACS results of binding of the IgG isotype control and h-UMG1 mAb to activated T cells, Figure 34a shows the binding of the IgG isotype control to CD25 positive cells, and Figure 34b shows the binding of the IgG isotype control to CD69 positive cells. Binding of an IgG isotype control to cells is shown, FIG. 34C shows binding of h-UMG1 mAb to CD25 positive cells, and FIG. 34D shows binding of h-UMG1 mAb to CD69 positive cells.
35 shows the results of epitope mapping of binding of h-UMG1 mAb (humanized anti-CD43 mAb) to human CD43 peptide microarray.
Figure 36 shows the results of epitope mapping of binding of h-UMG1 mAb (humanized anti-CD43 mAb) to PPSTSINEGSPLWTS (SEQ ID NO: 51) peptide microarray.
37A-37C show heat map, substitution matrix and amino acid plot showing conserved amino acids for h-UMG1 mAb binding, FIG. 37A shows heat map, FIG. 37B shows substitution matrix, and FIG. 37C shows amino acid plot. indicates
38A-38C show UMG1 mAb binding to human tissue, FIG. 38A shows binding of UMG1 to thymocytes characterized by increased presence of benign cells in the cortical thymus (entrance), and FIG. 38B shows human tonsils. shows the UMG1 mAb membrane and cytoplasmic binding on the phase, and FIG. 38C shows the cytoplasmic binding observed in macrophages in tissue in the lung.
39A and 39B show UMG1 mAb binding to diffuse large B-cell lymphoma and T-lymphoma, FIG. 39A shows UMG1 binding to diffuse large B-cell lymphoma and FIG. 39B UMG1 binding to T-cell lymphoma. indicates Representative images of Ly2084 tissue microarrays.
40A and 40B show UMG1 mAb binding to melanoma and seminal tumor tissue, FIG. 40A shows UMG1 binding to melanoma tissue (ME2081), and FIG. 40B shows UMG1 binding to seminal tumor tissue (TE2081). .

6.1. Justice

Unless defined otherwise, all technical, notational, and other scientific terms used herein are intended to have the meanings commonly understood by one of ordinary skill in the art.

The monoclonal antibody “ UMG1 ” is a murine anti-human CD43 antibody produced by a hybridoma cell line deposited under ICLC Accession No. ICLC PD n° 16001.

As used herein, unless otherwise limited, the term "antibody" has its art-recognized broadest and includes all known formats including, without limitation: a bivalent monospecific monoclonal antibody; Bivalent bispecific antibody, trivalent trispecific antibody, F(ab) fragment, F(ab)'2 fragment, scFv fragment, diabody, single domain antibody, camelid VHH single domain antibody, tandab , and including flexibodies.

As used herein, the terms “treat” or “treatment” are used in their broadest accepted clinical sense. The term includes, without limitation, alleviation of signs or symptoms of a disease; improvement of signs or symptoms of the disease; relief of symptoms; reduction in disease severity; stabilization of the disease state (ie, not worsening); delaying or slowing disease progression; amelioration or alleviation of the disease state; remission (partial or total), detectable or non-detectable; cure; Including prolonged survival compared to expected survival if not treated. Unless explicitly stated otherwise, “treat” or “treatment” is not intended to prevent or prevent disease.

"Subject" or "individual" means any subject, particularly a mammalian subject, for which diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like. Unless otherwise stated, "patient" means a human "subject".

The term "sufficient amount" means an amount sufficient to produce a desired effect, such as an amount sufficient to modulate protein aggregation in a cell.

The term “therapeutically effective amount” means an amount effective to treat a disease. A “prophylactically effective amount” is an amount effective to delay or prevent the onset of a disease.

In this disclosure, "comprises,""comprising,""containing,""having,""includes,""including. .

As used herein, the singular forms "a,""an," and "the " include plural referents unless the context clearly dictates otherwise. The terms "include", "such as" and the like are intended to convey inclusion without limitation, unless specifically indicated otherwise.

Ranges provided herein are to be understood as shorthand for all values within the range, including the recited endpoints. For example, a range from 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, It is intended to include any number, combination of numbers, or sub-ranges of the group consisting of 47, 48, 49, and 50.

Unless specifically stated or clear from context, the term "about" as used herein is understood within the normal tolerance of one of ordinary skill in the art.

6.2. general overview

The present disclosure provides novel humanized and murine CD43 antibodies and binding molecules derived therefrom that have different expression and binding properties compared to those of other previously disclosed and commercially available CD43 antibodies.

6.3. CD43 binding protein

6.3.1. Mouse monoclonal UMG1 antibody

In a first aspect, the present invention relates to a monoclonal mouse antibody produced by hybridoma cells deposited under ICLC PD n° 16001.

The hybridoma cells were obtained from Centro Biotecnologie Avanzate (CBA), Interlab Cell Line Collection (ICLC), Largo Rosanna, 10, 16132 Genova, under Accession No. ICLC PD n° 16001 on August 4, 2016 under the Budapest Treaty, deposited in Italy. Antibodies were tested in the Examples below. As shown in the Examples, the antibody binds to a specific epitope of CD43 in a portion of the protein that can be sialoglycosylated.

In this first aspect, the invention also relates to an antibody comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1 , CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are amino acid sequences GFTFSSFGMH (SEQ ID NO: 1), YISSGSGNFYYVDTVKG (SEQ ID NO: 43), STYYHGSRGAMDY (SEQ ID NO: 3), SASSSVSSMYWY (SEQ ID NO: 4), respectively , DTSKMAS (SEQ ID NO: 5), and QQWSSYPPIT (SEQ ID NO: 6). This sequence is also described in SEQ ID NO. 1 to 6 are given.

In some embodiments, the antibody comprises all three heavy chain complementarity determining regions (CDRs) and all three light chain CDRs from said antibody.

The above-mentioned CDR sequences are CDR sequences from monoclonal mouse antibodies generated by hybridoma cells deposited under ICLC PD n° 16001, as determined by sequencing.

As used herein, the term "CDR" or "complementarity determining region" refers to a non-contiguous antigen binding site found within the variable regions of both heavy and light chain polypeptides. Such specific regions are described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), wherein the definition includes overlaps or subsets of amino acid residues when compared to each other. The amino acid residues comprising the CDRs as defined by each of the references cited above are presented for comparison. Preferably, the term "CDR" is a CDR as defined by Kabat based on sequence comparison. CDRH1, CDRH2 and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 represent the light chain CDRs.

Such monoclonal antibodies may have framework sequences of any species. Preferably, it may have a mouse or human framework.

The term “framework (FR) amino acid residues” as used herein refers to these amino acids in the framework region of an immunoglobulin chain. The term “framework region” or “FR region” as used herein includes amino acid residues that are part of a variable region but not part of a CDR (eg using the Kabat definition of CDR).

Methods for producing monoclonal antibodies having CDR sequences as mentioned above are known in the art and comprise the introduction of nucleic acid sequences encoding the CDRs into a suitable expression vector encoding the desired framework sequences. Additional methods are detailed below.

In a second aspect, the present invention relates to an antibody which recognizes the same epitope as the antibody according to the first aspect.

Typically, and as is generally known in the art, an antibody is a protein belonging to the protein family of immunoglobulins and consists of the framework regions of the antibody as defined above and the variable regions of complementarity determining regions. In nature, antibodies are produced by plasma cells in response to specific antigens. Generally, each antibody has two identical heavy chain immunoglobulins and two identical light chain immunoglobulins. Each heavy chain and each light chain may have a variable region and a constant region. The constant region of a heavy chain can be one of five types of mammalian Ig heavy chains: α, δ, ε, γ and μ. The types of heavy chains commonly present define the classes (isotypes) of antibodies: IgA, IgD, IgE, IgG and IgM antibodies, respectively. Similarly, the constant region of a light chain can be one of two types of mammalian Ig light chains: κ and λ. The variable regions of the heavy and light chains are usually prepared from a unique combination of numerous protein sequences that allow for binding to a specific antigen.

According to the present invention, the term “antibody” also encompasses an isolated antibody.

Generally, each heavy chain is linked to one of the light chains, whereby the variable regions of the heavy and light chains combine to form one of two identical antigen-binding sites, and their constant regions combine to form the constant region of an antibody. do. Furthermore, constructs of one heavy chain and one light chain can both be linked via the constant regions of their heavy chains to form a “Y”-shaped molecule, whereby the two arms engage the antigen-binding variable region and the stem shows the constant region.

The antibody according to the second aspect may be a complete antibody, which usually means that such an antibody comprises each variable domain as well as a heavy chain of 3 or 4 constant domains and a light chain of 1 constant domain, whereby Each domain may contain further modifications, such as mutations, deletions or insertions, and such modifications do not change the overall domain structure.

Furthermore, the antibody according to the second aspect of the invention may form homodimers or heterodimers, or homomultimers or heteromultimers, whereby "dimer" and "multimer" are two and at least three This means that each of the antibodies can be combined to form a complex. The prefix "homo" means that the complexes can be formed of the same antibody molecule, whereby the prefix "hetero" means that the complexes can be formed of different antibody molecules.

In general, the term "antibody" is intended to encompass all of the above-mentioned immunoglobulin isotypes, i.e. the antibody may be an IgA, IgD, IgE, IgG or IgM antibody and includes any subclass of these isotypes. . Preferably the antibody is an IgG antibody. As antibodies may be expressed and produced recombinantly, such antibodies may also comprise two different constant regions of a heavy chain, for example one IgG1 and one IgG2 heavy chain, or heavy chains from different species. However, the heavy chains are preferably from the same species. Moreover, the antibody may comprise a lambda or kappa light chain.

An antibody recognizing the same epitope as one of the antibodies of the first aspect of the invention further comprises a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3 wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are amino acid sequences GFTFSSFGMH (SEQ ID NO: 1), YISSGSGNFYYVDTVKG (SEQ ID NO: 43), STYYHGSRGAMDY (SEQ ID NO: 3), SASSSVSSMYWY (SEQ ID NO: 4), DTSKMAS (SEQ ID NO: 5), and QQWSSYPPIT (SEQ ID NO: 6), respectively.

Moreover, an antibody recognizing the same epitope as one of the antibodies of the first aspect of the present invention may have at least one conservative amino acid exchange in the CDR compared to the above-mentioned sequence, e.g. chemical structure and properties similar to the original amino acid. and/or antibodies with similar amino acids having a function.

An antibody that recognizes the same epitope as one of the antibodies of the first aspect of the invention may also be an antibody with increased or decreased affinity or specificity compared to one of the antibodies of the first aspect of the invention. Such antibodies are readily obtained by methods known in the art and described further herein below.

In general, the antibody according to the second aspect of the invention comprises at least 75%, 80%, 85%, 90% of the sequence of a monoclonal mouse antibody produced by a hybridoma cell deposited under ICLC PD n° 16001; 95% or 100% (eg at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical sequences, particularly in the variable regions of such antibodies can

In some embodiments, the mouse antibody has 60-100% sequence identity with SEQ ID NO: 7, such as DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEW VAYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDY 80-100%, NO: 7, 90-100%, 90-100%, 80WGQGTSVTVSS a variable heavy chain to an amino acid sequence having between 100%, 95-100%, 97-100%, or 99-100% sequence identity. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% to SEQ ID NO: , an amino acid sequence having 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the mouse antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 7.

In some embodiments, the mouse antibody has 60-100% sequence identity to SEQ ID NO: 12, such as 70-100%, 85-100%, 85-100%, QIALTQSPAIMSASPGEKVTMTCSASSSVSSMYWYQLKPGSSPRLLIYDTSKMASGVPIRFSGSGSGTSFSLTVSRVEAEDAATYYCQQWSSYPPITFGAGSKLELK (SEQ ID NO: 12) a variable light chain to an amino acid sequence having 100%, 95-100%, 97-100%, or 99-100% sequence identity. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises SEQ ID NO: 12 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, amino acid sequences having 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the mouse antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 12.

In some embodiments, the mouse antibody has 60-100% sequence identity to SEQ ID NO: 7, such as 70-100% DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEWV AYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDYWG, NO: 7 to 100%, 85% to 100% SEQ ID NO: 7) A variable heavy chain to an amino acid sequence having 90-100%, 95-100%, 97-100%, or 99-100% sequence identity and 60-100% sequence identity to SEQ ID NO: 12, such as QIALTQSPAIMSASPGEKVTMTCSASSSVSSMYWYQLKPGSSPRLLIYDTSKMASGVPIRFSGSGSGTSFSLTVSRVEAEDAATYYCQQQQWSAEDAATYY to an amino acid sequence having 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to SEQ ID NO: 12) It contains a variable light chain for

6.3.2. UMG1 Monospecific, Bispecific, and Multispecific Antibodies

In general, the antibody according to the invention may be a monoclonal, bispecific or multispecific antibody. Such antibodies are known in the art.

As used in the context of the present invention, the term "monoclonal" can be understood in its broadest sense to describe an antibody produced by a single clone of B lymphocytes or an antibody having the same or similar amino acid sequence.

As used herein, the term “bispecific” may be understood in its broadest sense to describe an antibody that interacts with two different epitopes. Bispecific antibodies can be derived from two monoclonal antibodies. Optionally, these two different epitopes may be located on the same antigen, but these epitopes may also be located on two different antigens.

As used herein, the term “multispecific” may be understood in its broadest sense to describe an antibody that interacts with at least three different types of epitopes. Optionally, these epitopes may be located on the same antigen or on two or more antigens.

Preferably, the antibody according to embodiment 2 of the present invention is a monoclonal antibody.

Furthermore, the antibody according to embodiment 2 of the present invention is preferably a bispecific or multispecific antibody.

Methods for generating antibodies are well known to those skilled in the art. Preferably, the antibody is generated by preparing hybridoma cells. Methods for generating antibodies as well as methods for generating hybridoma cells with the aid of hybridoma cells are well known to those skilled in the art. In general, mice are injected with the desired antigen and euthanized several days later to isolate spleen cells secreting antibodies to the desired antigen. In general, the fusion of these antibody-secreting spleen cells with immortal, non-secreting myeloma cells results in hybridoma cells. Thereafter, these hybridoma cells are generally screened and hybridomas that produce the desired antibody are selected. The selected hybridomas can then be cultured in vivo or in vitro and the desired antibody isolated.

Bifunctional or bispecific antibodies may have antigen binding sites of different specificities. Various forms of bispecific antibodies and their production are known to those skilled in the art. For example, these antibodies are produced by chemical conjugation of BSIgG, an IgG molecule comprising two distinct heavy chains and two distinct light chains secreted by so-called "hybridomas", and antibodies or antibody fragments of different specificities. the resulting heteroantibody conjugates (Segal DM et al. Current Opin. Immunol. 1999, 11:558-562; Van Spriel AB et al. Immunology Today 2000, 21:391-397); each is incorporated by reference in its entirety).

Preparation: Bispecific antibodies can be generated to deliver cells, cytotoxins or drugs to specific sites. An important use may be the delivery of cytotoxic cells of the host, such as NK or cytotoxic T cells, to specific cellular targets (PJ Lachmann, Clin. Exp. Immunol. 1990, 79: 315), which in its entirety incorporated by reference). Another important use may be the delivery of cytotoxic proteins to specific cellular targets (V. Raso, T. Griffin, Cancer Res. 1981, 41:2073; S. Honda et al., Cytotechnology, 1990, 4:59] which is incorporated by reference in its entirety). A further important use may be the delivery of anti-cancer non-protein drugs to specific cellular targets (J. Corvalan et al., Intl. J. Cancer Suppl. 1988, 2:22; M. Pimm). et al., British J. of Cancer 1990, 61:508; each of which is incorporated by reference in its entirety). Such bispecific antibodies can be produced by chemical crosslinking (M. Brennan et al., 1985, Science 229:81; which is incorporated by reference in its entirety), disulfide exchange or hybrid-hybridoma (quadroma). It can be prepared by production. Quadromas can be constructed by fusing hybridomas that secrete two different types of antibodies to two different antigens (Milstein and Cuello, Nature, 1983, 305: 537-539; incorporated by reference).

As used in the context of the present invention, the term "epitope", in its broadest sense, is recognized and bound by an antibody produced by a hybridoma cell deposited under ICLC PD n° 16001 in one or more of the antigen binding regions of the antibody. It can be understood as part of a CD43 molecule that can be The portion of an antibody that binds to an epitope is referred to as a paratope. In many cases, epitopes have conformational properties that specifically result in a binding site for the paratope.

Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics.

Furthermore, it is understood and recognized by those skilled in the art that the interaction between an epitope and an antibody may generally be based on the primary structure of an antigen, ie, a contiguous sequence of amino acids. Ordinarily, the interaction may also be based on the secondary, tertiary or quaternary structure of the epitope, as well as post-translational modifications such as glycosylation. The interaction between the epitope and the antibody may further be based on the three-dimensional structure of the antigen and the resulting surface features, which may involve discrete partitions of the amino acid sequence comprising amino acids that will interact with the antibody at a distant location. have.

When the two antibodies recognize the same or sterically overlapping epitope, the antibody recognizes "the same epitope" as that recognized by the antibody according to the first aspect. In general, the most widely used and rapid method for determining whether two epitopes recognize identical or sterically overlapping epitopes is a competition assay, which can be constructed in any number of different formats using labeled antigen or labeled antibody. . For example, the antigen is immobilized on a 96-well plate, and the ability of the unlabeled antibody to block binding of the labeled antibody is measured using radioactive or enzymatic labeling.

An antibody that recognizes “the same epitope” as that recognized by the antibody according to the first aspect usually refers to an antibody that blocks binding of a reference antibody to an antigen by 50% or more in a competition assay, and conversely, the reference antibody usually competes At least 50% of the binding of the antibody to the antigen is blocked in the assay.

In general, an epitope recognized and bound by an antibody produced by a hybridoma cell deposited under ICLC PD n° 16001 is a glycoside in combination with an antibody produced by a hybridoma cell deposited under ICLC PD n° 16001. It can be identified by any suitable epitope mapping method known in the art.

Examples of such methods include screening peptides of varying lengths derived from CD43 for binding to antibodies produced by hybridoma cells deposited under ICLC PD n° 16001, thereby specifically binding the antibody. The smallest fragment capable of usually contains the sequence of an epitope recognized by the antibody. In general, CD43 peptides can be produced synthetically or by proteolytic digestion of CD43. Methods for identifying peptides that bind to antibodies, such as mass spectrometry analysis, are well known to those skilled in the art. In another example, NMR spectroscopy can be used to identify residues that interact with an antibody of the invention. For example, a CD43 peptide uniformly labeled by 15N and 2H can be mixed with an unlabeled antibody and amino acids in the labeled peptide that interact with the unlabeled antibody will be detected when the position of these amino acids changes within the NMR spectral range. can Typically, the difference between the two spectra allows the identification of amino acids in CD43 that are involved in the interaction with the antibody. Preferably, mass spectrometric analysis is used for identification of peptides that bind to the antibody.

Illustratively, epitopes recognized and bound by antibodies produced by hybridoma cells deposited under ICLC PD n° 16001 can also be obtained by amplifying various DNA fragments of CD43 DNA by polymerase chain reaction (PCR); These fragments can be identified by a method comprising integrating them into an expression vector comprising their ligation to a histidine fusion protein, and after expression of the protein, detecting the epitope by, for example, Western blot.

In a further example, in order to determine the sites on CD43 that are recognized and bound by antibodies produced by hybridoma cells deposited under ICLC PD n° 16001, deletion mutations in the expression vector cloned into CD43 were added by PCR method. By introduction, a series of mutants, such as E. coli ( Escherichia coli ) mutant series, can be prepared, which series express proteins with various deleted sites in CD43. These E. coli mutants can be cultured and induced for expression. Western blot analysis can be performed using cell lysates as antigens.

Additional methods for identifying epitopes recognized and bound by antibodies produced by hybridoma cells deposited under ICLC PD n° 16001 include detection via immunoassays such as enzyme-linked immunosorbent assays (ELISA). can do.

As used in the context of the present invention, the term "affinity" can be understood in its broadest sense as the strength of the interaction between an epitope and the epitope-binding site of an antibody. Methods for determining the absolute value for antibody affinity, ie, the affinity constant, are well known to those skilled in the art. However, the relative values of antibody affinity can also generally be determined, ie, the affinities of two antibodies are compared without determining their absolute values. Methods for comparing the affinity of antibodies are well known to those skilled in the art. For example, flow cytometry can be used, whereby cells with a desired epitope can be independently contacted with different antibodies, which are subsequently displayed using an immunofluorescent secondary antibody. Usually, after detection using flow cytometry, the intensity of the signal of the antibody can be compared.

Screening method : Methods for identifying an antibody according to the second aspect that recognize the same epitope as that recognized by the antibody according to the first aspect are well known to the person skilled in the art. For example, the antibody according to the second aspect can be identified by phage display based on the antibody library.

Consequently, an antibody of the invention that recognizes the same epitope may also be a human antibody.

In another preferred embodiment, the antibody according to the second aspect is a chimeric antibody. In a more preferred embodiment, the antibody according to the second aspect is a chimeric antibody according to the first aspect.

A chimeric antibody is an antibody in which at least one region of an immunoglobulin of one species is genetically fused to another region of an immunoglobulin of another species to reduce the immunogenicity of the antibody (see, e.g., U.S. Pat. 4,816,567 and US Pat. No. 4,816,397).

6.3.3. Humanized UMG1 Antibody

In another preferred embodiment, the antibody according to the second aspect is a humanized antibody. In a more preferred embodiment, the antibody according to the second aspect is a chimeric or humanized antibody according to the first aspect.

Generally, humanized antibodies are a particular type of chimeric antibody. For example, humanized antibodies can be generated by grafting DNA of a human antibody into DNA encoding a mouse antibody framework or by grafting DNA of a mouse antibody into DNA encoding a human antibody framework. Preferably, the DNA of the human antibody is grafted into the DNA encoding the mouse antibody framework. In general, grafting of DNA involves grafting one or more DNA sequences into target antibody framework encoding DNA. Optionally, the heavy and light chains as well as the variable and constant regions may be partially or fully humanized. Preferably, the heavy and light chain variable regions of the mouse antibody are humanized. More preferably, the heavy and light chain variable regions of the mouse antibody are humanized by changing the DNA sequence encoding 1 to 50, preferably 1 to 30, more preferably 1 to 20 amino acids. Transplanted DNA may comprise regions of DNA of six hypervariable loops that determine antigen specificity, generally referred to as complementarity determining regions (CDRs), or regions of DNA that do not contain CDRs, or both. Preferably, humanization comprises grafting of DNA that does not comprise CDRs.

In general, the resulting DNA construct can then be used to express and generate antibodies that are usually less immunogenic or not immunogenic compared to the non-human parental antibody. This includes the production of deglycosylated antibodies or modified antibodies such as afucosylated antibodies. Such methods are well known in the art. Consequently, an antibody of the invention that recognizes the same epitope may also be a deglycosylated antibody or an afucosylated antibody.

6.3.4. Engineered Humanized Antibodies

The present disclosure also provides engineered humanized antibodies that recognize CD43. The h-UMG1 antibody may comprise one or more of the variable heavy and light chain regions provided as SEQ ID NOs: 8-11 , and SEQ ID NOs: 13-16 , respectively. Those skilled in the art can create various embodiments by making one or more conservative substitutions of amino acid residues provided by the present disclosure. A “conservative substitution” or “conservative amino acid substitution” refers to the substitution of an amino acid with a chemically or functionally similar amino acid.

In some embodiments, the antibody is IgG1, IgG2, IgG4, or IgM. In some embodiments, the antigen binding protein is an Fv fragment, Fab fragment, F(ab') ₂ fragment, Fab' fragment, scFv fragment, scFv-Fc fragment, and/or single-domain antibody.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity with SEQ ID NO: 8, such as 70-100% EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVSYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWG ID: 90-100%) an amino acid sequence having 100%, 95-100%, 97-100%, or 99-100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 8.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity with SEQ ID NO: 9, such as 70-100% EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVSYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWG ID: 90-100%) 100%, 95-100%, 97-100%, or 99-100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO:9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO:9.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity to SEQ ID NO: 10, such as 70-100% EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWG ID: 10-100% an amino acid sequence having 100%, 95-100%, 97-100%, or 99-100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 10.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity to SEQ ID NO: 11, such as QVQLVESGGGVVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTYY, 70% to 100% HGSRGAMDYWG (ID NO: 11) to 100%, HGSRGAMDYWGQGT 100%, 95-100%, 97-100%, or 99-100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 11.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity to SEQ ID NO: 13, such as EIVLTQSPATLSLSPGERATLSCSASSSVSSMYWYQQKPGLAPRLIYDTSKMASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSYPPITFGQGTRLEIK (SEQ ID NO: 13 to 90-100%, 70-100%, 80-100%) 100%, 95-100%, 97-100%, or 99-100% sequence identity.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 13 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 13.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity to SEQ ID NO: 14, such as EIALTQSPATLSLSPGERATLSCSASSSVSSMYWYQLKPGLAPRLIYDTSKMASGIPIRFSGSGSGTDFTLTVSRVEPEDFAVYYCQQWSSYPPITFGQGTRLEIK (SEQ ID NO: 14) to 100%, 70-100%, 80-100% an amino acid sequence having 100%, 95-100%, 97-100%, or 99-100% sequence identity.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 14 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 14.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity with SEQ ID NO: 15, such as 70-100% sequence identity QVVMTQSPAFLSVTPGEKVTITCSASSSVSSMYWYQQKPDQAPKLLIYDTSKMASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSYPPITFGGGTKVEIK (SEQ ID NO: 15-100%, 80-100%, 90- 100%) an amino acid sequence having 100%, 95-100%, 97-100%, or 99-100% sequence identity.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 15.

In some embodiments, the h-UMG1 antibody has 60-100% sequence identity with SEQ ID NO: 16, such as 70-100% sequence identity QVVMTQSPAFLSVTPGEKVTITCSASSSVSSMYWYQLKPDQAPKLLIYDTSKMASGVPIRFSGSGSGTDFTFTVSSVEAEDAATYYCQQWSSYPPITFGGGTKVEIK (SEQ ID NO: 16-100%, 70-100%, 90- 100%) an amino acid sequence having 100%, 95-100%, 97-100%, or 99-100% sequence identity.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 13 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 13 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 13.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence with 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 16 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 8 and SEQ ID NO: 16 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 8 and SEQ ID NO: 16.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 13 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 13 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 13.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 14.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 16 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 9 and SEQ ID NO: 16 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 16.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 13 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 13 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 13.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 14.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 15.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 16 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 10 and SEQ ID NO: 16 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10 and SEQ ID NO: 16.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 13 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 13 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 13.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 14 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 14.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 15 and 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 15.

In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 16 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises SEQ ID NO: 11 and SEQ ID NO: 16 with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11 and SEQ ID NO: 16.

In another preferred embodiment, the monoclonal antibody according to the antibody of aspect 2 is directed against the EGIL T3 subgroup of T cell acute lymphoblastic leukemia (T-ALL) cells, against T cell lymphoblastic lymphoma cells, and May induce antibody-dependent cellular cytotoxicity (ADCC) against Waldenstrom macroglobulinemia (WM) cells.

Lymphocytes belong to the group of white blood cells and are mediators of humoral and cell-mediated immunity. There are two lymphocyte populations, B-cells and T-cells.

Like many other cell types, B-cells and T-cells can develop abnormally into B-cell and T-cell tumors. There are many different types of tumors due to the numerous developmental stages that develop B-cells and T-cells. Both B-cells and T-cells originate from lymphoid progenitor cells.

In the case of B-cells, these lymphoid progenitor cells each develop through many stages of B cell development, including specific definable cell types until a plasma cell is formed. One of these stages involves so-called "IgM-secreting B cells", which eventually develop into antibody-producing plasma cells. Tumors originating from "IgM-secreting B-cells" are referred to as "Waldenstrom's macroglobulinemia" (WM). WM is a rare, indolent, incurable disease. These cells are characterized by bone marrow accumulation of clonal IgM secreting lymphoplasmic cells.

T-cells develop from lymphoid progenitor cells to mature T-cells in only a few developmental stages. Tumors can especially evolve from mature T-cells or lymphoid progenitor cells, the latter resulting in B- or T-cell acute lymphoblastic leukemia, (B-ALL) and (T-ALL), respectively. T-cell phenotype T-ALL accounts for approximately 20% of all acute lymphoblastic leukemia cases and occurs more frequently in adults than in children. T-ALL is closely related to T-cell lymphoblastic lymphoma (T-LBL) and the differential diagnosis between the two diseases is the predominant localization at specific sites, such as the bone marrow in T-ALL or the bone marrow in T-LBL. It is based on secondary lymphoid organs. The European Group for the Immunological Characterization of Leukemias (EGIL) divided T-ALL into four subgroups according to their immunological phenotypes (Bene MC, Leukemia 1995; 9:1783]):

1) characterized by cytoplasmic positivity for EGIL T1 (pro-), CD3 (cCD3) and surface expression of CD7;

2) characterized as positive for EGIL T2 (pre-) cCD3, CD7 and positive for CD2 or CD5;

3) characterized by the presence or absence of EGIL T3 (cortical) cCD3, positive for CD1a and surface CD3 (sCD3), and

4) Characterized as positive for EGIL T4 (mature leukemia), cCD3 and sCD3 and negative for CD1a.

As used herein, the term “antibody-dependent cellular cytotoxicity (ADCC)” is the killing of cells bound and displayed by an antibody by cytotoxic effector cells, such as natural killer (NK) cells.

To test whether an antibody is capable of inducing ADCC, the following assay can be used. The degranulation assay is performed by co-culturing peripheral blood mononuclear cells (PBMCs) from healthy donors, including effector cells, with target cells expressing the epitope in the presence of different antibody concentrations. 4 x 10 ⁴ target cells were seeded into 96-well round bottom plates and in the presence of different concentrations of antibody (0, 10, 50, 100 and 200 μg/ml) or control IgG1 at 37° C. 5% CO ₂ for 30 min. incubate Subsequently, 0.4 x 10 ⁶ PBMCs from the same donor (fixed effector cells (E): target cells (T) = 10:1) were added to each well with 20 μl/ml phycoerythrin (PE)- Conjugated anti-CD107a monoclonal antibody (mAb) (BD) is added together after which cells are incubated at 37° C. 5% CO ₂ for 3 hours. After 1 hour, 6 μg/ml monensin is added to each well (GolgiStop, BD). At the end of the incubation period, cells are stained with allophycocyanin (APC)-conjugated anti-CD56 and peridinine chlorophyll protein complex (PerCp)-conjugated anti-CD3 and analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific). By detecting CD3 ⁻ /CD56 ⁺ /CD107a ⁺ cells, NK cells (CD3 ⁻ /CD56 ⁺ ) that induce target cell lysis (CD107a ⁺ ) are measured. Thus, the increase of CD3 ⁻ /CD56 ⁺ /CD107a ⁺ cells with increasing antibody concentration confirms the potential of the antibody to induce ADCC. The data generated allow the design of immune targeting approaches, which are urgent and unmet clinical needs, for example in T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma. Additional methods for testing whether an antibody is capable of inducing ADCC may also be used and are well known to those of skill in the art.

6.3.5. UMG1 binding molecule

In a third aspect, the present invention provides a binding molecule derived from the UMG1 antibody according to aspect 1 or aspect 2.

According to the present invention, the binding molecule is a molecule derived from the monoclonal mouse UMG1 antibody produced by hybridoma cells deposited under ICLC PD n° 16001. Preferably, the binding molecule is an immunoglobulin comprising molecule, ie, the binding molecule comprises at least one immunoglobulin (Ig) domain.

In a preferred embodiment, the binding molecule of the invention is selected from the group consisting of single chain antibodies. In a more preferred embodiment, the binding molecule is a single chain variable fragment (scFv), a multimer of scFv, such as a diabody, a triabody, a tetrabody, an antibody fragment, preferably a Fab, tandab. (tandab) and flexibody (flexibody) is selected from the group consisting of.

The structure of an antibody and in particular the function of its CDRs are generally known in the art (Carter PJ. Potent antibody therapeutics by design. Nature Rev. Immunol. 6:343-357, 2006), which is incorporated in its entirety. is included). Single chain Fv (scFv) and their multimers, tandabs, diabodies and flexibodies are generally known in the art, eg, International Publication No. WO 1988/001649 A1, each of which is incorporated by reference in its entirety. It is a standard antibody format known from WO 1993/011161 A1, International Publication WO 1999/057150 A2 and European Patent EP1293514B1.

In scFvs, the two antigen-binding variable regions (VH Fv and VL Fv) of the light and heavy chains of an antibody are artificially linked, usually by a linker peptide, and are designated as single-chain variable fragments or single-chain antibodies (Bird, et al. (1988) Science 242:423-426; Orlandi, et al (1989) Proc Natl Acad Sci USA 86:3833-3837; Clarkson et al., Nature 352: 624-628 (1991) , each of which is incorporated by reference in its entirety). The antigen binding site can be prepared from the variable domains of the light and heavy chains of monoclonal antibodies. Several investigations have shown that scFv fragments can have virtually the fully intrinsic antigen binding affinity of one binding site of an entire antibody.

In the context of the present invention, diabodies are scFvs with two binding specificities and may be monospecific and bivalent or bispecific and bivalent.

Tandab and Flexibodies are additional antibody formats as defined, respectively, in, for example, US Patent Publication No. US2007031436 and European Patent EP1293514B1, each of which is incorporated by reference in its entirety.

Antibody fragments containing proteins of the idiotype can be generated by techniques known in the art. For example, such fragments include, but are not limited to, F(ab')2 fragments, which may be produced by pepsin digestion of antibody molecules; Fab' fragments, which may be generated by reducing the disulfide linkages of F(ab')2 fragments; Fab fragments, which can be generated by treating the antibody molecule with papain and a reducing agent; and Fv fragments.

6.3.6. Antibody-Drug Conjugates (ADCs)

The antibody or binding molecule of the invention may further be linked to an active ingredient, preferably a toxin, nanoparticle, cytokine or radionuclide. Such antibody drug conjugates (ADCs) are known in the art (Wu AM, Senter PD. Nature Biotechnol. 23:1137-1146, 2005, Pastan et al. Annu. Rev. Med. 58:221-237, 2007], International Publication No. WO 1990/012592 A1, International Publication No. WO 2007/030642 A2, International Publication No. WO 2004/067038 A1, International Publication No. WO 2004/003183 A1, US Patent Application Publication No. US 2005/0074426 A1, International Publication No. WO 1994/004189 A1, each of which is incorporated by reference in its entirety). See also Yaghoubi et al., " Potential drugs used in the antibody-drug conjugate (ADC) architecture for cancer therapy ," J Cell Physiol. 2019 Jun 18. doi: 10.1002/jcp.28967. [Epub ahead of print]]; Arlotta et al., " Antibody and antibody derivatives as cancer therapeutics ," Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2019 Apr 9:e1556. doi:10.1002/wnan.1556. [Epub ahead of print]]; See Wolfka-Washer et al., " Safety and Tolerability of Antibody-Drug Conjugates in Cancer ," Drug Saf. 2019 Feb;42(2):295-314]; See Johnston et al., " Antibody conjugated nanoparticles as a novel form of antibody drug conjugate chemotherapy ," Drug Discov Today Technol. 2018, 30:63-69]; Lyon, "Drawing lessons from the clinical development of antibody-drug conjugates," Drug Discov Today Technol. 2018 Dec;30:105-109]; and Abdollahpour-Alitappeh et al., " Antibody-drug conjugates (ADCs) for cancer therapy: Strategies, challenges, and successes ," J Cell Physiol. 2019 May;234(5):5628-5642, the disclosure of which is incorporated herein by reference in its entirety.

In various embodiments, the binding molecule is conjugated with a therapeutic agent (ie, drug) to form a binding molecule-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (such as radioisotopes), immune modulators (such as cytokines, chemokines, or checkpoint inhibitors), and toxins (such as cytotoxic agents). In certain embodiments, the therapeutic agent is attached to the binding molecule via a linker peptide, as discussed in more detail below in section 6.7.3.

Methods for making antibody-drug conjugates (ADCs) that may be adapted for conjugating drugs to the binding molecules disclosed herein are described, for example, in US Pat. No. 8,624,003 (pot method), US Pat. No. 8,163,888 (1- one-step), U.S. Patent No. 5,208,020 (two-step method), U.S. Patent No. 8,337,856, U.S. Patent No. 5,773,001, U.S. Patent No. 7,829,531, U.S. Patent No. 5,208,020, US Patent No. 7,745,394, International Publication No. WO 2017/136623, International Publication No. WO 2017/015502, International Publication No. WO 2017/015496, International Publication No. WO 2017/015495, International Publication No. WO 2004/010957, International Publication WO 2005 /077090, International Publication No. WO 2005/082023, International Publication No. WO 2006/065533, International Publication No. WO 2007/030642, International Publication No. WO 2007/103288, International Publication No. WO 2013/173337, International Publication WO 2015/057699 International Publication No. WO 2015/095755, International Publication No. WO 2015/123679, International Publication No. WO 2015/157286, International Publication No. WO 2017/165851, International Publication No. WO 2009/073445, International Publication No. WO 2010/068759, International Publication No. WO 2010/138719, International Publication No. WO 2012/171020, International Publication No. WO 2014/008375, International Publication No. WO 2014/093394, International Publication No. WO 2014/093640, International Publication No. WO 2014/160360, International Publication No. WO 2015/054659, International Publication No. WO 2015/195925, International Publication No. WO 2017/160754, Storz ( MAbs . 2015 Nov-Dec; 7(6): 989-1009), Lambert et al. ( Adv Ther , 2017 34: 1015), Diamantis et al. ( British Journal of Cancer , 2016, 114, 362-367), Carrico et al. ( Nat Chem Biol , 2007. 3: 321-2), We et al. ( Proc Natl Acad Sci USA , 2009. 106: 3000-5)], Rabuka et al. ( Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. ( Angew Chem Int Ed Engl ., 2012: 4161-5), Rabuka et al. ( Nat Protoc. , 2012 7:1052-67), Agarwal et al. ( Proc Natl Acad Sci USA ., 2013, 110: 46-51)], Agarwal et al. ( Bioconjugate Chem ., 2013, 24: 846-851), Barfield et al. ( Drug Dev. and D., 2014, 14:34-41), Drake et al. ( Bioconjugate Chem ., 2014, 25:1331-41), Liang et al. ( J Am Chem Soc ., 2014, 136:10850-3), Drake et al. ( Curr Opin Chem Biol ., 2015, 28:174-80), and York et al. ( BMC Biotechnology , 2016, 16(1):23), each of which is incorporated herein by reference in its entirety for all that the literature teaches.

6.3.7. Chimeric Antigen Receptor (CAR)

The present disclosure also provides a chimeric antigen receptor (CAR) comprising the binding molecule of aspect 3 linked to an intracellular domain comprising one or more signaling domains.

Preferably, the present invention relates to a scFv of a preferred embodiment of the binding molecule of embodiment 3 linked to an intracellular region comprising a CD3ζ chain, a signaling region of a T cell receptor, and two co-stimulatory domains CD28 and 4-1BB A chimeric antigen receptor (CAR) comprising

The CAR according to the present invention is a suitable tool for targeting malignant cells having an epitope recognized and bound by the monoclonal antibody of embodiment 1 or 2 when expressed on T-cells or NK cells. As used herein, the term “chimeric antigen receptor” (CAR) refers to a synthetic receptor comprising a targeting moiety associated with one or more signaling domains within a single fusion molecule. Generally, the binding moiety of a CAR comprises an scFv, but it may also contain other binding entities. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domain for the CAR may be derived not only from the cytoplasmic region of the CD3ζ or Fc receptor gamma chain, but also from other cytoplasmic regions. First-generation CARs have been shown to successfully redirect T-cell cytotoxicity. Transmembrane and hinge domains as well as signaling domains from co-stimulatory molecules have been added to form second and third generation CARs, which have led to some successful therapeutic trials in humans, where T-cells express CD19. may be redirected to malignant cells (Porter DL et al., N Eng J Med, 2011).

6.3.7.1. CAR-T embodiment

Those skilled in the art will recognize that the CAR-T provided by the present disclosure can be designed for the particular application provided by the present disclosure (D. Xu et al. Oncotarget . 2018 Mar 2; 9(17) )]), which is incorporated herein by reference in its entirety.

In various embodiments, the CAR is a first-generation CAR (Eshhar et al. Proc Natl Acad Sci USA (1993) 90(2)); In various embodiments, the CAR is a co-stimulatory CAR (Krause et al. J ExpMed. (1998) 188(4)); in various embodiments, the CAR is a second-generation CAR (Finney et al. J Immunol (1998) 161(6).; Maher et al. Nat Biotechnol (2002) 20(1); Finney et al. (2004) J Immunol.172(1).); Imai et al. (2004) Leukemia 18(4)); in various embodiments, the CAR is a third-generation CAR (Pule et al. (2005) MolTher. 12(5); Geiger et al. Blood (2001) 98; Wilkie et al. (2008) J Immunol. 180(7)); in various embodiments, the CAR is a fourth-generation TRUCKS CAR (Chmielewski et al. Cancer Res (2011)). 71.]); in various embodiments, the CAR is an Armored CAR generation CAR (Pegram et al. (2012) Blood 119; Curran et al. (2015) MolTher. 2015 Apr;23(4)) ]); in various embodiments, the CAR is an engineered co-stimulatory generation CAR (Zhao et al. (2015) Cancer Cell 28); in various embodiments, the CAR is a SynNotch/sequential AND gated generation CAR ( (Roybal et al. (2016) Cell 164); in various embodiments, the CAR is a co-stimulatory generation CAR in cis and trans (Stephan et al. (2007) Nat Med 13(12)); In various embodiments, the CAR is a dual-targeted generation CAR (Wilkie et al. (2012) J Clin Immunol. 32(5)); in various embodiments, the CAR is a Combinatorial CAR/A ND gate generation CAR (Kloss et al. (2013) Nat Biotechnol 31(1)]); In various embodiments, the CAR is a TanCAR generation CAR (Ahmed et al. (2013) MolTher Nucleic Acids. 2:e105); In various embodiments, the CAR is a Go-CART generation CAR (Foster et al, (2014)); The disclosure of which is incorporated herein by reference in its entirety.

In certain embodiments, the CAR is a pCAR as described in US pre-grant publication US 2019/0002521, which is incorporated herein by reference in its entirety.

6.3.7.2. CAR construct with a primary intracellular signaling domain (CAR-UMG1)

In some embodiments, the CAR construct comprises a primary intracellular signaling domain. The primary intracellular signaling domain generates an intracellular signal when an extracellular domain fused thereto, such as an antigen binding domain, binds to a cognate antigen. A primary intracellular signaling domain is derived from a primary stimulatory molecule, such as comprising the intracellular sequence of the primary stimulatory molecule. The primary intracellular signaling domain comprises, for example, a primary stimulatory molecule sequence sufficient to generate an intracellular signal when the antigen binding domain to be fused binds to a cognate antigen.

A primary stimulatory molecule is a molecule that mediates an immune effector response upon binding to a cognate ligand, such as in the cell in which it is expressed. Typically, it produces an intracellular signal that is dependent on binding to a cognate ligand comprising an antigen. The TCR/CD3 complex is an exemplary primary stimulatory molecule; It generates an intracellular signal when a cognate ligand, such as a peptide, binds to the loaded MHC molecule. Typically, such as for TCR/CD3 primary stimulatory molecules, generation of an intracellular signal by a primary intracellular signaling domain is dependent on binding of the primary stimulatory molecule to an antigen.

Primary stimulation may mediate altered expression of certain molecules, such as downregulation of TGF-β, and/or reorganization of cytoskeletal structures.

Stimulation can result in optimization, eg, increase, of immune effector function of CART cells, eg, in the presence of co-stimulation. For example, in the context of CART cells, stimulation can mediate T cell responses such as proliferation, activation, differentiation, and the like.

In some embodiments, the primary intracellular signaling domain comprises a signaling motif, such as an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine-based activation motif (ITAM). Primary intracellular signaling domains include (eg) TCR zeta (CD3 zeta, CDζ), consensus FcR gamma, (FCER1G), Fc gamma Rlla, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5 , CD22, CD79a, CD79b, CD278 (also known as “ICOS”), FcsRI, DAP10, DAP 12, and ITAM containing cytoplasmic signaling sequences from CD66d.

The primary intracellular signaling domain comprises a functional fragment, or analog, of a primary stimulatory molecule (eg CD3 zeta, CD3ζ). A primary intracellular signaling domain may comprise an entire intracellular region or a fragment of an intracellular region sufficient to generate an intracellular signal when the antigen binding domain to which it is fused binds to a cognate antigen. In some embodiments, the primary intracellular signaling domain is an intracellular signal of a naturally occurring primary stimulatory molecule, such as an intracellular signal of a primary stimulatory molecule in a human, or other mammal, such as a non-human species, such as a rodent, monkey, primate, or murine cell. has at least 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity to the entire intracellular region, or a fragment of the intracellular region, sufficient to produce

In some embodiments, the primary intracellular signaling domain is at least an entire intracellular region, or an intracellular region, sufficient to produce an intracellular signal of a naturally occurring human primary stimulatory molecule, such as a naturally occurring human primary stimulatory molecule disclosed herein. has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the corresponding residue of a fragment of, or 30, 25, 20, 15, 10, 5, 4, 3, differ by 2, or no more than 1 amino acid residue.

6.3.7.3. CAR construct with costimulatory signaling domain (CAR-UMG1)

In some embodiments, the CAR construct comprises an extracellular domain to which it is fused, such as a costimulatory signaling domain, which generates an intracellular signal when the antigen binding domain binds to a cognate antigen. The costimulatory signaling domain is derived from a costimulatory molecule. The costimulatory signaling domain comprises a primary costimulatory molecule sequence sufficient to generate an intracellular signal when the extracellular domain to which it is fused, such as an antigen binding domain, binds a cognate ligand.

The costimulatory domain may be one that optimizes the performance, such as persistence, or immune effector function, of a T cell comprising a CAR comprising the costimulatory domain.

Costimulatory molecules are cell surface molecules other than antigen receptors or their counter ligands that promote an immune effector response. In some cases, this requires an efficient or enhanced immune response. Typically, the costimulatory molecule generates, in certain embodiments, an intracellular signal that is dependent on binding to an antigen, such as a cognate ligand other than an antigen that is recognized by the antigen binding domain of a CART cell. Typically, signaling from primary and costimulatory molecules contributes to an immune effector response, and in some cases both are required for efficient or enhanced generation of an immune effector response.

A costimulatory domain comprises a functional fragment, or analog, of a costimulatory molecule (eg, ICOS, CD28, or 4-1BB). This may include, for example, the entire intracellular region or a fragment of the intracellular region of the intracellular region sufficient to generate an intracellular signal when the antigen binding domain to which it is fused binds to a cognate antigen. In certain embodiments, the costimulatory domain is an entire intracellular region sufficient to produce an intracellular signal of a naturally occurring costimulatory molecule, such as a costimulatory molecule in a human, or other mammalian, such as a non-human species, such as a rodent, monkey, primate or murine cell. , or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a fragment of an intracellular region.

Exemplary co-stimulatory domains include, but are not limited to, CD27, CD27, CD28, 4-1BB (CD137), QX40, CD30, CD40, ICQS (CD278), ICAM-1, LFA-1 (CD11a/CD18), CD2, CD7 , LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD8, CDS, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD19, CD4, CD8 alpha, CD8 beta , IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, ITGAM, CDllb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAMl (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRTAM, Ly9 (CD229), PSGLl, ClOO (SEMA4D), CD69 , SLAMF6 (NTB-A, Ly108), SLAM (SLAMFl, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, and PAG/Cbp.

In some embodiments, the costimulatory signaling domain is the corresponding entire intracellular region, or a fragment of the intracellular region, sufficient to produce an intracellular signal of a naturally occurring human costimulatory molecule, such as a naturally occurring human costimulatory molecule disclosed herein. have at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the residue, or 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 The following amino acid residues are different.

6.3.7.4. Immune effector cells comprising a chimeric antigen receptor (CAR)

In a sixth aspect, the present invention provides CD3 ⁺ lymphocytes, NK lymphocytes, Cytokine induced killer (CIK) cells, gamma- comprising the chimeric antigen -ch-UMG1 according to aspect 4 or the expression vector according to aspect 5 delta lymphocytes, NKT cells or another immune effector cell.

In general, CD3 is a complex of four signaling chains involved in the α:β heterodimer of the T-cell receptor in a functional T-cell receptor complex. The CD3 complex is normally required for T-cell receptor signaling. In general, the CD3 ⁺ lymphocyte population exclusively includes thymocytes and T-cells. Detection of CD3 ⁺ cells can be accomplished, for example, by flow cytometry.

6.3.8. Bispecific T-cell engager (BiTE)

The two main approaches for T-cell redirection involve genetic modification using chimeric antigen receptors (CARs), or the use of recombinant proteins designated as bispecific T-cell agents (BiTEs).

The present disclosure provides various embodiments of the BiTE-UMG1 construct (Huehls AM et al ., " Bispecific T-cell engagers for cancer immunotherapy " Immunol Cell Biol. 2015 Mar;93(3):290-6) ; _ do.

In general, BiTEs are constructed of two single-chain variable fragments (scFvs) linked side by side by a flexible linker. One scFv binds a T-cell-specific molecule, usually CD3, while a second scFv binds a tumor-associated antigen. This structure and specificity allows BiTE to physically link T cells to tumor cells, ultimately stimulating T-cell activation, tumor death and cytokine production.

In some embodiments, the BiTE-UMG1 construct targets a hematologic cancer. In some embodiments, the BiTE-UMG1 construct targets a solid tumor cancer type. In some embodiments, the BiTE-UMG1 construct targets tumor-associated macrophages in a solid tumor.

6.4. Pharmaceutical composition of CD43 binding protein

In a seventh aspect, the present invention provides a monoclonal UMG1 antibody according to aspect 1 or 2 or a UMG1 binding molecule according to aspect 3 or CD3 ⁺ lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma- according to aspect 6 Pharmaceutical compositions comprising delta lymphocytes, NKT cells or other immune effector cells are provided.

As used herein, the term “pharmaceutical composition” may be used interchangeably with the term “drug”.

Some embodiments of the pharmaceutical composition are antibodies or antigen-binding fragments thereof. In some embodiments, the antibody is monoclonal. In some embodiments, the monoclonal antibody is a chimeric antibody. In some embodiments, the monoclonal antibody is a humanized antibody. In some embodiments, the monoclonal antibody is a human antibody. In some embodiments, the pharmaceutical composition is an antibody-drug conjugate.

In various embodiments, the pharmaceutical composition is disclosed in U.S. Patent No. 8,961,964, U.S. Patent No. 8,945,865, U.S. Patent No. 8,420,081, U.S. Patent No. 6,685,940, U.S. Patent No. 6,171,586, U.S. Patent No. 8,821,865, U.S. Patent No. 9,216,219, US Patent Publication No. 10/813,483, International Patent WO 2014/066468, International Patent WO 2011/104381, and International Patent WO 2016/180941 are described in more detail, each of which is incorporated herein in its entirety. do.

6.5. Manufacturing method

UMG1 binding molecules (antibodies, proteins, antigens, etc.) provided by the present disclosure can be prepared using standard methods known in the art.

For example, UMG1 binding molecules can be prepared by expression using standard cell free translation, transient transfection, and stable transfection approaches currently used for antibody production. In certain embodiments, Expi293 cells (ThermoFisher) are prepared using the protocol and reagents of ThermoFisher, such as ExpiFectamine, or as described in Fang et al. ( Biological Procedures Online , 2017, 19:11)] can be used for the generation of binding molecules using other reagents known in the art, such as polyethyleneimine. The expressed protein can be readily purified using standard methods known in the art, such as, for example, a CH1 affinity resin such as CaptureSelect CH1 resin and protocols provided by ThermoFisher. Further purification can be accomplished using ion exchange chromatography commonly used in the art.

6.6. administration

The UMG1 pharmaceutical compositions provided by the present disclosure may be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, parenteral administration including subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intranasal, and intrapulmonary routes.

6.7. combination therapy

The present disclosure also provides combination therapies. In some embodiments, a pharmaceutical composition provided herein is provided in combination with another therapy treatment. Therapy Treatment may be surgical, radiation, holistic, cellular therapy, tissue regeneration, or other pharmaceutical compositions known for the treatment of cell proliferative diseases or cancers.

A therapeutically effective dosage varies in some embodiments when a pharmaceutical composition provided by the present disclosure is used in a therapeutic combination. Methods for experimentally determining therapeutically effective dosages of drugs and other agents for use in combination therapy regimens include the use of regular dosages, i.e., providing more frequent and lower dosages to minimize toxic side effects. include

A combination treatment regimen is wherein administration of a compound described herein is initiated prior to, during, or after treatment with said second agent and continues during treatment with said second agent or until any time after termination of treatment with said second agent. do. Such therapy also includes treatment in which a compound described herein or a second agent used in combination is administered at the same time or at different times and/or at decreasing or increasing intervals during the treatment period.

Combination therapy also includes periodic treatments that start and stop at various times to aid in the clinical management of the patient. For example, in combination therapy, a compound described herein is administered weekly at the start of treatment, reduced every other week, and, if appropriate, further reduced.

6.8. formulation

The present disclosure also provides various UMG1 pharmaceutical formulations comprising an effective amount of a UMG1 antigen, antibody, or binding molecule or protein.

In some embodiments, the pharmaceutical composition is formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. do. Suitable formulations depend on the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are optionally suitably selected. Pharmaceutical compositions comprising a UMG1 antibody or UMG1 binding molecule are prepared in a conventional manner, for example by conventional mixing, dissolving, granulating, dragee preparation, levigating, emulsifying, encapsulating, capturing or compression processes. do.

The UMG1 pharmaceutical composition may optionally contain other pharmaceuticals or pharmaceutical agents, carriers, adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure, buffers, and/or other therapeutically valuable substances. have. Methods of preparing a composition comprising a compound described herein include formulating the compound with one or more inert pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid.

Solid dosage forms of the composition include, but are not limited to, powders, tablets, dispersible granules, capsules, sachets, and suppositories.

Liquid formulation compositions include solutions in which the compounds are dissolved, emulsions comprising the compounds, or solutions containing liposomes, micelles, or nanoparticles comprising the compounds as disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions and creams. Forms of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to use, or emulsions. Such compositions also optionally contain minor amounts of non-toxic, auxiliary substances such as wetting or emulsifying agents, pH buffers and the like.

The content of the antibody, binding molecule or CD3+ lymphocyte in the pharmaceutical composition is not limited as long as it is useful for treatment or prevention, but preferably contains 0.0000001 to 10% by weight per total composition. In addition, the antibodies, binding molecules or CD3+ lymphocytes described herein are preferably used in a carrier. The choice of carrier may vary depending on the route of administration and the concentration of the active agent, and the carrier may be in the form of a lyophilized composition or an aqueous solution. In general, a suitable amount of a pharmaceutically acceptable salt is employed as the carrier to render the composition isotonic. Examples of carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. Preferably, acceptable excipients, carriers, or stabilizers are non-toxic at the dosages and concentrations employed, and include buffers such as citrate, phosphate, and other organic acids; salt-forming counter-ions such as sodium and potassium; low molecular weight (greater than 10 amino acid residues) polypeptides; proteins such as serum albumin, or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrin; monosaccharides; disaccharides; other sugars such as sucrose, mannitol, trehalose or sorbitol; chelating agents such as EDTA; non-ionic surfactants such as Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and/or preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. The composition may also contain at least one additional active compound, such as a chemotherapeutic agent.

Preferably, the antibody, binding molecule, CD3 ⁺ lymphocyte and/or active compound are included in an effective amount. The term “effective amount” refers to an amount sufficient to induce a detectable therapeutic response in a subject to which the pharmaceutical composition will be administered.

6.9. Polynucleotide encoding CD43 binding protein

In an eighth aspect, the present invention provides a nucleic acid or polynucleotide encoding a UMG1 antibody according to aspect 1 or 2 or a UMG1 binding molecule according to aspect 3.

Also provided are polynucleotides encoding antibodies that are optimized, such as by codon/RNA optimization, replacement with heterologous signal sequences, and removal of mRNA labile elements. Methods for generating an optimized nucleic acid encoding an antibody or fragment thereof (such as a light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or removing inhibitory regions within the mNRA, such as those US Pat. No. 5,965,726, which is incorporated by reference in its entirety; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and labile elements in RNA (e.g., A/T or A/U rich elements) can be used to increase the stability of the RNA for recombinant expression without altering the amino acids encoded by the nucleic acid sequence. can be mutated. Such alterations take advantage of the degeneracy of the genetic code, for example by using alternative codons for the same amino acid. In some embodiments, it may be desirable to change one or more codons to encode a conservative mutation, eg, a similar amino acid having a similar chemical structure and properties and/or function as the original amino acid. Such methods increase the expression of the antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, It can be increased by 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times.

In certain embodiments, an optimized polynucleotide sequence encoding an antibody or fragment thereof (eg, VL domain and/or VH domain) described herein comprises an antibody or fragment thereof (eg, VL domain and/or VH domain) described herein. or VH domain) to an antisense (eg complementary) polynucleotide of a non-optimized polynucleotide sequence. In a specific embodiment, an optimized nucleotide sequence encoding an antibody or fragment described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an antibody or fragment thereof described herein. In a specific embodiment, an optimized nucleotide sequence encoding an antibody or fragment thereof described herein is subjected to high stringency, moderate stringency or low stringency hybridization conditions, a non-optimized polynucleotide encoding an antibody or fragment thereof described herein. hybridizes to the antisense polynucleotide of the sequence. Information regarding hybridization conditions is described, see, for example, US Patent Application Publication No. US 2005/0048549 (eg, paragraphs 72-73).

The polynucleotides of the present invention can be obtained, and the nucleotide sequence of such polynucleotides can be determined by methods known in the art. The nucleotide sequences encoding the antibodies described herein, and modified versions of these antibodies, can be determined using methods well known in the art, i.e., nucleotide codons known to encode a particular amino acid are assembled in such a way as to generate nucleic acids. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6; is incorporated by reference), which, briefly, comprises the steps of synthesizing overlapping oligonucleotides containing a portion of the sequence encoding the antibody, annealing and linking these oligonucleotides, and then amplifying the linked oligonucleotides by PCR It entails making

Alternatively, polynucleotides encoding the antibodies described herein can be prepared from nucleic acids from suitable sources (eg, hybridomas) using methods well known in the art (eg, PCR and other molecular cloning methods). can be created For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding the light and/or heavy chains of an antibody. This PCR amplification method can be used to obtain a nucleic acid comprising a sequence encoding a variable light and/or variable heavy chain region of an antibody. Amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, eg, to generate chimeric and humanized antibodies.

If clones containing the nucleic acid encoding the particular antibody are not available but the sequence of the antibody molecule is known, then the nucleic acid encoding the immunoglobulin can be obtained from a suitable source, e.g., to identify cDNA clones from a cDNA library encoding the antibody. (For example, an antibody cDNA library or cDNA library generated from any tissue or cell expressing an antibody, such as a hybridoma cell selected to express an antibody described herein, or a nucleic acid isolated therefrom, preferably poly A+ RNA), by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

DNA encoding the antibodies of the invention described herein can be readily isolated using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody) and can be sequenced. Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, after which E. coli cells , simian COS cells, Chinese hamster ovary (CHO) cells (e.g., that do not otherwise produce immunoglobulin proteins) For example, CHO cells from CHO GS System™ (Lonza), or myeloma cells are transfected into host cells to obtain synthesis of the antibody in the recombinant host cells.

To generate whole antibodies, in scFv clones or other clones, PCR primers comprising VH or VL nucleotide sequences, restriction sites, and flanking sequences to protect these restriction sites are used. VH or VL sequences can be amplified. Using cloning techniques known to those of ordinary skill in the art, the PCR amplified VH domain can be cloned into a vector expressing a heavy chain constant region, e.g., a human gamma 4 constant region, wherein the PCR amplified VL domain comprises a light chain constant region, For example, it can be cloned into a vector expressing a human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain comprises a promoter, a secretion signal, a cloning site for the variable region, a constant domain, and a selectable marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the essential constant regions. The heavy chain conversion vector and light chain conversion vector are then co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, eg, IgG, using techniques known to those skilled in the art.

DNA can also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains in place of murine sequences, or by covalently splicing all or part of the coding sequences for non-immunoglobulin polypeptides to immunoglobulin coding sequences. have.

Site-specific or high-density mutagenesis or other mutagenesis methods of variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody. In particular, affinity maturation strategies and chain shuffling strategies are known in the art (Marks et al., 1992, Bio/Technology 10:779-783, each incorporated by reference in their entirety) and high affinity It can be used to generate human antibodies.

6.10. Hybridoma cells producing UMG1 monoclonal antibody

In a ninth aspect, the present invention provides a hybridoma cell producing a monoclonal antibody according to the antibody of aspect 1 or 2.

6.11. Hybridoma composition

The present invention also provides a hybridoma composition deposited under ICLC PD n° 16001.

6.12. UMG1 monoclonal antibody production method

In an eleventh aspect, the present invention provides a method for producing a monoclonal antibody according to aspect 1 or 2, said method comprising isolating said antibody from a hybridoma cell deposited under ICLC PD n° 16001 do.

6.13. Cell Isolation Using UMG1 Antibodies and/or Binding Molecules

In a twelfth aspect, the present invention relates to a method for identifying or isolating T-cell acute lymphoblastic leukemia cells, T lymphocyte cells, Waldenstrom's macroglobulinemia cells or tumor-associated macrophages, said method comprising: contacting the cell sample comprising the monoclonal antibody according to embodiment 1 or 2 or the binding molecule according to embodiment 3.

In general, macrophages are the most representative non-malignant cells in the tumor microenvironment. Tumor-associated macrophages (TAMs) are believed to acquire a pro-tumoral inflammatory and immuno-suppressive phenotype and favor chemo-resistance, angiogenesis, cell migration and intravasation/extravasation. . Thus, targeting TAMs may represent new treatments and still unexplored clinical options to improve the efficacy of current anti-cancer treatments.

Methods for identifying or isolating specific cells, such as T-cell acute lymphoblastic leukemia cells, T lymphocyte cells, Waldenstrom's macroglobulinemia cells or tumor-associated macrophages, based on the antibody or binding molecule, such as for flow cytometry Methods based on fluorescent cell sorting by means of fluorescence, magnetic cell isolation or single cell sorting by, for example, cell sorters are generally well known to the person skilled in the art.

6.14. Methods for producing immune effector cells

In a thirteenth aspect, the present invention provides CD3 ⁺ lymphocytes, NK lymphocytes, cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or other immune effector cells expressing a chimeric antigen receptor according to the chimeric antigen receptor of aspect 4 It provides a method for producing an expression vector according to the expression vector of aspect 5, wherein the CD3 ⁺ lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or other immune effector cells introducing it into

6.15. Expression vector composition

In a fifth aspect, the present invention provides an expression vector comprising a nucleic acid sequence encoding a chimeric antigen receptor according to aspect 4, an antibody according to aspects 1 and 2 or a binding molecule according to aspect 3 .

Generally, an expression vector is a plasmid used to introduce a desired nucleic acid sequence, such as a gene, into a target cell, resulting in the transcription and translation of a protein encoded by the nucleic acid sequence, such as a chimeric antigen receptor, antibody or binding molecule. Thus, expression vectors generally contain regulatory sequences, such as promoter and enhancer regions, as well as polyadenylation sites to direct efficient transcription of the nucleic acid sequences of the expression vector. Expression vectors may also contain additional essential or useful regions, such as selectable markers for selection in eukaryotic or prokaryotic cells, purification tags for purification of the resulting protein, multiple cloning sites or origins of replication.

Typically, the expression vector is a viral or non-viral vector. In general, various types of viral vectors, such as retroviral vectors, such as lentiviral or adenoviral vectors, or plasmids can be used. In a preferred embodiment, the expression vector according to aspect 5 is a viral vector. In a more preferred embodiment, the expression vector is a lentiviral vector.

6.16. treatment method

In another aspect, a method of treatment is provided, comprising administering to the patient a binding molecule or antibody as described herein in an amount effective to treat the patient.

In some embodiments, the method comprises administering to the patient a binding molecule or antibody as described herein in an amount effective to treat the patient with a CAR or CAR-T.

In some embodiments, the method comprises administering to the patient a binding molecule or antibody as described herein in an amount effective to treat the patient with BiTE.

In some embodiments, the method comprises administering to the patient a binding molecule or antibody as described herein in an amount effective to treat the patient with the antibody-drug conjugate.

6.16.1. indication

In some embodiments, an antibody or binding molecule of the present disclosure may be used to treat a proliferative disease or cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is, but is not limited to, T-cell malignancy, T-cell leukemia, T-cell lymphoma, T-cell acute lymphoblastic leukemia, multiple myeloma, B-cell malignancy, myeloid malignancy, acute myeloid leukemia and hematologic cancers, including chronic myelogenous leukemia.

In some embodiments, the cancer or proliferative disease is bladder, blood, blood immune cells (such as T-cells or B-cells, monocytes, etc.), bone, bone marrow, brain, breast, colon, colorectal, esophagus, stomach, gums, cancer from the head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, pancreas, skin, stomach, testis, tongue or uterus.

In some embodiments, the cancer or tumor to be treated with an antibody or binding molecule of the present disclosure is a malignant neoplasm; non-malignant; carcinoma; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary cancer; squamous cell carcinoma; lymphepithelial carcinoma; basal cell carcinoma; mammary gland carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; six-week adenocarcinoma; adenoid cystic cancer; adenocarcinoma in neoplastic polyps; adenocarcinoma, familial polyposis of the colon; solid carcinoma; malignant carcinoid tumors; broncho-alveolar adenocarcinoma; papillary adenocarcinoma; nucleophilic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granule cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; earwax adenocarcinoma; mucoepidermoid carcinoma; cystic adenocarcinoma; papillary cystic adenocarcinoma; papillary serous cystic adenocarcinoma; mucinous cystic adenocarcinoma; mucinous adenocarcinoma; ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; breast Paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant oocystoma; malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant Leedich cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary adrenal adenoma; pheochromocytoma; glomerular angiosarcoma; malignant melanoma; melanin deficiency melanoma; superficial dilated melanoma; malignant melanoma in nevus giant pigmentosa; epithelial cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic carcinoma rhabdomyosarcoma; alveolar rhabdomyosarcoma; epileptic sarcoma; malignant mixed tumor; Mullerian mixed tumor; neoblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal; malignant Brenner's tumor; malignant lobular tumor; synovial sarcoma, malignant mesothelioma; undifferentiated germ cell tumor; embryonic carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant melanoma; hemangiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; bone giant cell tumor; Ewing's sarcoma; malignant gingival tumors; eosinophilic odoosarcoma; malignant stromal blastoma; eosinophilic fibrosarcoma; malignant pineal tumor; Chordoma; malignant glioma; ependymocytoma; astrocytoma; protoplasmic astrocytoma; fibrillar astrocytoma; astroblastoma; glioblastoma; oligodendrocytes; glioblastoma oligodendroma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinocytoma; posterior neurogenic tumors; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granule cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's disease; paragranuloma; malignant small lymphocytic lymphoma; malignant diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestine disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia, and/or Waldenstrom's macroglobulinemia.

In a thirteenth aspect, the present invention relates to CD3+ lymphocytes, NK lymphocytes, cytokine-induced death (CIK) cells, gamma-delta lymphocytes, NKT cells or other immune effector cells expressing a chimeric antigen receptor according to the chimeric antigen receptor of aspect 4. A method of production is provided, comprising introducing an expression vector according to the expression vector of aspect 5 into said CD3+ lymphocytes, NK lymphocytes, cytokine-induced death (CIK) cells, gamma-delta lymphocytes, NKT cells or other immune effector cells. do.

In some embodiments, the cancer to be treated by the antibodies disclosed herein is a peripheral B cell or It is a non-Hodgkin's lymphoma derived from peripheral T cells.

In some embodiments, the cancer to be treated by the antibodies disclosed herein is multiple myeloma.

In some embodiments, the cancer to be treated by an antibody disclosed herein is melanoma.

In some embodiments, the cancer to be treated by the antibodies disclosed herein is testicular cancer, including seminal carcinoma, embryonic carcinoma, yolk tumor, and teratoma.

In some embodiments, the cancer to be treated by the antibodies disclosed herein is a pediatric malignancy such as nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma. ), medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immature teratoma Immaturity teratoma, or leiomyosarcoma.

6.17. Example

The following examples are provided to illustrate the invention, but should not be construed as limiting the invention. It will be understood that the embodiments include technical features and the present invention also relates to any combination of technical features presented in the embodiments.

6.17.1. Example 1: UMG1 Binding Specificity—UMG1 Antibody Binds to Lymphocytes in PBMCs and Not to Bone Marrow-Derived Cells

Binding of UMG1 to human peripheral blood mononuclear cells (PBMCs) from healthy donors was tested.

Methods: Peripheral blood mononuclear cells (PBMCs) from different healthy donors were obtained by Ficoll gradient separation. Subsequently, cells were seeded into 5 ml tubes and 1 μg/ml UMG1 antibody or 1 μg/ml negative in 100 μl binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)). Stained with a control “scrambled” murine IgG1 antibody and incubated at 4° C. for 30 min. The cells were then washed twice in binding solution and stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody in the dark at 4°C for 30 min. Subsequently, cells were washed twice in binding solution. Cells were analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific). One tube for each donor was left unstained and one tube for each donor was stained with only FITC-conjugated secondary antibody as a negative control.

Results : The UMG1 antibody was able to recognize lymphocyte subpopulations with varying prevalence (range: 0-15%) in different human donors. The UMG1 antibody did not show any reactivity with all other cell populations within PBMC, including bone marrow-derived cells, demonstrating that bone marrow-derived cells in PBMCs from healthy subjects were negative for expression of the UMG1 epitope ( FIG. 1A ). and FIG. 1B ).

In contrast, when we assayed the same PBMCs for CD43 expression using a commercial anti-CD43 antibody (S7 from Becton Dickinson), all lymphocytes and bone marrow cells were found to be positive (see FIG. 1B ).

Consequently, the epitope on CD43 recognized by the UMG1 antibody exhibits a specific and restricted expression pattern in PBMC cells that differs from that of the epitope recognized by the commercial anti-CD43 antibody (S7).

6.17.2. Example 2: UMG1 binding specificity - PBMC T lymphocyte subset bound by UMG1 antibody

This example further characterizes the lymphocyte sub-populations detected by the UMG1 antibody using immune-magnetic sorting of individual lymphocytes.

Methods : Briefly, 15 μg of UMG1 antibody was mixed with the components provided by the manufacturer (EasySep ^™ “Do-it-yourself” Selection Kit, STEMCELL Technologies) to obtain a solution prepared for immunomagnetic separation. This solution was added to PBMCs from 3 different donors with at least 10% lymphocytes detected by antibody after FcR blocking and cells were incubated for 15 min at room temperature (rt). Subsequently, EasySep® Magnetic Nanoparticles were added to the solution and cells were incubated for an additional 10 min at rt. After that, the solution was placed on a magnet and unbound cells were removed.

Results : The cells detected by the UMG1 antibody were almost all CD45 ⁺ CD3 ⁺ CD4 ⁺ CD8 ^- CD127 ⁺ CCR7 ⁺ T lymphocytes ( see FIGS. 2A to 2D and Table 1 ).

UMG1 antigen-positive cell marker marker +/- CD45 + CD3 + CD4 + CD8 - CD127 + CCR7 + CD45ra + CD45ro +/- (40% positive; see Figure 2) CD56 -

6.7.3. Example 3: UMG1 Binding Specificity—Only T-ALL and Waldenstrom's Macroglobulinemia Cancer Cell Lines Express UMG1 Epitope

In this experiment, various hematopoietic and non-hematopoietic cancer cell lines were evaluated for expression of the UMG1 epitope.

Method: Briefly, cells were seeded into 5 ml tubes and scrambled at 1 μg/ml of mAb UMG1 or 1 μg/ml in 100 μl of binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)). Stained with murine IgG1 antibody and incubated at 4° C. for 30 min. The cells were then washed twice in binding solution and stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody in the dark at 4°C for 30 min. Subsequently, cells were washed twice in binding solution and acquired on an ATTUNE NxT flow cytometer (THERMO Scientific). One tube for each cell line was left unstained and one tube for each cell line was stained with FITC-conjugated secondary antibody alone.

Results: T-ALL cell lines belonging to EGIL T3 classification and Waldenstrom macroglobulinemia ( FIGS. 3A to 3B ) were all positive for the expression of the UMG1 epitope, while the other cell lines analyzed were observed to be negative for the UMG1 epitope ( FIGS. 3A to 3B ). See Table 2 ). The UMG1 antibody recognizes T-ALL and Waldenstrom's macroglobulinemia cell lines, but not other hematopoietic cancers and non-hematopoietic tumors.

UMG1 epitope expression in various cancer cell lines cell line cancer type UMG1 +/- H929 Multiple myeloma (MM) + AMO MM - U266 MM - KMS11 MM - KMS26 MM + 8226 MM - BCWM.1 Waldenstrom's macroglobulinemia (WM) + MWCL.1 W.M. + H9 T lymphoma + HPB-ALL T-ALL + MOLT-4 T-ALL + JURKAT T-ALL - CEM T-ALL + HSB2 T-ALL - THP-1 monocytic leukemia - MOJ glioblastoma - SKMEL melanoma - HCC breast - MCF-7 breast - 5637 bladder - CAPAN pancreas - BXPC 3 pancreas - TCAM2 testicular cancer +

6.17.4. Example 4: UMG1 binding specificity - UMG1 binds to T-ALL human cell line with a different binding pattern than commercially available CD43 antibody

This example demonstrates the unique binding properties of the UMG1 antibody compared to the commercially available CD43 antibody in two different T-ALL human cell lines, ALL-SIL and KE-37.

Methods : Commercially available CD43 antibody: clones, CD43 1G10 (Becton Dickinson), CD43 MEM-59 (Invitrogen), and CD43 L-10 (Invitrogen) were compared with the UMG1 antibody.

Cells from various human cell lines (100,000 cells/tube) were collected. Wash the cells by adding 2 mL of cold staining buffer, centrifuge the cells for 5 min at 1,200 rpm at room temperature and discard the supernatant. Primary antibody UMG1 was added at a concentration (1 μg/ml) in a final staining volume of 100 μL of cells. Cells were gently mixed with a pulse vortex. Next, the cells were incubated at 2-8° C. for 15 minutes, protected from light. The cells were washed twice by adding 2 mL of staining buffer, the cells were centrifuged at 1,200 rpm for 5 min at room temperature, and the supernatant was discarded. Secondary fluorochrome-labeled antibody was diluted according to the manufacturer's instructions to a final volume of 100 µL of cells, protected from light, and incubated at 2-8 °C for 15 min. Cells were washed twice as indicated above, resuspended in 500 μL of PBS IX and analyzed by flow cytometry.

Results: The present inventors differed by FACS analysis for UMG1 antibody, and CD43 1G10 (Becton Dickinson), CD43 MEM-59 (Invitrogen), and CD43 L-10 (Invitrogen) in both ALL-SIL and KE-37 cell lines. Expression density and intensity were observed. See Figures 12A and 12B. These observations suggest that the UMG1 antibody has three different CD43 commercial antibodies and different binding sites for CD43.

6.17.5. Example 5: UMG1 Binding Specificity—UMG1 is Reactive with Tumor Immune Infiltrates

This example demonstrates the unique binding properties and expression of m-UMG1 in human colon, lung, and breast cancer tissues compared to other characterized CD43 antibodies.

Methods : Paraffin-embedded tissue samples from three different human cancers were excised, deparaffinized and then analyzed immunohistochemistry for expression of the UMG1 epitope using the following protocol.

The samples were placed in a heater at 65° C. for 30 minutes to remove paraffin. Next, sections were immersed in (1) xylene for 10 min, (2) xylene for 5 min and then rehydrated with graded alcohol: 90% ethanol for 2 min; in 70% ethanol for 2 min. The slides were washed with running tap water followed by a final wash in deionized water.

Antigen unmasking was performed using Novocastra Epitope Retrieval Solutions, pH 9 (Leica Biosystems) in an incubator at 98° C. for 30 min. Endogenous peroxidase was neutralized for 10 min using a Peroxidase Block. Peroxidase Block. 3/4%, (v/v) H ₂ O ₂ . Next, the samples were washed twice in PBS for 5 minutes in each wash. After washing, samples were incubated with Protein Block for 8 minutes. Protein Block, 0.4% casein in phosphate-buffered saline. After blocking, slides were washed twice in PBS for 5 min for each wash.

At a dilution of 1:300, sections were stained with primary antibody UMG1 ("m-UMG1") overnight at 4°C. Next, the stained sections were washed twice in PBS for 5 min for each wash. After washing, samples were incubated with rabbit anti-mouse IgG for 30 min and then washed twice in PBS samples for 5 min in each wash. After washing, samples were incubated with Novolink Polymer, anti-rabbit Poly-HRP-IgG for 30 min, then washed twice in PBS for 5 min for each wash.

Staining of sections was revealed by AEC (3-amino-9-ethylcarbazole) matrix-chromosome (Dako) followed by rinsing with running tap water. Sections were counterstained with hematoxylin for 5 min and then rinsed again with running tap water. Sections were mounted with Ultramount Aqueous Permenent Mounting Medium (Dako). Tissue sections were analyzed for UMG1 staining under a light microscope (Leica Microsystems) and micrographs were collected using a digital camera (Leica).

Results : We observed UMG1 staining in immunoinfiltrates of various solid tumors. More particularly, we have seen significant reactivity with tumor-associated macrophages in lung, colorectal, and breast cancer tissues. See FIG. 14A (Colorectal Adenocarcinoma (Grade 2, G2), 14B (Lung Adenocarcinoma) and 14C (Breast, Triple Negative Tubular Infiltrating Breast Cancer (G2, Basal-like)).

Significantly, UMG1 failed to directly stain cancer cells, unlike other previously described CD43 antibodies such as UN1 (UN1 staining in De Laurentiis, A. et al ., Molecular Cellular Proteomics , 2011, see Figure 9). .

These results demonstrate that UMG1 antibodies have different binding profiles for other characterized CD43 antibodies, particularly UN1 and CD43, which were previously shown to bind to CD43 in cancer cells.

6.17.6. Example 6: UMG1 binding specificity—UMG1 epitope is expressed in tumor-associated macrophages, and UMG1 epitope expression is elevated when macrophages are co-cultured to interact with cancer cells

In this example, samples from different types of cancer were evaluated for expression of the UMG1 epitope by immunohistochemistry and found that the specific CD43 epitope bound by UMG1 is highly expressed by tumor-associated macrophages (TAM). did.

Methods : Various types of cancer were stained as outlined in the Examples provided herein.

Results: By evaluating various types of cancer specimens through immunohistochemistry ( Table 3, Fig. 4 and Figs. 14A-14C ), UMG1 ⁺ macrophages are highly invasive components of most tumors, and bone marrow- Specific and specific high-grade infiltration was observed in pancreatic and ovarian cancers despite the absence of the UMG1 epitope in the derived cells.

UMG1+ TAM Infiltration in Various Tumors cancer type UMG1+ TAM Infiltration
(+: low; ++: medium; +++: high) Hodgkin's Lymphoma + Non-Hodgkin's Lymphoma ++ plasmacytoma ++ colorectal cancer + pancreatic cancer +++ lung cancer ++ prostate cancer + breast cancer ++ ovarian cancer +++ bladder cancer ++ melanoma ++

To better understand the importance of the UMG1 epitope in macrophages, in a second experiment, changes in UMG1 epitope expression were evaluated in a macrophage differentiation model in the presence or absence of co-cultured cancer cells. For this, THP-1 monocytic leukemia cells were used; As shown in Example 3 , these cells do not express the UMG1 epitope.

Methods : To obtain differentiated human unpolarized M0 macrophages (THP-1M), cells were cultured for 48 hours in complete appropriate medium in the presence of 50 ng/ml of phorbol 12-myristate 13-acetate (PMA). . The medium was then replaced with fresh medium without PMA. Next, PANC1 pancreatic cancer cell line cells were added to selected wells in a 1:1 ratio and incubated for 48 hours.

The cells were then prepared for immunofluorescence analysis. Briefly, after fixation, THP-1M cells were stained with chimeric antibodies derived from UMG1, ch-UMG1, or human IgG1 control further described in Example 16 and incubated overnight at 4°C. FITC anti-human secondary mAb was then added to the cells for 2 h. After washing, an anti-fading mounting medium containing DAPI (Vectashield®, Vectorlabs) was added to the cells and coverslips and then analyzed.

Results : As shown in Fig. 5a, THP-1-derived macrophages stained with control IgG1 were completely negative, whereas macrophages stained with ch-UMG1 were weakly (slightly) positive. Interestingly, in the presence of PANC1 cells, THP1-derived macrophages showed strong (bright) UMG1 expression. One specific interaction between THP-1-derived macrophages (white arrow) and PANC1 cells (red arrow) is shown ( FIG. 5A , left).

These findings demonstrate that UMG1-specific epitopes are significantly upregulated (ie, elevated) when macrophages interact with cancer cells within the co-cultured and reconstituted tumor microenvironment. This elevated expression means that the UMG1 epitope is a suitable target for therapeutic approaches focused on tumor-associated macrophage clearance. In addition to this relevant potential as a therapeutic tool, UMG1 may also be useful in detection, prognostic analysis roles, and predictive studies.

6.17.7. Example 7: UMG1 binding specificity - competitive binding assay suggests that the UMG1 binding site on CD43 is unique compared to the commercially available CD43 antibody

To determine whether the binding site of UMG1 is identical to or different from that of a commercially available CD43 antibody, (i) h-UMG1 (a humanized version of the UMG1 antibody, further described in Example 19 below) and phycoerythrin- Competitive binding assays between conjugated h-UMG1 (h-UMG1-PE) and (ii) h-UMG1 and three commercially available CD43 antibodies were performed in two different cell lines, CEM and HPB-ALL.

Methods : Competitive binding assays were performed and analyzed by FACS analysis using the following antibodies: unconjugated h-UMG1, h-UMG1-PE, and commercially available CD43 antibodies: MEM-59 PE (Invitrogen), L- 10 PE (Invitrogen) and 1G10 PE (Becton Dickinson). Briefly, CEM and HPB-ALL cells were treated with increasing concentrations (0.016 μg/ml, 0.08 μg/ml, 0.4 μg/ml) in the presence of either 1 μg/ml of CD43 clone or h-UMG1-PE (positive control). , 1 μg/ml, 2 μg/ml) with unconjugated h-UMG1 in the dark for 20 min on ice.

Approximately 500,000 cells were collected and stained for each test. Cells were analyzed and measured with FACS Canto (Becton Dickinson) and analyzed with DIVA software (BD FACSDiva™ software). For each measurement, 10,000 events were gated using DIVA software. Each experiment was performed in triplicate.

Results : As expected, unconjugated h-UMG1 competes with h-UMG1-PE binding in both CEM and HPB-ALL cell lines. That is, the number of stained cells labeled with h-UMG1-PE is decreased by increasing the concentration of unstained h-UMG1. See Figures 13A and 13B (circled lines).

In contrast, unconjugated h-UMG1 does not compete with the binding of other commercially available CD43 antibodies (MEM-59 (Invitrogen), L-10 (Invitrogen), and 1G10 (Becton Dickinson)). Indeed, with anti-CD43 The indicated number of stained cells was not reduced by increasing the concentration of unconjugated h-UMG1 antibody, see Figure 13a and Figure 13b (line with upward triangle, line with downward triangle, square with line).

These results suggest that the h-UMG1 antibody has a different binding site than the commercially available CD43 antibody.

6.17.8. Example 8: UMG1 binding specificity - flow cytometry profile of h-UMG1 in cell lines of hematopoietic lineage compared to UN1 historical published data

As mentioned above, UN1 has been reported in the literature to bind directly to various cancer lines, whereas UMG1 does not bind in the experiments reported in Examples 3 and 5 above. Instead, UMG1 binds to tumor-associated macrophages and invades solid tumors.

Since no UN1 antibody-secreting hybridomas have been deposited in biological repositories and no UN1 hybridoma master cell bank or working cell bank has been created, this does not preclude a side-by-side experimental comparison between the native UN1 antibody and UMG1, and therefore the inventors reported in Tassone et al. , Tissue Antigens 44:73-82, 1994] were repeated to further investigate the similarities and differences between UN1 and UMG1 binding. In these references, the binding of various cell lines JURKAT, MOLT-4, CEM and HPB-ALL lineages of the hematopoietic lineage was evaluated by flow cytometry expression.

Methods: Cells of the human cell line were collected at about 100,000 cells/tube. Cells were then washed by adding 2 mL of cold staining buffer, pelleted by centrifuging the cells at 1,200 rpm for 5 min at room temperature, and the supernatant discarded.

h-UMG1 primary antibody was added at a concentration of 1 μg/ml in a final staining volume of 100 μL cells. Next, cells were mixed by pulse vortex, protected from light and incubated at 2-8° C. for 15 min. The excess primary antibody was then washed twice by adding 2 mL of staining buffer, pelleted by centrifuging the cells at 1,200 rpm for 5 min at room temperature, and the supernatant discarded. Secondary fluorochrome-labeled antibody was added at the recommended dilution in a final volume of cells of 100 μL, protected from light and incubated at 2-8° C. for at least 15 min.

The excess secondary antibody was then washed twice by adding 2 mL of staining buffer, pelleted by centrifuging the cells at 1,200 rpm for 5 min at room temperature, and the supernatant discarded. The washed pelleted cells were resuspended in 500 μL of PBS IX and analyzed by flow cytometry.

Results: FIG. 17A shows the past of UN1 performed in 1994 by Tassone lab (Tassone et al. , Tissue Antigens 44:73-82, 1994) in JURKAT, MOLT-4, CEM, and HPB-ALL cell lines. Flow cytometry profiles are shown. 17B shows the results of flow cytometry profiles of h-UMG1 antibodies in JURKAT, MOLT-4, CEM, and HPB-ALL cell lines.

Comparison shows that both UMG1 and UN1 do not bind to JURKAT cells, but bind to MOLT-4, CEM, and HPB-ALL cell lines.

In particular, the flow cytometry profile of UMG1 in the CEM cell line shows approximately 1 log shift in the curve compared to UN1. The difference between the UN1 and UMG1 curves suggests that there is a difference in binding affinity to CEM cells.

6.17.9. Example 9: UMG1 binding specificity - h-UMG1 mAb binding to activated neutrophils

In this example, UMG1 epitope expression was assessed on inactivated and activated neutrophils isolated from human healthy donor peripheral blood.

Methods : Human whole blood from two healthy donors was collected in heparin anticoagulated vacuum vessels, diluted 1:1 with PBS, and Ficoll-Paque density gradient centrifugation (600 rcf, 15 min, RT, no brakes). ) was separated. RBCs (erythrocyte pellet) were suspended in 1x PBS followed by addition of 6% dextran solution. Dextran sedimentation was performed in the dark at RT for 30 min. The neutrophil supernatant was collected and centrifuged (5 min, RT, 600 rcf). To obtain a pure neutrophil population, RBC lysis was performed using 10x lysis buffer for 20 seconds, and lysis was stopped using 1x PBS. Samples were centrifuged for 10 min at 400 rcf without brake, the supernatant discarded, and the granulocyte pellet resuspended in culture medium. For each healthy donor granulocyte sample, half was incubated in growth medium only, while the other half was incubated with PMA (5 ng/ml) and inomycin (250 ng/ml) at 37° C., 5% CO ₂ for 15 min. stimulated. Samples were collected, washed in staining solution and Fc blocking was performed. Cells were incubated with CD11b Pe-Cy7 (pan-granulocyte marker), CD55 (neutrophil activation marker) and UMG1-APC or APC-conjugated IgG1 human isotype controls and acquired by flow cytometry h- on inactivated and activated granulocytes UMG1 expression was assessed.

Results : h-UMG1 mAb binding to inactivated and activated neutrophil populations was similar to that of isotype control (IgG1). Only a few neutrophils expressed the specific CD43 epitope targeted by the h-UMG1 mAb ( FIG. 33 ). CD43 is normally expressed on neutrophils and these results enhance the uniqueness of the UMG1 mAb target.

6.17.10. Example 10: UMG1 binding specificity - h-UMG1 mAb binding to activated T lymphocytes

In this example, UMG1 epitope expression was assessed on activated T lymphocytes from peripheral blood of healthy donors.

Methods : Whole healthy donor (HD) peripheral blood was collected in EDTA anti-coagulated vacuum vessels. Peripheral blood mononuclear cells (PBMCs) were isolated according to the Ficoll-Paque density gradient centrifugation protocol. Lymphocytes from 3 HDs were activated with PMA (25 ng/ml) and inomycin (1 μg/ml) at 37° C., 5% CO ₂ for 24 h. After activation, cells were harvested, washed with 1x PBS and stained with early and late T cell activation markers, specifically CD25-BV515, CD69-PE and h-UMG1-APC or IgG isotype control-APC. Flow cytometry was performed to evaluate h-UMG1 expression on CD25 ⁺ T cells and CD69 ⁺ T cells.

Results : No significant difference was observed in h-UMG1 mAb binding in activated T lymphocytes compared to the IgG isotype control ( FIGS. 34A -D ). Thus, h-UMG1 mAb does not target activated T lymphocytes.

6.17.11. Example 11: UMG1 Binding Specificity—Immunohistochemical Analysis of mUMG1 Binding in Healthy and Neoplastic Tissues

Methods: Tissue microarrays (TMA) used in immunohistochemical analysis included: Dog Female Normal Organ (DGF281); Rat Normal Organ (RAT901a); Mouse Normal Organ (MO541c); Rhesus Monkey Normal Organ (RhFDA1a); Cynomolgus Monkey Normal Organ (CyFDA1c); Multiple Organ Cancer and Normal Tissue (MC5003c); Malignant Melanoma and Skin Tissue (ME2081); Lymphoma Survey Tissue (Ly2084); breast cancer tissue (BR1505d); testicular disease (TE2081); embryonic carcinoma tumor test (T001a); human digestive system (GI1441); human brain tumor (GL2082); Human Pediatric Malignant Tumor (PC701) produced by US Biomax, Inc, and FDA Standard Paraffin Tissue Array Human Normal Organ (Cat. No. T8234701) produced by BioChain. A description of the selected TMA is provided below.

FDA Standard Tissue Array (Human Tissue, T8234701-5): Normal human tissue microarrays were provided on 5 slides containing 30 different human normal tissue types and 3 donors per tissue type.

Multi-organ cancer and normal tissue (MC5003c): high-density multi-organ tumor with normal tissue microarrays containing 20 types of tissue, each organ from 25 individuals (20 cases for tumors and 5 normal tissues) A single core was harvested per case.

Malignant Melanoma (ME2081): Malignant Melanoma and Skin Tissue Microarray, 88 cases of malignant melanoma, 16 skin tissues, 2 duplicate cores per case.

Lymphoma Investigation Tissue (Ly2084): Lymphoma Tumor Investigation Tissue Microarray (Slide 4 of 520 Cases of Lymphoma Investigation Slide Set), Malignant Tumor (64 B-cell Lymphoma, 24 Mucosal Associated Lymphoma Tissue, 6 T-cell Lymphoma) , including 104 cases of 4 Hodgkin's lymphomas, 4 anaplastic giant cell lymphomas, 1 each of mantle cell lymphoma, and Burkitt's lymphoma), with 2 duplicate cores per case.

Testicular disease (TE2081): testicular tumor tissue microarray, 46 cases of seminaloma, 8 yolk tumor, 16 embryonic carcinoma, 5 teratoma, 3 tuberculosis, 6 atrophy, 15 adjacent normal tissue and 5 With normal tissue, 2 duplicate cores per case.

Human pediatric malignancy (PC701): pediatric malignant tissue microarray with normal tissue, nephroblastoma in 21 cases, neuroblastoma in 13 cases, endodermal sinus carcinoma in 7 cases, and endodermal sinus carcinoma in 4 cases. Retinoblastoma, 3 hepatoblastoma, 2 medulloblastoma, 4 lymphoma, choroid plexus papilloma, glioblastoma, adrenocortical carcinoma, embryo Carcinomas Embryonal rhabdomyosarcoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma, respectively , and 7 normal tissues, single core per case.

For immunostaining, tissue sections were dewaxed and rehydrated. The antigen unmasking technique was performed using Novocastra Epitope Retrieval Solutions, pH 9 EDTA-based buffer in a thermostat (FALC Instruments S.r.L, Treviglio (BG) Italy, Model WB-MD 5) at 98° C. for 30 minutes.

TMA was incubated overnight at 4° C. with primary UMG1 mAb (m-UMG1 mAb was used in this application, 1:300 dilution). Immunostaining was performed using either a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd product number: RE7280-K) or a ready-to-use AEC (3-amino-9-ethylcarbazole, Dako, Ref K3464) substrate-chromogen ( chromogen). Slides were counterstained with Harris Hematoxylin (Novocastra, Leica Biosystems).

All sections were analyzed under a Zeiss Axio Scope A1 optical microscope (Zeiss, Germany) and photomicrographs were collected with ZEN2 imaging software (Zeiss Germany) using an Axiocam 503 Color digital camera.

Assessment of UMG1 expression: Sections were scanned using an Aperio CS2 Leica. Protein expression levels were scored passively by measuring signal intensity using a 4-grade scale: negative (0), weak (1), medium (2) and strong (3). Micke P, et al. , 2014, Int J Cancer , 135:2206-2214., the entire contents of which are incorporated by reference.

Results : In normal human tissues, positive binding of UMG1 mAb was observed on the thymus ( FIG. 38A ) and tonsil lymph nodes ( FIG. 38B ). Membrane staining was positive with moderate/strong intensity (scores 2-3) in >50% of lymphocytes and a small fraction of elements with monocyte/macrophage morphology. Although rare associations were observed in immune cells scattered within other organs, expression in these cells was more compatible with cytoplasmic expression without membrane enhancement (see Figure 38c with examples from lung tissue).

In the lymphoma disease spectrum, cytoplasmic and membrane staining was frequently observed and highlighted by variable intensities ( FIGS. 39A and 39B , Table 6 ). Membrane expression of UMG1 mAb target epitopes had different degrees of intensity and distribution, but was also evident in malignant cells at multiple observation points.

With respect to malignant melanoma, the UMG1 mAb target was expressed on the malignant cell membrane and TAM ( FIG. 40A , Table 6 ). In addition, some samples stained positively for intratumoral leukocyte infiltration, as expected by preliminary data on epitope expression of tumor-associated macrophages.

Among testicular neoplasms, greater positivity in neoplastic clones was observed in seminal tumors ( FIG. 40B , Table 6 ), followed by embryonic carcinoma and yolk tumors. Healthy tissue around the tumor and normal healthy testes were both negative.

Evaluation of UMG1 mAb Binding for Lymphoma, Melanoma, and Testicular Cancer tumor Analytical sample (N°) ^One UMG1+
(N°) ² More than 10% cell staining
(N°) ³ Staining intensity 2+ or 3+ (N°) ⁴ UMG1+ Immune Infiltrate
(N°) ⁵ lymphoma 167 94 73 45 N/A ⁶ Diffuse large B-cell lymphoma 117 64 53 30 N/A MALT 29 21 14 9 N/A T-lymphoma 12 5 4 2 N/A Anaplastic giant cell lymphoma 6 3 One 3 N/A Etc 3 One One One N/A melanoma 203 15 N/A 2 98 testicular cancer 137 126 112 69 132 semenoma 83 78 73 32 78 yolk tumor 16 16 12 8 16 embryonic carcinoma 32 26 23 23 32 Teratoma (mature and immature) 6 6 4 6 6

¹ Number of samples available for evaluation (i.e., those considered to be adequately stained)

² Number of samples marked by UMG1 antibody on neoplastic cell membrane

³ Number of samples with >10% neoplastic cells marked by UMG1 antibody

⁴ Number of samples with neoplastic cells marked by UMG1 antibody with an intensity score of 2+ or 3+

⁵ Number of samples with >10% immune cells infiltrating the tumor microenvironment marked by UMG1 antibody

⁶ Not Applicable or Not Rated

Evaluation of UMG1 mAb Binding in Pediatric and Embryonic Carcinomas tumor Analytical sample (N°) ^One UMG1+
(N°) ² More than 10% cell staining
(N°) ³ Staining intensity 2+ or 3+ (N°) ⁴ UMG1+ Immune Infiltrate
(N°) ⁵ adrenocortical adenocarcinoma One 0 0 0 0 Alveolar rhabdomyosarcoma One One One One One Burkitt's Lymphoma One One One One N/A Choroidal plexus papilloma One 0 0 0 One Diffuse B-cell lymphoma 2 One One 0 N/A Embryonic Carcinoma Rhabdomyosarcoma 0 0 0 0 0 endoderm sinus tumor 5 2 0 0 3 ependymocytoma One One 0 One One glioblastoma One One One One One hepatoblastoma 2 2 2 0 2 Hodgkin's Lymphoma One 0 0 0 N/A immature teratoma One 0 0 0 One leiomyosarcoma One One One 0 One medulloblastoma 2 One 0 0 One neoblastoma 21 7 4 3 5 neuroblastoma 13 10 6 6 7 primitive neuroectodermal tumor One One One One 0 retinoblastoma 4 3 3 2 3

¹ Number of samples available for evaluation (i.e., those considered to be adequately stained)

² Number of samples marked by UMG1 antibody on neoplastic cell membrane

³ Number of samples with >10% neoplastic cells marked by UMG1 antibody

⁴ Number of samples with neoplastic cells marked by UMG1 antibody with an intensity score of 2+ or 3+

⁵ Number of samples with >10% immune cells infiltrating the tumor microenvironment marked by UMG1 antibody

⁶ Not Applicable or Not Rated

Another set of cancers exhibiting expression of the UMG1 mAb epitope in both neoplastic cells and immune infiltrates are pediatric tumors of different origin (see Table 7 ).

In addition, UMG1 epitope expression on cancer cells or immune cell infiltrates (both tumor associated macrophages (TAMs) and other immune cell infiltrates) was observed at some points representing multiple solid tumors of different origins (MC5003c tissue microarray). UMG1 mAb binding in these samples was characterized by different levels of distribution and intensity (data not shown).

6.17.12. Example 12: UMG1 binding specificity - epitope binding site for CD43

Various CD43 protein variants were tested for binding of the h-UMG1 antibody by Western blot and FACS analysis to determine the binding site of h-UMG1 to CD43 in HEK293T-wild-type cells that do not express CD43.

CD43 protein variants : The sequences of the tested CD43 protein clones are provided as a sequence listing in FIG. 15A , Table 4 and SEQ ID NOs: 17-24. Wild-type CD43, designated "CD43 #1", was generated using the entire 400 amino acid region. To engineer CD43 protein variants, the N-terminal domain was cleaved sequentially. The first CD43 truncated variant, "CD43 #2", was generated using aa from 31 to 400 of full length CD43. A second CD43 variant, designated "CD43 #3", was generated using aa from 41 to 400 of full length CD43. A third CD43 variant, designated "CD43 #4", was generated using aa from 61 to 400 of full length CD43. The fourth CD43 variant, designated "CD43 #5", consists of aa 91 to 400 of full-length CD43. The fifth CD43 variant, designated "CD43 #6", has aa 64-78 deleted.

In addition, single amino acid deletion variants were also tested. The sixth CD43 variant, designated "CD43 #7", has a single amino acid deletion at aa 69, which is thought to be the GalNac site. The seventh CD43 variant, designated "CD43 #8", has a single amino acid substitution in which T is changed to N in aa69, or "T69N".

CD43 protein variants tested for UMG1 antibody binding variant Sequence region used
(based on wild-type CD43) SEQ ID NO: CD43 #1 (wt) aa 1-400 17 CD43 #2 aa 31-400 18 CD43 #3 aa 41-400 19 CD43 #4 aa 61-400 20 CD43 #5 aa 91-400 21 CD43 #6 aa 1-63 | 79-400 22 CD43 #7 aa 1-68 | 70-400 23 CD43 #8 aa 1-400 (T69N) 24

Construct: CD43 protein construct was obtained from Applied Biological Materials (ABM) Inc. Expression was performed using the pLenti-CMV-(insert)-Histag-GFP-2A-Puro expression vector from the service (Vancocver, Canada). His-Tag and/or GFP detection served as a positive control for successful transfection and/or protein expression.

Transfection : Each vector was transiently expressed in HEK293T cells using Lipofectamine LTX (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. HEK293T cells were maintained at 37° C. and 5% CO ₂ in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (ThermoFisher Scientific, MA, USA). 72 hours after transfection, cells were subjected to Western blot or flow cytometry (FACS) analysis.

Western Blot : Western blot using UMG1 antibody was performed to determine if UMG1 antibody was able to bind and detect CD43 wild-type and CD43 protein variants at the expected kDa size. His-tagged antibody was used as a positive control.

Briefly, whole cell protein extracts were obtained using NP40 lysis buffer supplemented with a Halt ^™ protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, MA, USA). Protein concentrations were estimated using a Bradford assay (Bio-Rad Laboratories, Berkeley, CA, USA). Cell lysates were loaded at a concentration of 60 μg per lane and separated using NuPAGE ^™ 3-8% Tris-Acetate Protein Gels (Invitrogen, Thermo Scientific, MA, USA). Proteins were electro-transferred for 30 min using a Trans-Blot® Turbo ^TM Transfer System (Bio-Rad Laboratories, Berkeley, CA, USA) and an anti-actin antibody purchased from Cell Signaling (data are not shown), anti-His-Tag antibody from abm (Vancouver, Canada) (#G020) and h-UMG1 primary antibody (both at 1:500 dilution). Goat anti-mouse and earthenware anti-human HRP-conjugated antibodies (Invitrogen) were used as secondary antibodies (1:3,000 dilution). Immunoactive bands were revealed by enhanced chemiluminescent detection using SuperSignal ^™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, MA, USA).

Flow cytometry (FACS) analysis: FACS was performed to determine whether the antibody detected CD43 wild-type and CD43 variants, as expressed in HEK293T cell line cells.

FACS analysis was performed according to standard procedures, using 1 μg/ml of h-UMG1-PE conjugated antibody to detect the percentage of h-UMG1 positive cells among GFP positive cells. Samples were acquired by flow cytometry (LSRFortessa ^™ X-20, BD) and analyzed with DIVA software (BD FACSDiva ^™ software). A minimum of 20,000 events were gated for each measurement.

Results : The results of Western analysis suggest that the UMG1 epitope binding site is located between 61 and 91 (numbered in wild-type CD43). Reference is made to the boxchin sequence in FIG. 15A , which indicates the hypothesized binding site for the h-UMG1 antibody. In addition, these studies show that the UMG1 antibody specifically binds to UMG1 rather than the CD43 His-tagged protein, which lacks a specific extracellular region recognized by UMG1. 15c and 15e .

FACS observations confirmed the results observed on western blots that the epitope binding site UMG1 was located between 61 and 91 aa of the CD43 wild-type because it was detected in GFP-expressing cells of CD43 #5 and CD43 #6 protein variants. because it cannot be 15D and 15F . In addition, Western blot and FACS studies presented in FIGS. 15D and 15F showed that the CD43 aa region from aa 61-91 when threonine 69 was deleted (CD43 #7) or substituted with a non-O-glycosylated amino acid (CD43 #8). This suggests that it is recognized by the h-UMG1 antibody even in HEK293T cells having transgenic expression of CD43. Due to this, an epitope that should not have any sugar group is bound. As expected, wild-type HEK293T cells that were not transfected with CD43 did not show any reactivity with the UGM1 antibody.

6.17.13. Example 13: UMG1 binding specificity-h-UMG1 mAb-linear epitope mapping of CD43 epitope

Method : Microarray Content: Human CD43 ( SEQ ID NO: 17 ) sequence was extended with neutral GSGSGSG ( SEQ ID NO: 46 ) linkers at the C- and N-terminus to avoid truncated peptides. The extended antigen sequence was translated into a linear 15 amino acid peptide with a peptide-peptide overlap of 14 amino acids. The resulting CD43 peptide microarray contained 400 different peptides (800 peptide spots) printed in duplicate and framed by an additional HA (YPYDVPDYAG ( SEQ ID NO: 47 ), 82 spots) control peptide. Microarray synthesis and analysis were performed by PEPperPRINT GmbH, Heidelberg.

Pre-staining of CD43 peptide microarray copies was performed with a secondary antibody (goat anti-human IgG (H+L) DyLight680 (0.2 μg/ml)) and a control antibody (mouse monoclonal anti-HA (12CA5) DyLight800 (0.5 μg). /ml)). After 15 min pre-swelling in wash buffer (PBS, pH 7.4 with 0.05% Tween 20) and 30 min incubation in blocking buffer (Rockland blocking buffer MB-070), CD43 peptide microarrays were incubated for 45 min at room temperature (RT). Background interactions with linear CD43 peptides that may interfere with the main assay were investigated by initial incubation with secondary and control antibodies in incubation buffer (wash buffer with 10% blocking buffer).

Another copy of the CD43 peptide microarray with humanized monoclonal anti-CD43 antibody (h-UMG1 mAb) at concentrations of 10 μg/ml and 100 μg/ml in incubation buffer was subsequently incubated (16 h at 4° C. and 140 rpm). ) and then stained with secondary and control antibodies. Readings were performed with a LI-COR Odyssey Imaging System at a scan intensity of 7/7 (red = 700 nm/green = 800 nm), a scan offset of 0.65 mm, and a resolution of 21 μm. An additional HA control peptide framing the peptide microarray was simultaneously stained with a control antibody as an internal quality control to confirm assay quality and peptide microarray integrity.

At scan intensities of 7/7 (red/green), we observed no background interaction of the linear CD43 peptide with secondary and control antibodies despite significant increases in brightness and contrast. Therefore, data quantification using the PepSlide ^® analyzer was omitted. Staining with control antibody generated a predicted well-defined HA control spot pattern framing the peptide microarray (green) to verify overall microarray integrity and assay quality.

Quantification of spot intensities and peptide annotations were based on 16-bit grayscale tiff files that exhibit higher dynamic range than 24-bit colored tiff files. Microarray image analysis was performed with a PepSlide ^® analyzer. A software algorithm classifies the fluorescence intensity of each spot into raw, foreground and background signals, and calculates the spot-to-spot deviation of the averaged central foreground intensity and duplicate spots. Based on the averaged central foreground intensities, intensity maps were generated and the interactions of the peptide maps were highlighted with intensity color codes, where red is high spot intensity and white is low spot intensity. We allowed a maximum spot-to-spot deviation of 40%, otherwise the corresponding intensity value was zero.

We further plotted the average spot intensity of the assay with humanized monoclonal antibodies against the N-terminus to C-terminus antigen sequence of CD43 to visualize the overall spot intensity and signal-to-noise ratio. . Intensity plots were correlated with peptide and intensity maps as well as visual inspection of microarray scans to identify epitopes of humanized monoclonal anti-CD43 antibodies. If it is not clear whether a particular amino acid contributes to antibody binding, the corresponding letter is written in gray. For a better overview of the data, the baseline of the intensity plot was leveled.

Results : We observed a weak antibody response to an epitope-like spot pattern formed by adjacent peptides with the consensus motif INEGSPLW ( SEQ ID NO: 48 ; aa 71-78 on human CD43); In addition, the present inventors have identified two more potent peptides with the highly basic consensus RRRQKR ( SEQ ID NO: 49 ) and RRPTLTTFFGRRK ( SEQ ID NO: 50 ), most likely due to non-specific ionic binding of the antibody. Interactions were observed ( FIG. 35 ). A suitable signal-to-noise ratio was well defined by the simultaneous staining of the HA control peptide.

6.17.14. Example 14: UMG1 binding specificity - linear epitope mapping of h-UMG1 mAb - epitope substitution scan

Methods : Microarray Content: Epitope substitution scans of wild-type peptide PPSTSINEGSPLWTS ( SEQ ID NO: 51 ) were based on exchange of all amino acid positions by the 20 major amino acids. The resulting peptide microarray consisted of 300 different peptide variants of wild-type peptide printed in triplicate (900 peptide spots), 9 spots of custom control peptide PPSTSVNEGSPLGTS ( SEQ ID NO: 52 ) and an additional HA control peptide (YPYDVPDYAG, 82 spots ( SEQ ID NO: 47 )). Microarray synthesis and analysis were performed by PEPperPRINT GmbH, Heidelberg.

Pre-staining of the peptide microarray copies was performed with secondary and control antibodies. After 15 min pre-swelling in wash buffer and 30 min incubation in blocking buffer, the peptide microarray copies were initially incubated with secondary and control antibodies for 45 min at room temperature with variants of the wild-type peptide that might interfere with the main assay. background interactions were investigated.

Subsequent copies of other peptide microarrays with humanized monoclonal anti-CD43 antibody at concentrations of 1 μg/ml, 10 μg/ml, 100 μg/ml (data not shown) and 250 μg/ml in incubation buffer After incubation, they were stained with secondary and control antibodies. After washing, readings were taken at a scan intensity of 7/7 (red/green). An additional HA control peptide framing the peptide microarray was stained simultaneously as an internal quality control to confirm assay quality and peptide microarray integrity.

At scan intensities of 7/7 (red/green), we observed no background interactions of secondary and control antibodies with 300 variants of the wild-type peptide or additional custom peptides, despite significant increases in brightness and contrast ( See Adjusted Scan). Therefore, data quantification using the PepSlide ^® analyzer was omitted. Staining with control antibody generated a predictive well-defined HA control spot pattern framing the peptide microarray to verify overall microarray integrity and assay quality.

Quantification of spot intensities and peptide annotations were based on 16-bit grayscale tiff files that exhibit higher dynamic range than 24-bit colored tiff files. Microarray image analysis was performed with a PepSlide ^® analyzer. The software algorithm classifies the fluorescence intensity of each spot into raw, foreground and background signals, and calculates the spot-to-spot deviation of the averaged central foreground intensity and three duplicate spots. Based on the averaged central foreground intensities, intensity maps were generated and the interactions of the peptide maps were highlighted with intensity color codes, where red is high spot intensity and white is low spot intensity. A very weak signal with a blurry spot shape went to zero.

We plotted the averaged spot intensity of our assays with humanized monoclonal antibodies against microarray content from top left to bottom right of the chip in a row fashion to visualize overall spot intensity and signal-to-noise ratio. Intensity plots were correlated with peptide and intensity maps, as well as visual inspection of microarray scans to identify variants of wild-type peptides interacting with humanized monoclonal anti-CD43 antibodies.

To provide an in-depth view of the epitope substitution scans of wild-type peptides, we further generated heat maps of microarray scans as well as substitution matrices and amino acid plots reflecting amino acid preference at a given position. Data sets were analyzed to identify conservative and variable amino acid positions of wild-type peptides.

Highlight the preference for a given amino acid by color code (red for preferred amino acids, blue for less preferred) against the substitution matrix and average the spot intensity of a given peptide over all 20 peptides that were substituted at the same position It was calculated by dividing by the applied spot intensity. Amino acid plots were calculated by dividing the spot intensity of a given peptide by the spot intensity of the wild-type peptide. Therefore, the position of the amino acid reflected the intensity ratio compared to the amino acid of the native wild-type peptide.

Results : A substitution scan of the wild-type peptide PPSTSINEGSPLWTS ( SEQ ID NO: 51 ) revealed a weak but typical epitope substitution pattern with conserved (few or single spots in a row) amino acid positions and variable (consecutive rows of spots) amino acid positions ( Fig. see 36 ). A low signal-to-noise ratio was observed and well-defined with simultaneous counterstaining of frames of the HA control peptide.

Heat maps, substitution matrices and amino acid plots of humanized monoclonal anti-CD43 antibody analyzed for substitution scans of the wild-type peptide PPSTSINEGSPLWTS ( SEQ ID NO: 51 , aa 66-80 of human CD43) are N- and C-terminal Conserved core motif EGSPLW ( SEQ ID NO: 53 ) framed by variable stretch PPSTSIN ( SEQ ID NO: 54 , aa 66-72 of human CD43) and TS ( SEQ ID NO: 55 , aa 79-80 of human CD43) , aa 73-78 of human CD43) were highlighted (see FIGS. 37a-c ). This finding was in accordance with the proposed epitope INEGSPLW of this epitope mapping to the full-length human CD43 protein.

Amino acid positions 76 (P) and 77 (L) of human CD43 were essential for antibody binding and did not allow any amino acid exchange at all. Amino acid positions 73 (E) and 78 (W) were highly conserved and allowed only conservative exchange by D and F, respectively. The exchange of W by F at position 78 even resulted in an apparent increase in antibody binding. Amino acid position 74(G) had a low degree of sequence conservation and was susceptible to substitution with M, acidic amino acids D and E, as well as aromatic amino acids W, F and Y.

All other amino acid positions, including amino acid position 75(S), exhibited variable properties. At variable amino acid positions, we observed a general preference for acidic and aromatic amino acids such as E, D, F and W. In addition, the variable amino acid positions exhibited rejection of substitution with the basic amino acids K and H, but surprisingly R did not.

6.17.15. Example 15a: UMG1 binding specificity - binding to the deglycosylated extracellular portion of CD43 compared to other CD43 antibodies

This example shows the determination of binding affinity between humanized-UMG1 (h-UMG1) (H3-L4) and the extracellular portion of CD43 (aa 20-253). The results are reported as the dissociation constant K _D .

Methods: Antibodies dissolved in water were manually printed on a pure gold-coated (47 nm thick) PlexArray Nanocapture Sensor Chip (Plexera Bioscience, Seattle, WA, US) at 40% humidity. Different concentrations of analyte (CD43) were tested for affinity. Each concentration was printed in duplicate, and each spot contained 0.2 uL of sample solution. The chips were incubated overnight at 4° C. at 80% humidity and rinsed twice with 10Х PBST for 10 minutes, 1Х PBST for 10 minutes, and deionized water for 10 minutes. The chips were then blocked overnight in water with 5% (w/v) nonfat milk, washed twice with 10Х PBST for 10 minutes, 1Х PBST for 10 minutes, and with deionized water for 10 minutes and under a nitrogen stream prior to use. dried. SPRi measurements were performed with a PlexAray HT (Plexera Bioscience, Seattle, WA, US). Collimated light (660 nm) passes through a coupling prism and is reflected off the SPR-activated gold surface and is received by a CCD camera. Various concentrations of analyte (human recombinant CD43 extracellular portion (20-253 aa) produced in E. coli vector; SEQ ID NO: 42 ) were used in this experiment (various concentrations of analyte are shown in different colors in FIG. 19 ). is shown by the line of ). Buffer and samples were injected by a non-pulsatile piston pump into a 30 μL flow cell mounted on a coupling prim. Each measurement cycle included 4 steps: washing with PBST running buffer at a constant rate of 2 μL/s to obtain a stable baseline, injecting the sample at 5 μL/s for binding, at 2 μL/s Surface washing with PBST for 300 s, and regeneration with 0.5% (v/v) H3PO4 at 2 μL/s for 300 s. All measurements were performed at 25°C. Changes in signal (AU) after binding and washing were recorded as assay values.

Selected protein-grafted regions in the SPR images were analyzed, and the average reflectance change of the selected regions was plotted as a function of time. Real-time binding signals were recorded and analyzed by a Data Analysis Module (DAM, Plexera Bioscience, Seattle, WA, US). Kinetic analysis was performed using BIAevaluation 4.1 software (Biacore, Inc.).

Results : SPR binding results showed a K _D value of 99.4 nM between the extracellular portion of CD43 and h-UMG1 ( FIG. 19 ). These results indicate strong binding affinity to the target.

In addition, binding of CD43 to the deglycosylated extracellular portion (produced in E. coli , no mammalian glycosylation) binds UMG1 only to glycosylated or neuraminidase-sensitive epitopes such as UN1 and MEM-59, respectively. Distinguish from other anti-CD43 antibodies that bind (de Laurentiis A, et al ., Mol Cell Proteomics. 2011 May;10(5)).

6.17.16. Example 15b: UMG1 binding specificity - comparison of UMG1 binding properties to UN1 historical data

As demonstrated in the examples above, UMG1 and its chimeric and humanized derivatives, ch-UMG1 and h-UMG1, each exhibit some different binding properties compared to other anti-CD43 antibodies, including data previously reported for UN1. have Table 5 compares the characteristics of UMG1 against historically reported data for UN1.

UMG1 properties compared to known anti-CD43 antibodies characteristic UN1 UMG1
hybridoma No deposit, no frozen master cell bank, no frozen working cell bank Deposited under ICLC Deposit No. ICLC PD n° 16001 CDR sequence never sequenced known
SEQ ID NO: 1, 43, 3-6 Binding to glycosylated CD43 has exist has exist Binding to aglycosylated CD43 doesn't exist
(de Laurentiis et al ., Molecular & Cellular Proteomics 10:1-12, 2011; Fig. 8.) has exist

Western blot and FACS data for CD43 in the form of deletion or mutated form of residue T69 (Example 12).

Example 15a, Figure 19, the SPR data

Linear epitope mapping (Example 13, Figure 35) and amino acid substitution scan (Example 14, Figure 36 and Figure 37A-C) Binding to PBMC positive for the lymphocyte subpopulation; negative for monocytes and granulocytes

(Tassone et al. , Tissue Antigens 44:73-82, 1994) positive for a subpopulation of lymphocytes; Negative for bone marrow-derived cells.

Minimal binding observed in inactivated and activated neutrophils.

No binding observed in activated T lymphocytes

See Example 1, Example 2, Example 9, Example 10, Table 1, FIGS. 2A-2D, 33, and 34A-34D. Binding to T lymphoma and T-ALL Yes, observed only on cell lines

(Tassone et al. , Tissue Antigens 44:73-82, 1994) Yes, observed on cell lines and tissue microarrays

See Example 3, Example 11, Table 2, FIG. 39
Binding to Waldenstrom's Macroglobulinemia Cells unnotice has exist

See Example 3, Table 2, Figures 3a to 3b binding to cancer tissue has exist

(Cecco et al ., Tissue Antigens 51:528-535, 1998; Tassone et al. , Int. J. Oncology 20:707-711, 2002; Tuccillo et al ., Mol. Cancer Ther . 13(3)]). Yes, membrane expression observed on some neoplastic cells in multiple tissues (reduced intensity and distribution compared to UN1)

See Example 4, Table 3, and FIGS. 4 and 5, Example 11, FIGS. 14A-14C, 39A-39B, 40A-40B. Binding to tumor-associated macrophages (TAMs) and lymphocytes doesn't exist

de Laurentiis et al ., Molecular & Cellular Proteomics 10:1-12, 2011); Fig. 9. has exist

Example 4, Example 11, Table 3, Figures 4 and 5A-5B; Example 5, see FIGS. 14A to 14C binding to fetal tissue has exist

(Tassone et al. , Tissue Antigens 44:73-82; Tassone et al. , Int. J. Oncology 20:707-711, 2002). not explained Antibody-dependent cytotoxicity (ADCC) in HPB-ALL cell lines has exist

(Tuccillo et al ., Mol. Cancer Ther . 13(3), 2014). has exist

Example 17 Flow Cytometry Profiles in Cell Lines of Hematopoietic Lineage Binds to MOLT-4, CEM, and HPB-ALL but not Jurkat

(Tassone et al. , Tissue Antigens 44:73-82, 1994) Binds to MOLT-4, CEM, and HPB-ALL but not Jurkat, but with different affinities in CEM cells

Example 8

6.17.17. Example 16: Construction of a chimeric antibody with binding specificity of ch-UMG1-UMG1

A chimeric antibody with the binding specificity of UMG1 by fusing murine UMG1 VH ( SEQ ID NO: 34 ) to human VH constant region and murine UMG1 VL ( SEQ ID NO: 35 ) to human light chain constant region using standard techniques ( ch-UMG1) was constructed.

6.17.18. Example 17: ch-UMG1 - ch-UMG1 induces antibody-dependent cell-mediated cytotoxicity (ADCC) of T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma

To determine the potential activity of the mAb ch-UMG1 as an immunotherapeutic tool, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was tested in Example 3 against the two cell lines found to express the UMG1 epitope. tested.

PBMCs from healthy donors (effector cells) were co-cultured with T-ALL cell line HPB-ALL or T lymphoma cell line H9 (target cells) in the presence of different concentrations of ch-UMG1 as follows.

Method: 4 × 10 ⁴ target cells were seeded in 96-well round bottom plates and chimeric negative at different concentrations of mAb ch-UMG1 (0, 10, 50, 100, 200 μg/ml) or highest dose (200 μg/ml) or in the presence of positive control (NC and PC, respectively, 200 μg/ml) IgG1 at 37° C. 5% CO ₂ for 30 minutes. Subsequently, 0.4 × 10 ⁶ PBMCs from the same donor (fixed E:T=10:1) were added to each well along with 20 μl/ml of PE-conjugated anti-CD107a mAb (Becton Dickinson), followed by cells was incubated for 3 hours at 37° C. 5% CO ₂ . After 1 h, 6 μg/ml monesin was added to each well (GolgiStop, BD). At the end of the incubation period, cells were stained with APC-conjugated anti-CD56 and PerCp-conjugated anti-CD3 and analyzed by FACS using an ATTUNE NxT flow cytometer (THERMO Scientific).

Results: It was found that CD3 ⁻ /CD56 ⁺ /CD107a ⁺ cells significantly increased with the concentration of ch-UMG1 antibody, confirming the potential of ch-UMG1 antibody as an ADCC inducer ( FIGS. 6A and 6B ). .

The chimeric mAb ch-UMG1 is an active immunotherapeutic tool against T cell acute lymphoblastic leukemia/lymphoblastic lymphoma. These data enable the design of immune targeting approaches, an urgent and unmet clinical need in T cell acute lymphoblastic leukemia/lymphoblastic lymphoma.

6.17.19. Example 18: ch-UMG1 - ch-UMG1 induces antibody-dependent cell-mediated cytotoxicity (ADCC) of Waldenstrom's macroglobulinemia cells

To further investigate the immunotherapeutic potential of the ch-UMG1 antibody, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) of Waldenstrom's macroglobulinemia was evaluated.

Methods: We performed a degranulation assay by co-cultivating purified NK cells from a healthy donor (effector cells) and a BCWM.1 cell line (target cells) in the presence of different concentrations of ch-UMG1 antibody or negative/positive control. did. We selected mAb cetuximab as negative control and mAb rituximab as positive control.

Specifically, 10 ⁵ target cells were seeded in 96 well round bottom plates and different concentrations of ch-UMG1 antibody (0, 10, 50, 100, 200 μg/ml), 200 μg/ml cetuximab, or 200 μg/ml In the presence of rituximab, 37° C. 5% CO ₂ was incubated for 30 minutes. Subsequently, 10 ⁵ NK cells (fixed E:T=1:1) from the same donor were added to each well with 20 μl/ml of PE-conjugated anti-CD107a mAb (BD) and then the cells were seeded at 37 Incubated for 2 hours at °C 5% CO ₂ . After 1 h, 6 μg/ml monesin was added to each well (GolgiStop, BD). At the end of the incubation period, cells were stained with APC-conjugated anti-CD56 and PerCp-conjugated anti-CD3 and analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific).

Results : CD3 ⁻ /CD56 ⁺ /CD107a ⁺ cells were found to increase significantly with ch-UMG1 antibody concentration, reaching exactly the same effect as obtained with rituximab. These results confirm the potential of the ch-UMG1 antibody as an ADCC inducer ( FIG. 7 ). The chimeric mAb ch-UMG1 is an active immunotherapeutic tool for Waldenstrom's macroglobulinemia.

6.17.20. Example 19: h-UMG1 - Humanized UMG1 Monoclonal Antibody Construction

Humanized UMG1 antibodies were constructed using the combination of human heavy chain SEQ ID NO: 8 to SEQ ID NO: 11 and human light chain SEQ ID NO: 13 to SEQ ID NO: 16 provided herein.

Expression and purification of the antibody was performed as follows: The corresponding cDNA of the antibody was cloned into a vector system using conventional (non-PCR based) cloning techniques. The vector plasmid was a synthesized gene. Plasmid DNA was prepared under low-endotoxin conditions based on anion exchange chromatography. The DNA concentration was determined by measuring the absorption at a wavelength of 260 nm. Sequence accuracy was verified with a Sanger sequencer (with up to two sequencing reactions per plasmid, depending on the size of the cDNA).

Suspension-adapted CHO K1 cells (originally obtained from ATCC and adapted to serum-free growth in suspension culture) were used for production. For the afucosylated antibody (a-h-UMG1), GlymaxX technology was used (ProBioGen), which was transiently expressed in CHO cells (Evitria). Seeds were grown in a chemically defined, animal-free, serum-free medium. Cells were transfected with custom proprietary transfection reagents, and after transfection, cells were grown in serum-free medium free of animal components.

The supernatant was harvested by centrifugation and subsequently filtered (0.2 μm filter). Antibodies were purified using MabSelect ^™ SuRe ^™ . Purity was determined by analytical size exclusion chromatography on an Agilent AdvanceBio SEC column (300A 2.7 um 7.8×300 mm) and DPBS as running buffer at 0.8 ml/min.

Endotoxin content was determined with a Charles River Endosafe PTS system. Titers were determined with a ForteBio Protein A biosensor (dynamic analysis) and calculated based on human IgG1 standards.

Putative h-UMG1 antibodies (constructed as described in Example 19 ) were tested for their affinity to HPB-ALL and H9 cell lines known to be positive for the UMG1 epitope.

Methods : Four humanized heavy chain (H 1-4) and four humanized light chain L 1-4) variants were identified to identify murine complementarity determining regions (CDRs) and grafted these CDRs into human antibody frameworks to provide framework regions generated by substituting selected residues in the closest human germline sequence of Sixteen humanized antibodies were constructed by combining each of the four humanized heavy chains ( SEQ ID NOs: 8-11 ) with each of the four humanized light chains ( SEQ ID NOs: 13-16 ) . The IgG1 isotype was used for all heavy chain variants.

Additionally, eight hybrid CHL(1-4) and H(1-4)CL variants were generated. Eight hybrid variants have a mouse heavy chain and 4 with a human light chain selected between L1-4 ( SEQ NOs: 13-16 ) and a mouse light chain with a human heavy chain selected between H1-4 ( SEQ NOs: 8-11 ) Includes 4

The recombinant gene was placed in an Evitria vector plasmid and transfected into CHO K1 cells (by eviFect, Evitria). After transfection, cells were grown in serum-free medium (eviMake2, Evitria) without animal components. The supernatant was harvested by centrifugation and subsequently sterile filtered (0.2 μm filter).

6.17.21. Example 20: h-UMG1 - h-UMG1 antibody screening for binding to HPB-ALL and H9 cell lines

Selection: Each humanized antibody was screened for its affinity for its target (mean fluorescence intensity, estimated as MIF) in two different cell lines (HPB-ALL and H9) and by flow cytometry (Attune NxT, Thermo Scientific). Binding of chimeric (ch-UMG1) and hybrid mAbs was compared. Each screening was performed twice and repeated 4 times in total. All tests were performed under identical conditions: all mAbs were used at a final concentration of 1 μg/ml; Rituximab (Roche) was used as an IgG1 negative control; FITC mouse anti-human IgG (BD Biosciences) was used as a secondary mAb.

Results: All evaluated antibodies were able to bind the target with at least the same affinity of the chimeric mAb (ch-UMG1). See FIG. 16 . One humanized antibody (H3-L4) in screening achieved the highest MFI and was selected for further development under the designation UMG1. See FIG. 16 .

6.17.22. Example 21: Reduction of HPB-ALL tumors in an NSG mouse model by h-UMG1 - humanized UMG1 (h-UMG1) and afucosylated h-UMG1 (a-h-UMG1)

This example reports the tumor volume curves of an in vivo experiment comparing the humanized version of UMG1-mAb (h-UMG1) and the afucosylated version of UMG1-mAb (ah-UMG1) versus control IgG1.

Methods : In this experiment, 15 NOD-SCID-g-chain-null (NSG) mice were subcutaneously transplanted with 5 × 10 ⁶ HPB-ALL cells. Mice were then randomized to receive weekly intraperitoneal administration of 15 mg/kg of control IgG1, h-UMG1 or ah-UMG1 from day 1 to death, tumor volume >2000 mm^3, or unacceptable toxicity. Tumor volumes were assessed every other day and average tumor volumes for each treatment group at each time point were reported and summarized in FIG. 11 .

RESULTS : From day 29, both the h-UMG1 (line with square) and ah-UMG1 (line with triangle) antibody-treated cohorts showed significantly reduced disease burden compared to the IgG1 control (line with circle) cohort. See FIG. 11 . These results suggest that both antibodies have potent anti-tumor activity.

6.17.23. Example 22: CAR-UMG1 - UMG1-targeted chimeric antigen receptor-T cells (CAR-UMG1) induce T-cell activation in the presence of H9 cells

To further refine the potential of UMG1 antibodies as immunotherapeutic tools, a third-generation CAR was developed.

Method: An extracellular domain consisting of an scFv derived from the sequence of the UMG1 antibody ( SEQ ID NO: 7 for heavy chain and SEQ ID NO: 12 for light chain) was combined with a CD3ζ chain (the transduction region of TCR), and two co- A third-generation CAR was designed by coupling with an intracellular region consisting of the stimulatory domains CD28 and 4-1BB, thus mimicking physiological T-cell activation. A map of the CAR construct is shown in FIG. 20 (circularization map) and the complete sequence of the CAR construct is provided in SEQ ID NO:41 .

The construct was cloned as a CAR cassette in a lentiviral vector (Qin DY et al., Anticancer Drugs. 2016 Sep;27(8):711-22). Subsequently, viral particles were used to transduce CD3 ⁺ lymphocytes from healthy donors at a multiplicity of infection (MOI) of 5 and the transduction efficiency was assessed by flow cytometry (approximately 38%). These CAR-T cells were analyzed for their ability to release IFNγ and IL-2 and selective cytotoxicity in the presence of target cells.

Results: As shown in FIGS . 8 and 9 , CAR-UMG1 was able to release significantly higher amounts of interferon gamma (IFNγ) and interferon 2 (IL-2) only in the presence of H9 T cell lymphocyte cells. . Additionally, only CAR-UMG1 was able to induce selective killing of H9 cells (see FIG. 10 ). These results demonstrate the ability of CAR-UMG1 to recognize H9 cells and induce T-cell activation.

The chimeric antigen receptor CAR-UMG1 induces significant cytotoxicity against cells expressing the UMG1 epitope.

6.17.24. Example 23: UMG1-CD3 bispecific antibody

To test the specificity of the UMG1-CD3 bispecific antibody, and its ability to redirect T-cell cytotoxicity to UMG1 positive cells, KE37 expressing the UMG1 CD43 epitope but negative for CD3 (UMG1 ⁺ , CD3 ⁻ ) Assays were performed on cell lines and ALL-SIL cell lines negative for both the UMG1 antigen and CD3 (UMG1 ⁻ , CD3 ⁻ ).

Methods: UMG1-CD3 constructs comprising SEQ ID NO: 40 were used to generate UMG1-CD3 bispecific antibodies. Redirected T-cell cytotoxicity was analyzed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and KE37 cell lines (UMG1 ⁺ , CD3 ⁻ ) and ALL-SIL cell lines (UMG1 ⁻ , CD3 ⁻ ).

Increasing concentrations of UMG1-CD3 bispecific antibody were incubated with CFSE (Invitrogen)-labeled target cells and effector cells at a PBMC E:T cell ratio of 10:1 or 20:1. Cell lysis was assessed 72 hours post treatment by flow cytometry as loss of target-cell membrane integrity reflected by nuclear uptake of 7AAD.

Representative FACS images from experiments using 1 μg/ml UMG1-CD3 bispecific antibody and an E:T cell ratio of 20:1 are shown in FIGS. 18A and 18B .

Results: Increased killing was observed in both cell lines treated with UMG1-CD3 bispecific antibody, KE37 (see FIG. 18a ) and ALL-SIL (see FIG. 18b ) compared to untreated cells (indicated by NT). .

In addition, the KE37 cell line expressing the UMG1 antigen showed higher apoptosis, about 86% of the cell population analyzed, whereas the ALL-SIL cell line not expressing the UMG1 antigen showed lower cells, about 22% of the cell population analyzed. have a mortality %. These results demonstrate that T cell death can be directed to UMG1 ⁺ cells with UMG1-CD3 bispecific antibodies. 18A and 18B .

6.17.25. Example 24: UMG1-CD3 bispecific binding activity

This example tests the binding of UMG1-CD3 bispecific antibodies to KE37 cell lines (UMG1 ⁺ , CD3 ⁻ ), CCRF-CEM cells (UMG1 ⁻ , CD3 ⁺ ) and Jurkat cell lines (UMG1 ⁻ , CD3 ⁺ ).

Methods: Binding activity was analyzed by flow cytometry using KE37 cell lines (UMG1 ⁺ , CD3 ⁻ ), CCRF-CEM cells (UMG1 ⁻ , CD3 ⁺ ) and Jurkat cell lines (UMG1 ⁻ , CD3 ⁺ ).

T-ALL cell lines were incubated with increasing concentrations of UMG1-CD3 bispecific antibody for 20 min. After two 1x PBS pH 7.4 (Gibco, 10010-015) washes (5 min, RT, 1300 rpm), T-ALL cells were stained with AF647 anti-human IgG (PE) for 20 min. After two 1x PBS pH 7.4 (Gibco, 10010-015) washes (5 min, RT, 1300 rpm), binding activity was assessed by flow cytometry as a percentage of PE positive cells in treated cells compared to negative control.

Results: As shown in Figure 32, UMG1-CD3 bispecific binding reached the maximal level of KE37 at a concentration of 0.001 μg/ml, whereas more gradual binding was observed in CCRF-CEM cells and 90% of binding was observed. A concentration of 10 μg/ml was reached. Conversely, very low UMG1-CD3 bispecific binding was observed on Jurkat cells in all conditions tested in the experimental conditions of the present invention. These results indicate that the UMG1-CD3 bispecific antibody can bind to UMG1 ⁺ or CD3 ⁺ positive cells. These results also suggest a higher UMG1-CD3 bispecific affinity for UMG1 than CD3.

6.17.26. Example 25: UMG1-CD3 bispecific antibody mediates T-cell cytotoxicity on T-ALL cells

This example tests the efficacy of UMG1-CD3 bispecific antibodies against T-ALL cell lines and patient-derived primary T-ALL cells.

Methods: UMG1-CD3 constructs comprising SEQ ID NO: 40 were used to generate UMG1-CD3 bispecific antibodies. Redirected T-cell cytotoxicity was analyzed by flow cytometry using a panel of human PBMCs (peripheral blood mononuclear cells) and T-ALL cell lines and T-ALL primary blast cells.

Increasing concentrations of UMG1-CD3 bispecific antibody were administered to Far-Red (Invitrogen)-labeled target cells as well as effectors of different PBMC E:T cell ratios of 1:1, 5:1, 10:1 or 20:1. Incubated with cells. After 48 hours of incubation, cell mixtures were evaluated by flow cytometry as loss of target-cell membrane integrity reflected by nuclear uptake of 7AAD (BD Pharmingen).

Results: Increased killing was mainly observed in UMG1-positive cells after treatment with UMG1-CD3 bispecific antibody compared to untreated cells ( FIGS. 21A and 21B ; FIGS. 22A-22C ). In addition, CCRF-CEM expressing UMG1 antigen (see FIGS. 21A and 22A ), the KE37 cell line (see FIG. 22B ) and EGILIII T-ALL blast cells (see FIG. 21B ) of (80-95%) of the analyzed cell populations While exhibiting higher cell death, the ALL-SIL cell line that does not express the UMG1 antigen has a lower death (10%) of the analyzed cell population (see FIG. 22C ).

6.17.27. Example 26: UMG1-CD3 bispecific antibody—identification of UMG1-CD3 bispecific antibody-mediated T-cell cytotoxicity against T-ALL cells

T-ALL cell lines were co-cultured with total human PBMCs or PBMCs depleted of CD8 ⁺ T cells or CD4 ⁺ T cells by immunomagnetic beads.

Methods : Redirected T-cell cytotoxicity was analyzed by flow cytometry using total human PBMC or CD8 ⁺ T cell- or CD4 ⁺ T cell-immunomagnetically depleted (by Miltenyi magnetic beads) PBMC and CCRF-CEM cell lines. did.

UMG1-CD3 bispecific antibody (1 μg/ml) was incubated with Far-Red (Invitrogen)-labeled target cells as well as effector cells at a PBMC E:T cell ratio of 10:1. After 48 h incubation, cell mixtures were evaluated by flow cytometry as loss of target-cell membrane integrity reflected by nuclear uptake of 7AAD (BD Pharmingen).

Results: Reduced target cell cytotoxicity was observed in lymph-depleted samples compared to total PBMC, thus confirming T cell-mediated activity of UMG1-CD3 bispecific cytotoxicity against T-ALL cells (see FIG. 24 ). ).

6.17.28: Example 27: UMG1-CD3 bispecific antibody induced apoptosis in T-ALL cell line

This example demonstrates the ability of UMG1-CD3 bispecific antibodies to induce apoptosis in T-ALL cell lines.

Methods: Apoptosis was assessed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and CCRF-CEM cells.

Increasing concentrations of UMG1-CD3 bispecific antibody were incubated with Far-Red (Invitrogen)-labeled target cells as well as effector cells at a PBMC E:T cell ratio of 10:1. Apoptosis was assessed after 24 h treatment by flow cytometry as a percentage of annexin positivity in target cells.

Results : After treatment with the UMG1-CD3 bispecific antibody in a dose-dependent manner, increased apoptosis was observed (see FIG. 23 ).

6.17.29. Example 28: UMG1-CD3 Bispecific Antibody Induced Activation of PMBCs

In this example, the ability of the UMG1-CD3 bispecific antibody to induce activation of PBMCs by binding CD3-positive T-cells was tested.

Methods : Redirected T-cell cytotoxicity was assessed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) from different healthy donors.

Increasing concentrations of UMG1-CD3 bispecific antibody were incubated with CFSE (Invitrogen)-labeled PBMCs. Cell proliferation was assessed after 96 h treatment with Cell-Titer Glo (Promega) and flow cytometry. PBMC activation was assessed by flow cytometry analysis of CD69 and CD25 positivity on CD4 and CD8 cells after 24 h treatment with UMG1-CD3 bispecific antibody or negative control. Protein expression of phospho-NFκB-p65 (A-8, Santacruz) was also assessed by Western blot analysis 96 hours after treatment with UMG1-CD3 bispecific antibody in PBMC alone or CCRF-CEM alone. Cytokine release was assessed after 4 h incubation of PBMCs with Brefeldin A (Santacruz) as intracellular IFNγ and TNFα (BD Pharmingen) positivity for CD4 and CD8 cells at 24 h from UMG1-CD3 bispecific antibody treatment.

Results : UMG1-CD3 bispecific antibody treatment increased PBMC proliferation ( FIGS. 25 and 26A and 26B ). Upregulation of early and late T cell activation markers CD25 and CD69 on T cells was observed in the presence of UMG1-CD3-bispecific antibody compared to negative control (NC) ( FIGS. 27A and 27B ). Activation induced by the UMG1-CD3 bispecific antibody resulted in increased IFNγ and TNFα release and T cell proliferation ( FIGS. 28A -D ). The NFκB pathway was activated by treatment with the UMG1-CD3 bispecific antibody only in PBMCs, but not in the CCRF-CEM cell line ( FIG. 29 ). The NFκB pathway is known to be constitutively activated in the CCRF-CEM cell line.

6.17.30. Example 29: UMG1-CD3 bispecific antibody—of UMG1-CD3 bispecific antibody against multiple myeloma cell lines in vitro efficacy

The efficacy of the UMG1-CD3 bispecific antibody was evaluated in three different myeloma cell lines.

Methods : Redirected T-cell cytotoxicity was analyzed by flow cytometry using human PBMC (peripheral blood mononuclear cells) and multiple myeloma (MM) cell lines. Two UMG1 positive MM cell lines (H929, Kms26) and one UMG1 target epitope negative (delta 47) were tested.

UMG1-CD3 bispecific antibody (1 μg/ml) was incubated with Far-Red (Invitrogen)-labeled target cells as well as effector cells at a PBMC E:T cell ratio of 10:1. After 48 hours of treatment, cell mixtures were evaluated by flow cytometry as loss of target-cell membrane integrity reflected by nuclear uptake of 7AAD (BD Pharmingen).

Results : Increased killing was mainly observed in UMG1-positive cells after treatment with UMG1-CD3 bispecific antibody compared to untreated cells (NC, indicated as negative control). Additionally, H929 and Kms26 UMG1 positive cells exhibited 80-95% apoptosis ( FIGS. 30A-30D ), whereas the delta47 cell line not expressing UMG1 antigen had 10% apoptosis ( FIGS. 30E-30F ). .

6.17.31. Example 30: UMG1-CD3 bispecific antibody - of UMG1-CD3 bispecific antibody against testicular cancer (spermatoma) cell line in vitro efficacy

The efficacy of the UMG1-CD3 bispecific antibody was evaluated in testicular cancer (spermatoma) cell lines.

Methods : Redirected T-cell cytotoxicity was analyzed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and seminal tumor cell lines. In particular, the TCAM2 seminal tumor UMG1 epitope positive cell line was tested.

UMG1-CD3 bispecific antibody (1 μg/ml) was incubated with Far-Red (Invitrogen)-labeled target cells as well as effector cells at a PBMC E:T cell ratio of 10:1. After 48 hours of treatment, cell mixtures were evaluated by flow cytometry as loss of target-cell membrane integrity reflected by nuclear uptake of 7AAD (BD Pharmingen). Using similar conditions but adding a group treated with a-h-UMG1 mAb (15 mg/kg) and 24 h after treatment by flow cytometry as a loss of target-cell membrane integrity reflected by nuclear uptake of 7AAD (BD Pharmingen) A second experiment was performed by treating the cell mixture.

Results : Significant apoptosis was observed in TCAM2 cells after 48 hours treatment with UMG1-CD3 bispecific antibody compared to untreated cells ( FIGS. 31A to 31B ). The UMG1-CD3 bispecific antibody showed increased killing after 24 h treatment compared to the ah-UMG1 mAb ( FIG. 31C ).

6.18. order

▷ UMG1 heavy chain CDR1 [SEQ ID NO:1]:

▷ UMG1 heavy chain CDR2 [SEQ ID NO:2]:

▷ UMG1 heavy chain CDR3 [SEQ ID NO:3]:

▷ UMG1 light chain CDR1 [SEQ ID NO:4]:

▷ UMG1 light chain CDR2 [SEQ ID NO:5]:

▷ UMG1 light chain CDR3 [SEQ ID NO:6]:

▷ UMG1 VH (murine) (clone IGHV5-17*02) [SEQ ID NO: 7]:

○ CDRs are underlined and italicized

○ Germline mutations are in bold

■ V is A in the germline

▷ Humanized VH1 (clone IGHV3-48*01) [SEQ ID NO: 8]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ Humanized VH2 (clone IGHV3-48*01 with germline inversion) [SEQ ID NO: 9]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ Humanized VH3 (clone IGHV1-48*01 with germline inversion and conserved residue flanking CDRs) [SEQ ID NO: 10]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ Humanized VH4 (clone IGHV3-30*02 with germline inversion) [SEQ ID NO: 11]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ UMG1 VL (murine, kappa) (clone IG IGKV4-55*01 [SEQ ID NO: 12]:

○ CDRs are underlined and italicized

○ Germline mutations are in bold

■ A is V in the germline

■ L is Q in the germline

■ I is V in the germline

■ F is Y in the germline

■ V is I in the germline

■ V is M in the germline

▷ Humanized VL1 (clone IGKV3D-20*01) [SEQ ID NO: 13]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ Humanized VL2 (IGKV3D-20*01 with germline inversion) [SEQ ID NO: 14]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ Humanized VL3 (clone IGKV6D-41*01) [SEQ ID NO: 15]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ Humanized VL4 (clone IGKV6D-41*01 with partial germline inversion) [SEQ ID NO: 16]:

○ CDRs are underlined and italicized

○ Mutant versus original mouse sequence in bold

▷ CD43 clone #1 (wild-type CD43 with 400 aa) [SEQ ID NO: 17]:

▷ CD43 clone #2 (cleaved CD43) (31 to 400 aa) [SEQ ID NO: 18]:

▷ CD43 clone #3 truncated CD43 (41 to 400 aa) [SEQ ID NO: 19]:

▷ CD43 clone #4 truncated CD43 (61-400 aa) [SEQ ID NO: 20]:

▷ CD43 clone #5 truncated CD43 (91-400 aa) [SEQ ID NO: 21]:

▷ Deletion of CD43 clone #6 64 to 78 aa [SEQ ID NO: 22]:

▷ CD43 clone #7 69 aa deletion (O-glycosylation site for GalNac) [SEQ ID NO: 23]:

▷ CD43 clone #8 amino acid substitution T69N [SEQ ID NO: 24]:

▷ human nucleotide CD43 full length [SEQ ID NO: 25]:

▷ human protein CD43 full length [SEQ ID NO: 26]:

▷ UMG1 chimeric heavy chain, nucleic acid (clone NUC 7200_evi-5 UMG.1.CH-h1.HC) [SEQ ID NO: 27]:

▷ UMG1 chimeric light chain, nucleic acid (clone NUC 7201_evi-5 UMG.1.CH-hk.LC) [ SEQ ID NO: 28]

▷ Humanized heavy chain (VH3), nucleic acid (clone NUC 7683_evi-5 UMG.HUM3-h1.HC), nucleic acid [SEQ ID NO: 29]

▷ Humanized light chain (VL4), nucleic acid (clone NUC 7700_evi-5 UMG.HUM4-hk.LC) [ SEQ ID NO: 30 ]

▷ Mouse heavy chain, nucleic acid (clone NUC 29709_evi-5 UMG.VH-m1.HC) [SEQ ID NO: 31]

▷ Mouse light chain, nucleic acid (clone NUC 29710_evi-5 UMG.VL-mk.LC) [ SEQ ID NO: 32 ]

▷ Bispecific human heavy chain, nucleic acid (NUC 32827_evi-5 UMG.VH3-h1.HC-CD3.scFv) [SEQ ID NO: 33]

▷ Chimeric heavy chain, (clone PRO 7200_evi-5 UMG.1.CH-h1.HC) [SEQ ID NO: 34]

▷ Chimeric light chain (clone PRO 7201_evi-5 UMG.1.CH-hk.LC ) [SEQ ID NO: 35]

▷ Human heavy chain (VH3) (clone PRO 7683_evi-5 UMG.HUM3-h1.HC ) [SEQ ID NO: 36]

▷ Human light chain (VL4) (clone PRO 7700_evi-5 UMG.VL4-hk.LC) [SEQ ID NO:37]

▷ Mouse heavy chain (clone PRO 29709_evi-5 UMG.VH-m1.HC) [ SEQ ID NO: 38]

▷ Mouse light chain (clone PRO 29710_evi-5 UMG.VL-mk.LC) [SEQ ID NO: 39]

▷ Bispecific human heavy chain-CD3 (clone PRO 32827_evi-5 UMG.VH3-h1.HC-CD3.scFv) [SEQ ID NO: 40]

▷ Plasmid sequence for CAR-T, nucleic acid [SEQ ID NO: 41]:

▷ Recombinant human protein CD43 (20-253 aa) [SEQ ID NO: 42]:

▷ UMG1 heavy chain CDR2 - long chain [SEQ ID NO: 43]

▷ UMG1 VH (Murine) - an alternative sequence [SEQ ID NO: 44]

▷ Humanized VH1 - Alternative Sequence [SEQ ID NO: 45]

▷ Neutral peptide linker [SEQ ID NO: 46]

▷ Synthetic peptide from influenza hemagglutinin (HA) epitope [SEQ ID NO: 47]

▷ Linear epitope of human CD43 - 71 to 78 aa [SEQ ID NO: 48]

▷ Linear epitope of human CD43 - highly basic SEQ ID NO: 1 [SEQ ID NO: 49]

▷ Linear epitope of human CD43 - highly basic sequence 2 [SEQ ID NO: 50]

▷ Linear epitope of human CD43 used in the substitution scan [SEQ ID NO: 51]

▷ Linear epitope of non-human CD43 [SEQ ID NO: 52]

▷ Linear epitope of human CD43 - 73 to 78 aa [SEQ ID NO: 53]

▷ Linear peptide sequence of human CD43 - 66 to 72 aa [SEQ ID NO: 54]

▷ Linear peptide sequence of human CD43 - 79 to 80 aa [SEQ ID NO: 55]

7. Included are quoted

All publications, patents, patent applications, and other documents cited in this application are in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was incorporated by reference for all purposes. It is incorporated herein by reference.

8. Equivalent

While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be understood that various changes may be made without departing from the spirit and scope of the invention. Various modifications will become apparent to those skilled in the art upon review of this specification.

SEQUENCE LISTING <110> Universita Degli Studi Magna Graecia Catanzaro <120> Monoclonal antibody targeting a unique sialoglycoslyated cancer-associated epitope of CD43 <130> P136949WO <140> PCT/EP2020/067528 <141> 2020-06-19 <160> 85 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> murine <400> 1 Gly Phe Thr Phe Ser Ser Phe Gly Met His 1 5 10 <210> 2 <211> 16 <212> PRT <213> murine <400> 2 Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val Lys 1 5 10 15 <210> 3 <211> 13 <212> PRT <213> murine <400> 3 Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr 1 5 10 <210> 4 <211> 12 <212> PRT <213> murine <400> 4 Ser Ala Ser Ser Ser Val Ser Ser Met Tyr Trp Tyr 1 5 10 <210> 5 <211> 7 <212> PRT <213> murine <400> 5 Asp Thr Ser Lys Met Ala Ser 1 5 <210> 6 <211> 10 <212> PRT <213> murine <400> 6 Gln Gln Trp Ser Ser Tyr Pro Pro Ile Thr 1 5 10 <210> 7 <211> 122 <212> PRT <213> artificial sequence < 220> <223> artificial sequence <400> 7 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 8 <211> 122 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 8 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 Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser T yr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 122 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 9 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 Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr T rp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 122 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 10 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 Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 122 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 11 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 12 Gln Ile Ala Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Leu Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Val Pro Ile Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Phe Ser Leu Thr Va l Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Ala Gly Ser Lys Leu Glu Leu Lys 100 105 <210> 13 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 14 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 14 Glu Ile Ala Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr L eu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Leu Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Ile Pro Ile Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Arg Val Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 15 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 15 Gln Val Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys V al Glu Ile Lys 100 105 <210> 16 <211> 107 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 16 Gln Val Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Leu Lys Pro Asp Gln Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Val Pro Ile Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Phe Thr Val Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 400 <212> PRT <213> homo sapiens <400> 17 Met Ala Thr Leu Leu Leu Leu Leu Gly Val Leu Val Val Ser Pro Asp 1 5 10 15 Ala Leu Gly Ser Thr Thr Ala Val Gln Thr Pro Thr Ser Gly Glu Pro 20 25 30 Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser 35 40 45 Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Ala 50 55 60 Leu Pro Pro Ser Thr Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser 65 70 75 80 Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr Gln 85 90 95 Glu Val Ser Ile Lys Met Ser Ser Val Pro Gin Glu Thr Pro His Ala 100 105 110 Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser His 115 120 125 Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser Ser 130 135 140 Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Leu Glu Thr 145 150 155 160 Ser Arg Gly Thr Ser Gly Pro Leu Thr Met Ala Thr Val Ser Leu 165 170 175 Glu Thr Ser Lys Gly Thr Ser Gly Pro Pro Val Thr Met Ala Thr Asp 180 185 190 Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Val Thr Met Thr 195 200 205 Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln Val Ser 210 215 220 Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn Ala 225 230 235 240 Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly Met Leu 245 250 255 Pro Val Ala Val Leu Val Ala Leu Leu Ala Val Ile Val Leu Val Ala 260 265 270 Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly Ala Leu 275 280 285 Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala Trp Ala 290 295 300 Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr Val Gly 305 310 315 320 Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu Gly Ser 325 330 335 Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys Ser Arg 340 345 350 Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly Pro Ser 355 360 365 Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly Ala Val 370 375 380 Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp Gly Ala Ala Pro 385 390 395 400 <210> 18 <211> 370 <212> PRT < 213> homo sapiens <400> 18 Glu Pro Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr 1 5 10 15 Thr Ser Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr 20 25 30 Ser Ala Leu Pro Pro Ser Thr Ser Ile Asn Glu Gly Ser Pro Leu Trp 35 40 45 Thr Ser Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr 50 55 60 Tyr Gln Glu Val Ser Ile Lys Met Ser Ser Val Pro Gln Glu Thr Pro 65 70 75 80 His Ala Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly 85 90 95 Ser His Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr 100 105 110 Ser Ser Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Ser Ser Leu 115 120 125 Glu Thr Ser Arg Gly Thr Ser Gly Pro Leu Thr Met Ala Thr Val 130 135 140 Ser Leu Glu Thr Ser Lys Gly Thr Ser Gly Pro Val Thr Met Ala 145 150 155 160 Thr Asp Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Pro Val Thr 165 170 175 Met Thr Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln 180 185 190 Val Ser Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr 195 200 205 Asn Ala Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly 210 215 220 Met Leu Pro Val Ala Val Leu Val Ala Leu Leu Ala Val Ile Val Leu 225 230 235 240 Val Ala Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly 245 250 255 Ala Leu Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala 260 265 270 Trp Ala Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr 275 280 285 Val Gly Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu 290 295 300 Gly Ser Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys 305 310 315 320 Ser Arg Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly 325 330 335 Pro Ser Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly 340 345 350 Ala Val Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp Gly Ala 355 360 365 Ala Pro 370 <210> 19 <211> 360 <212> PRT <213> homo sapiens <400> 1 9 Ser Ser Lys Met Tyr Thr Thr Ser Ile Thr Ser Asp Pro Lys Ala Asp 1 5 10 15 Ser Thr Gly Asp Gln Thr Ser Ala Leu Pro Pro Ser Thr Ser Ile Asn 20 25 30 Glu Gly Ser Pro Leu Trp Thr Ser Ile Gly Ala Ser Thr Gly Ser Pro 35 40 45 Leu Pro Glu Pro Thr Thr Tyr Gln Glu Val Ser Ile Lys Met Ser Ser 50 55 60 Val Pro Gin Glu Thr Pro His Ala Thr Ser His Pro Ala Val Pro Ile 65 70 75 80 Thr Ala Asn Ser Leu Gly Ser His Thr Val Thr Gly Gly Thr Ile Thr 85 90 95 Thr Asn Ser Pro Glu Thr Ser Ser Arg Thr Ser Gly Ala Pro Val Thr 100 105 110 Thr Ala Ala Ser Ser Leu Glu Thr Ser Arg Gly Thr Ser Gly Pro 115 120 125 Leu Thr Met Ala Thr Val Ser Leu Glu Thr Ser Lys Gly Thr Ser Gly 130 135 140 Pro Val Thr Met Ala Thr Asp Ser Leu Glu Thr Ser Thr Gly Thr 145 150 155 160 Thr Gly Pro Pro Val Thr Met Thr Thr Gly Ser Leu Glu Pro Ser Ser 165 170 175 Gly Ala Ser Gly Pro Gln Val Ser Ser Val Lys Leu Ser Thr Met Met 180 185 190 Ser Pro Thr Thr Ser Thr Asn Ala Ser Thr Val Pro Phe Arg Asn Pro 195 200 205 Asp Glu Asn Ser Arg Gly Met Leu Pro Val Ala Val Leu Val Ala Leu 210 215 220 Leu Ala Val Ile Val Leu Val Ala Leu Leu Leu Leu Trp Arg Arg Arg 225 230 235 240 Gln Lys Arg Arg Thr Gly Ala Leu Val Leu Ser Arg Gly Gly Lys Arg 245 250 255 Asn Gly Val Val Asp Ala Trp Ala Gly Pro Ala Gln Val Pro Glu Glu 260 265 270 Gly Ala Val Thr Val Thr Val Gly Gly Ser Gly Gly Asp Lys Gly Ser 275 280 285 Gly Phe Pro Asp Gly Glu Gly Ser Ser Arg Arg Pro Thr Leu Thr Thr 290 295 300 Phe Phe Gly Arg Arg Lys Ser Arg Gln Gly Ser Leu Ala Met Glu Glu 305 310 315 320 Leu Lys Ser Gly Ser Gly Pro Ser Leu Lys Gly Glu Glu Glu Pro Leu 325 330 335 Val Ala Ser Glu Asp Gly Ala Val Asp Ala Pro Ala Pro Asp Glu Pro 340 345 350 Glu Gly Gly Asp Gly Ala Ala Pro 355 360 <210> 20 <211> 340 <212> PRT <213> homo sapiens <400> 20 Gln Thr Ser Ala Leu Pro Pro Ser Thr Ser Ile Asn Glu Gly Ser Pro 1 5 10 15 Leu Trp Thr Ser Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro 20 25 30 Thr Thr Tyr Gln Glu Val Ser Ile Lys Met Ser Ser Val Pro Gin Glu 35 40 45 Thr Pro His Ala Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser 50 55 60 Leu Gly Ser His Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro 65 70 75 80 Glu Thr Ser Ser Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser 85 90 95 Ser Leu Glu Thr Ser Arg Gly Thr Ser Gly Pro Leu Thr Met Ala 100 105 110 Thr Val Ser Leu Glu Thr Ser Lys Gly Thr Ser Gly Pro Val Thr 115 120 125 Met Ala Thr Asp Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Pro 130 135 140 Val Thr Met Thr Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly 145 150 155 160 Pro Gln Val Ser Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr 165 170 175 Ser Thr Asn Ala Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser 180 185 190 Arg Gly Met Leu Pro Val Ala Val Leu Val Ala Leu Leu Ala Val Ile 195 200 205 Val Leu Val Ala Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg 210 215 220 Thr Gly Ala Leu Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val 225 230 235 240 Asp Ala Trp Ala Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr 245 250 255 Val Thr Val Gly Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp 260 265 270 Gly Glu Gly Ser Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg 275 280 285 Arg Lys Ser Arg Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly 290 295 300 Ser Gly Pro Ser Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu 305 310 315 320 Asp Gly Ala Val Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp 325 330 335 Gly Ala Ala Pro 340 <210> 21 <211> 310 <212> PRT <213> homo sapiens <400> 21 Glu Pro Thr Thr Tyr Gln Glu Val Ser Ile Lys Met Ser Ser Val Pro 1 5 10 15 Gln Glu Thr Pro His Ala Thr Ser His Pro Ala Val Pro Ile Thr Ala 20 25 30 Asn Ser Leu Gly Ser His Thr Val Thr Gly Gly Thr Ile Thr Thr Asn 35 40 45 Ser Pro Glu Thr Ser Ser Arg Thr Ser Gly Ala Pro Val Thr Thr Ala 50 55 60 Ala Ser Ser Leu Glu Thr Ser Arg Gly Thr Ser Gly Pro Pro Leu Thr 65 70 75 80 Met Ala Thr Val Ser Leu Glu Thr Ser Lys Gly Thr Ser Gly Pro 85 90 95 Val Thr Met Ala Thr Asp Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly 100 105 110 Pro Pro Val Thr Met Thr Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala 115 120 125 Ser Gly Pro Gln Val Ser Ser Val Lys Leu Ser Thr Met Met Ser Pro 130 135 140 Thr Thr Ser Thr Asn Ala Ser Thr Val Pro Phe Arg Asn Pro Asp Glu 145 150 155 160 Asn Ser Arg Gly Met Leu Pro Val Ala Val Leu Val Ala Leu Leu Ala 165 170 175 Val Ile Val Leu Val Ala Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys 180 185 190 Arg Arg Thr Gly Ala Leu Val Leu Ser Arg Gly Gly Lys Arg Asn Gly 195 200 205 Val Val Asp Ala Trp Ala Gly Pro Ala Gln Val Pro Glu Glu Gly Ala 210 215 220 Val Thr Val Thr Val Gly Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe 225 230 235 240 Pro Asp Gly Glu Gly Ser Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe 245 250 255 Gly Arg Arg Lys Ser Arg Gln Gly Ser Leu Ala Met Glu Glu Leu Lys 260 265 270 Ser Gly Ser Gly Pro Ser Leu Lys Gly Glu Glu Glu Pro Leu Val Ala 275 280 285 Ser Glu Asp Gly Ala Val Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly 290 295 300 Gly Asp Gly Ala Ala Pro 305 310 <210> 22 <211> 385 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 22 Met Ala Thr Leu Leu Leu Leu Leu Gly Val Leu Val Val Ser Pro Asp 1 5 10 15 Ala Leu Gly Ser Thr Thr Ala Val Gln Thr Pro Thr Ser Gly Glu Pro 20 25 30 Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser 35 40 45 Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Thr 50 55 60 Ser Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr 65 70 75 80 Gln Glu Val Ser Ile Lys Met Ser Ser Val Pro Gln Glu Thr Pro His 85 90 95 Ala Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser 100 105 110 His Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser 115 120 125 Ser Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Ser Leu Glu 130 135 140 Thr Ser Arg Gly Thr Ser Gly Pro Pro Leu Thr Met Ala Thr Val Ser 145 150 155 160 Leu Glu Thr Ser Lys Gly Thr Ser Gly Pro Val Thr Met Ala Thr 165 170 175 Asp Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Pro Val Thr Met 180 185 190 Thr Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln Val 195 200 205 Ser Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn 210 215 220 Ala Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly Met 225 230 235 240 Leu Pro Val Ala Val Leu Val Ala Leu Leu Ala Val Ile Val Leu Val 245 250 255 Ala Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly Ala 260 265 270 Leu Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala Trp 275 280 285 Ala Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr Val 290 295 300 Gly Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu Gly 305 310 315 320 Ser Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys Ser 325 330 335 Arg Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly Pro 340 345 350 Ser Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly Ala 355 360 365 Val Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Gly Asp Gly Ala Ala 370 375 380 Pro 385 <210> 23 <211> 399 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 23 Met Ala Thr Leu Leu Leu Leu Leu Gly Val Leu Val Val Ser Pro Asp 1 5 10 15 Ala Leu Gly Ser Thr Thr Ala Val Gln Thr Pro Thr Ser Gly Glu Pro 20 25 30 Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser 35 40 45 Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Ala 50 55 60 Leu Pro Pro Ser Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser Ile 65 70 75 80 Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr Gln Glu 85 90 95 Val Ser Ile Lys Met Ser Ser Val Pro Gin Glu Thr Pro His Ala Thr 100 105 110 Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser His Thr 115 120 125 Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser Ser Arg 130 135 140 Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Ser Leu Glu Thr Ser 145 150 155 160 Arg Gly Thr Ser Gly Pro Leu Thr Met Ala Thr Val Ser Leu Glu 165 170 175 Thr Ser Lys Gly Thr Ser Gly Pro Val Thr Met Ala Thr Asp Ser 180 185 190 Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Val Thr Met Thr Thr 195 200 205 Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln Val Ser Ser 210 215 220 Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn Ala Ser 225 230 235 240 Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly Met Leu Pro 245 250 255 Val Ala Val Leu Val Ala Leu Leu Ala Val Ile Val Leu Val Ala Leu 260 265 270 Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly Ala Leu Val 275 280 285 Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala Trp Ala Gly 290 295 300 Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr Val Gly Gly 305 310 315 320 Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu Gly Ser Ser 325 330 335 Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys Ser Arg Gln 340 345 350 Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly Pro Ser Leu 355 360 365 Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly Ala Val Asp 370 375 380 Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp Gly Ala Ala Pro 385 390 395 <210> 24 <211> 400 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 24 Met Ala Thr Leu Leu Leu Leu Leu Gly Val Leu Val Val Ser Pro Asp 1 5 10 15 Ala Leu Gly Ser Thr Thr Ala Val Gln Thr Pro Thr Ser Gly Glu Pro 20 25 30 Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser 35 40 45 Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Ala 50 55 60 Leu Pro Pro Ser Asn Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser 65 70 75 80 Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr Gln 85 90 95 Glu Val Ser Ile Lys Met Ser Ser Val Pro Gin Glu Thr Pro His Ala 100 105 110 Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser His 115 120 125 Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser Ser 130 135 140 Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Leu Glu Thr 145 150 155 160 Ser Arg Gly Thr Ser Gly Pro Leu Thr Met Ala Thr Val Ser Leu 165 170 175 Glu Thr Ser Lys Gly Thr Ser Gly Pro Pro Val Thr Met Ala Thr Asp 180 185 190 Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Val Thr Met Thr 195 200 205 Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln Val Ser 210 215 220 Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn Ala 225 230 235 240 Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly Met Leu 245 250 255 Pro Val Ala Val Leu Val Ala Leu Leu Ala Val Ile Val Leu Val Ala 260 265 270 Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly Ala Leu 275 280 285 Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala Trp Ala 290 295 300 Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr Val Gly 305 310 315 320 Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu Gly Ser 325 330 335 Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys Ser Arg 340 345 350 Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly Pro Ser 355 360 365 Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly Ala Val 370 375 380 Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp Gly Ala Ala Pro 385 390 395 400 <210> 25 <211> 1203 <212> DNA < 213> homo sapiens <400> 25 atggccacgc ttctccttct ccttggggtg ctggtggtaa gcccagacgc tctggggagc 60 acaacagcag tgcagacacc cacctccgga gagcctttgg tctctactag cgagcccctg 120 agctcaaaga tgtacaccac ttcaataaca agtgacccta aggccgacag cactggggac 180 cagacctcag ccctacctcc ctcaacttcc atcaatgagg gatcccctct ttggacttcc 240 attggtgcca gcactggttc ccctttacct gagccaacaa cctaccagga agtttccatc 300 aagatgtcat cagtgcccca ggaaacccct catgcaacca gtcatcctgc tgttcccata 360 acagcaaact ctctaggatc ccacaccgtg acaggtggaa ccataacaac gaactctcca 420 gaaacctcca gtaggaccag tggagcccct gttaccacgg cagctagctc tctggagacc 480 tccagaggca cctctggacc ccctcttacc atggcaactg tctctctgga gacttccaaa 540 ggcacctctg gaccccctgt taccatggca actgactctc tggagacctc cactgggacc 600 actggacccc ctgttaccat gacaactggc tctctggagc cctccagcgg ggccagtgga 660 ccccaggtct ctagcgtaaa actatctaca atgatgtctc caacgacctc caccaacgca 720 agcactgtgc ccttccggaa cccagatgag aactcacgag gcatgctgcc agtggctgtg 780 cttgtggccc tgctggcggt catagtcctc gtggctctgc tcctgctgtg gcgccggcgg 840 cagaagcggc ggactggggc cctcgtgctg agcagaggcg gcaagcgtaa cggggtggtg 900 gacgcctggg ctgggccagc ccaggtccct gaggaggggg ccgtgacagt gaccgtggga 960 gggtccgggg gcgacaaggg ctctgggttc cccgatgggg aggggtctag ccgtcggccc 1020 acgctcacca ctttctttgg cagacggaag tctcgccagg gctccctggc gatggaggag 1080 ctgaagtctg ggtcaggccc cagcctcaaa ggggaggagg agccactggt ggccagtgag 1140 gatggggctg tggacgcccc agctcctgat gagcccgaag ggggagacgg ggctgcccct 1200 taa 1203 <210> 26 <211> 400 <212> PRT <213> homo sapiens <400> 26 Met Ala Thr Leu Leu Leu Leu Leu Gly Val Leu Val Val Ser Pro Asp 1 5 10 15 Ala Leu Gly Ser Thr Thr Ala Val Gln Thr Pro Thr Ser Gly Glu Pro 20 25 30 Leu Val Ser Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser 35 40 45 Ile Thr Ser Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Ala 50 55 60 Leu Pro Pro Ser Thr Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser 65 70 75 80 Ile Gly Ala Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr Gln 85 90 95 Glu Val Ser Ile Lys Met Ser Ser Val Pro Gin Glu Thr Pro His Ala 100 105 110 Thr Ser His Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser His 115 120 125 Thr Val Thr Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser Ser 130 135 140 Arg Thr Ser Gly Ala Pro Val Thr Thr Ala Ala Ser Leu Glu Thr 145 150 155 160 Ser Arg Gly Thr Ser Gly Pro Leu Thr Met Ala Thr Val Ser Leu 165 170 175 Glu Thr Ser Lys Gly Thr Ser Gly Pro Pro Val Thr Met Ala Thr Asp 180 185 190 Ser Leu Glu Thr Ser Thr Gly Thr Thr Gly Pro Val Thr Met Thr 195 200 205 Thr Gly Ser Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln Val Ser 210 215 220 Ser Val Lys Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn Ala 225 230 235 240 Ser Thr Val Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg Gly Met Leu 245 250 255 Pro Val Ala Val Leu Val Ala Leu Leu Ala Val Ile Val Leu Val Ala 260 265 270 Leu Leu Leu Leu Trp Arg Arg Arg Gln Lys Arg Arg Thr Gly Ala Leu 275 280 285 Val Leu Ser Arg Gly Gly Lys Arg Asn Gly Val Val Asp Ala Trp Ala 290 295 300 Gly Pro Ala Gln Val Pro Glu Glu Gly Ala Val Thr Val Thr Val Gly 305 310 315 320 Gly Ser Gly Gly Asp Lys Gly Ser Gly Phe Pro Asp Gly Glu Gly Ser 325 330 335 Ser Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys Ser Arg 340 345 350 Gln Gly Ser Leu Ala Met Glu Glu Leu Lys Ser Gly Ser Gly Pro Ser 355 360 365 Leu Lys Gly Glu Glu Glu Pro Leu Val Ala Ser Glu Asp Gly Ala Val 370 375 380 Asp Ala Pro Ala Pro Asp Glu Pro Glu Gly Gly Asp Gly Ala Ala Pro 385 390 395 400 <210> 27 <211> 1437 <212> DNA < 213> murine <400> 27 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtgacg tgcagctggt cgagagtggc ggagggctgg tgcagcccgg tggcagccga 120 aagctgtctt gcgtcgctag tggtttcacc ttttccagct tcggcatgca ctgggtgagg 180 caggcacctg agaaaggact ggaatgggtc gcctacatct ctagtggaag cgggaacttc 240 tactatgtgg acactgtcaa ggggaggttt accatttctc gggataaccc aaaaaataca 300 ctgtttctgc aaatgacttc actgagatcc gaagacaccg ccatgtacta ttgtgctaga 360 tcaac atact accacggctc caggggcgct atggactatt ggggtcaggg cacctctgtg 420 acagtctcga gcgctagcac aaagggccct agtgtgtttc ctctggctcc ctcttccaaa 480 tccacttctg gtggcactgc tgctctggga tgcctggtga aggattactt tcctgaacct 540 gtgactgtct catggaactc tggtgctctg acttctggtg tccacacttt ccctgctgtg 600 ctgcagtcta gtggactgta ctctctgtca tctgtggtca ctgtgccctc ttcatctctg 660 ggaacccaga cctacatttg taatgtgaac cacaaaccat ccaacactaa agtggacaaa 720 aaagtggaac ccaaatcctg tgacaaaacc cacacctgcc caccttgtcc tgcccctgaa 780 ctgctgggag gaccttctgt gtttctgttc ccccccaaac caaaggatac cctgatgatc 840 tctagaaccc ctgaggtgac atgtgtggtg gtggatgtgt ctcatgagga ccctgaggtc 900 aaattcaact ggtacgtgga 960 gtggagtggaa gaacagtaca attcaaccta cagagtggtc agtgtgctga ctgtgctgca tcaggattgg 1020 ctgaatggca aggaatacaa gtgtaaagtc tcaaacaagg ccctgcctgc tccaattgag 1080 aaaacaatct caaaggccaa gggacagcct agggaacccc aggtctacac cctgccacct 1140 tcaagagagg aaatgaccaa aaaccaggtg tccctgacat gcctggtcaa aggcttctac 1200 ccttctgaca ttgctgtgga gtgggagtca aatggacagc ctgagaacaa ctacaaaaca 1260 accccccctg tgctggattc tgatggctct ttctttctgt actccaaact gactgtggac 1320 aagtctagat ggcagcaggg gaatgtcttt tcttgctctg tcatgcatga ggctctgcat 1380 aaccactaca ctcagaaatc cctgtctctg tctcccggga aatgatagta aaagctt 1437 <210> 28 <211> 723 <212> DNA <213> murine <400> 28 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcaga tcgccctgac ccagagtcct gcaattatgt cagcctcccc gggcgagaag 120 gtgaccatga catgctccgc ttccagctct gtcagttcaa tgtactggta tcagctgaag 180 cccggctcct cccccaggct gctgatctac gacacaagca aaatggcatc tggcgtgccc 240 attcggttca gcggctctgg aagtgggact tcattttccc tgaccgtgtc cagagtcgag 300 gctgaagatg ccgctacata ctattgtcag ca gtggtcta gttatccccc tatcactttc 360 ggtgcaggca gcaagctcga gctgaaacgt acggtcgcgg cgccttctgt gttcattttc 420 cccccatctg atgaacagct gaaatctggc actgcttctg tggtctgtct gctgaacaac 480 ttctacccta gagaggccaa agtccagtgg aaagtggaca atgctctgca gagtgggaat 540 tcccaggaat ctgtcactga gcaggactct aaggatagca catactccct gtcctctact 600 ctgacactga gcaaggctga ttacgagaaa cacaaagtgt acgcctgtga agtcacacat 660 caggggctgt ctagtcctgt gaccaaatcc ttcaataggg gagagtgctg atagtaaaag 720 ctt 723 <210 > 29 <211> 1437 <212> DNA <213> artificial sequence <220> <223> artificial sequence <400> 29 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtgagg tgcagctggt ggaatctggc ggagggctgg tgcagcccgg tggcagcctg 120 agactgtctt gcgtcgccag tggattcacc ttttccagct tcgggatgca ctgggtcagg 180 caggcacctg gaaaggggct ggagtgggtg gcctacatct ctagtggttc cggcaacttc 240 tactatgtgg acactgtcaa gggcaggttt accattagcc gggataacgc taaaaattct 300 ctgtatctgc aaatgaatag tctgagagcc gaagacacag ccgtgtacta ttgtgctaga 360 tcaacttact atcatggttc ccgcggcgca atggattact ggggacaggg gaccctggtg 420 acagtctcga gcgctagcac aaagggccct agtgtgtttc ctctggctcc ctcttccaaa 480 tccacttctg gtggcactgc tgctctggga tgcctggtga aggattactt tcctgaacct 540 gtgactgtct catggaactc tggtgctctg acttctggtg tccacacttt ccctgctgtg 600 ctgcagtcta gtggactgta ctctctgtca tctgtggtca ctgtgccctc ttcatctctg 660 ggaacccaga cctacatttg taatgtgaac cacaaaccat ccaacactaa agtggacaaa 720 aaagtggaac ccaaatcctg tgacaaaacc cacacctgcc caccttgtcc tgcccctgaa 780 ctgctgggag gaccttctgt gtttctgttc ccccccaaac caaaggatac cctgatgatc 840 tctagaaccc ctgaggtgac atgtgtggtg gtggatgtgt ctcatgagga ccctgaggtc 900 aaattcaact ggtacgtgga tggagtggaa gtccacaatg ccaaaaccaa gcctagagag 960 gaacagtaca attcaaccta cagagtggtc agtgtgctga ctgtgctgca tcaggattgg 1020 ctgaatggca aggaatacaa gtgtaaagtc tcaaacaagg ccctgcctgc tccaattgag 1080 aaaacaatct caaaggccaa gggacagcct agggaacccc aggtctacac cctgccacct 1140 tcaagagagg aaatgaccaa aaaccaggtg tccctgacat gcctggtcaa aggcttctac 1200 ccttctgaca ttgctgtgga gtgggagtca aatggacagc ctgagaacaa ctacaaaaca 1260 accccccctg tgctggattc tgatggctct ttctttctgt actccaaact gactgtggac 1320 aagtctagat ggcagcaggg gaatgtcttt tcttgctctg tcatgcatga ggctctgcat 1380 aaccactaca ctcagaaatc 210 cctgtctctctg 213 cc 223 artificial sequence tctcccggga aatgg 212 <> aaagct 223 artificial attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcagg tggtcatgac ccagtctcct gctttcctgt ccgtgacacc gggcgagaag 120 gtcaccatca catgctccgc atccagctct gtcagttcaa tgtactggta tcagctgaag cacccaaact gctgatctac gatacatcta aaatggccag tggcgtcccc 240 attaggttct cgggatcggg gagcggaact gacttcactt ttaccgtgtc gagcgtcgag 300 gccgaagatg ccgctaccta ctattgtcag cagtggtcta gttatccccc tatcacattt 360 ggcggaggga ctaaggtgga gattaagcgt acggtcgcgg cgccttctgt gttcattttc 420 cccccatctg atgaacagct gaaatctggc actgcttctg tggtctgtct gctgaacaac 480 ttctacccta gagaggccaa agtccagtgg aaagtggaca atgctctgca gagtgggaat 540 tcccaggaat ctgtcactga gcaggactct aaggatagca catactccct gtcctctact 600 ctgacactga gcaaggctga ttacgagaaa cacaaagtgt acgcctgtga agtcacacat 660 caggggctgt ctagtcctgt gaccaaatcc ttcaataggg gagagtgctg atagtaaaag 720 ctt 723 <210> 31 <211> 1414 <212> DNA <213> murine <400> 31 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtgacg tgcagctggt cgagagtggc ggagggctgg tgcagcccgg tggcagccga 120 aagctgtctt gcgtcgctag tggtttcacc ttttccagct tcggcatgca ctgggtgagg 180 caggcacctg agaaaggact ggaatgggtc gcctacatct ctagtggaag cgggaacttc 240 tactatgtgg acactgtcaa ggggaggttt accatttct c gggataaccc aaaaaataca 300 ctgtttctgc aaatgacttc actgagatcc gaagacaccg ccatgtacta ttgtgctaga 360 tcaacatact accacggctc caggggcgct atggactatt ggggtcaggg cacctctgtg 420 acagtctcga gcgcaaaaac aacccctcca agcgtctacc ccctggcgcc tgggagcgcg 480 gcgcagacga actcgatggt cacgttgggg tgcctcgtca agggatattt cccggagcca 540 gtcacggtca cgtggaactc ggggagcctg tcgagcggcg tccacacgtt cccggcagtc 600 ctgcaaagcg acctgtacac gctgagctcg tcagtcacgg tcccgagctc gacgtggccg 660 tcggagacgg tcacgtgcaa cgtggcgcac ccggcgagct cgacgaaagt ggacaagaag 720 atcgtgccgc gggactgcgg gtgcaagcca tgcatatgca cggtcccgga agtgtcgagc 780 gtgttcatct tcccgccgaa gccgaaggac gtgctgacga tcacgctgac gccgaaagtc 840 acgtgcgtcg tcgtagacat ctcgaaggac gacccggaag tccagttctc gtggttcgtc 900 gacgacgtgg aagtccacac ggcgcagacg cagccgcggg aggagcagtt caactcgacg 960 ttcaggagcg tgtcggagct gccgatcatg caccaggact ggctgaacgg gaaggagttc 1020 aagtgccgcg tcaactcggc ggcgttccca gcgccaattg agaagacgat ctcgaagacg 1080 aaggggcggc cgaaagcgcc gcaagtctac acgatcccgc cgccgaagga gcaga tggcg 1140 aaggacaaag tctcgctgac gtgcatgatc acggacttct tcccggagga catcacggtc 1200 gagtggcagt ggaacgggca gcctgcagag aactacaaga acacgcagcc gatcatggac 1260 acggacggga gctacttcgt gtactcgaag ctgaacgtgc agaagtcgaa ctgggaggcg 1320 gggaacacgt tcacgtgctc agtcctgcac gaggggctgc acaaccacca cacggagaag 1380 agcctgtcgc actcgcccgg gaaatgataa gctt 1414 <210> 32 <211> 718 <212> DNA <213> murine <400> 32 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcaga tcgccctgac ccagagtcct gcaattatgt cagcctcccc gggcgagaag 120 gtgaccatga catgctccgc ttccagctct gtcagttcaa tgtactggta tcagctgaag 180 cccggctcct cccccaggct gctgatctac gacacaagca aaatggcatc tggcgtgccc 240 attcggttca gcggctctgg aagtgggact tcattttccc tgaccgtgtc cagagtcgag 300 gctgaagatg ccgctacata ctattgtcag cagtggtcta gttatccccc tatcactttc 360 ggtgcaggca gcaagctcga gctgaaacgg gctgacgcgg cgcctacagt ctccattttt 420 ccacctagta gcgaacagct gacatccggg ggggcttccg tcgtctgctt tctgaacaac 480 ttttacccca aggacatcaa cgtgaagtgg aaaattgatg gctccgag ag gcagaacgga 540 gtcctgaatt cttggaccga ccaggattct aaggacagta catattcaat gtccagcacc 600 ctgacactga ctaaagatga gtacgaacgg cacaatagct atacctgcga ggcaacccat 660 aaaacaagca caagcccaat cgtcaaatcc ttcaaccgta atgagtgttg ataagctt 718 <210> 33 <211> 2209 <212> DNA <213> homo sapiens <400> 33 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtgagg tgcagctggt ggaatctggc ggagggctgg tgcagcccgg tggcagcctg 120 agactgtctt gcgtcgccag tggattcacc ttttccagct tcgggatgca ctgggtcagg 180 caggcacctg gaaaggggct ggagtgggtg gcctacatct ctagtggttc cggcaacttc 240 tactatgtgg acactgtcaa gggcaggttt accattagcc gggataacgc taaaaattct 300 ctgtatctgc aaatgaatag tctgagagcc gaagacacag ccgtgtacta ttgtgctaga 360 tcaacttact atcatggttc ccgcggcgca atggattact ggggacaggg gaccctggtg 420 acagtctcga gcgctagcac aaagggccct agtgtgtttc ctctggctcc ctcttccaaa 480 tccacttctg gtggcactgc tgctctggga tgcctggtga aggattactt tcctgaacct 540 gtgactgtct catggaactc tggtgctctg acttctggtg tccacacttt ccctgctgtg 600 ctgcagtcta gtggac tgta ctctctgtca tctgtggtca ctgtgccctc ttcatctctg 660 ggaacccaga cctacatttg taatgtgaac cacaaaccat ccaacactaa agtggacaaa 720 aaagtggaac ccaaatcctg tgacaaaacc cacacctgcc caccttgtcc tgcccctgaa 780 ctgctgggag gaccttctgt gtttctgttc ccaccaaaac caaaagatac cctgatgatc 840 tctagaaccc ctgaggtgac atgtgtggtg gtggatgtgt ctcatgagga ccctgaggtc 900 aaattcaact ggtacgtgga tggagtggaa gtccacaatg ccaaaaccaa gcctagagag 960 gaacagtaca attcaaccta cagagtggtc agtgtgctga ctgtgctgca tcaggattgg 1020 ctgaatggca aggaatacaa gtgtaaagtc tcaaacaagg ccctgcctgc tccaattgag 1080 aaaacaatct caaaggccaa gggacagcct agggaacccc aggtctacac cctgccacct 1140 tcaagagagg aaatgaccaa aaaccaggtg tccctgacat gcctggtcaa aggcttctac 1200 ccttctgaca ttgctgtgga gtgggagtca aatggacagc ctgagaacaa ctacaaaaca 1260 accccccctg tgctggattc tgatggctct ttctttctgt actccaaact gactgtggac 1320 aagtctagat ggcagcaggg gaatgtcttt tcttgctctg tcatgcatga ggctctgcat 1380 aaccactaca ctcagaaatc cctgtctctg tctcctggca aaggcggcgg aggatccggg 1440 ggtgggggaa gcggcggagg aggtagc gac atcaaactgc agcagagtgg agccgaactg 1500 gctagacctg gtgcttctgt gaaaatgtcc tgtaaaacct ccggttacac ctttacccgg 1560 tacacaatgc attgggtgaa acagaggcct ggacaggggc tggaatggat cggatacatc 1620 aaccctagtc ggggatacac aaactacaac cagaaattca aagacaaggc caccctgaca 1680 accgacaaat cttcttctac tgcctacatg cagctgtcat ctctgacttc cgaggatagt 1740 gccgtctact actgtgctcg gtactacgat gatcattact gtctggacta ctggggccag 1800 ggaacaacac ttaccgtttc tagcgtcgag ggcggatctg gcggtagcgg tggatctgga 1860 ggctctggag gagtggatga tatccagctg acccagtctc ctgctatcat gtccgcttca 1920 cctggcgaaa aagtgaccat gacctgccgt gcttcatctt ccgtgtcata catgaattgg 1980 taccagcaga aatctggcac atctcccaaa cgatggatct acgacacctc aaaagtcgct 2040 agtggcgtgc cttaccgttt ctccggttcc ggatctggaa catcatactc cctgaccatc 2100 tcttctatgg aggctgagga tgctgccaca tactactgtc agcagtggag tagcaatcct 2160 ctgacctttg gtgctgggac aaaactggag ctgaaatgat aagctttga 2209 <210> 34 <211> 452 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 34 Asp Val Gln Leu Val Gl u Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Va l Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 35 <211> 214 <212> PRT <213> artificial sequence <220> <223> artificial sequence <40 0> 35 Gln Ile Ala Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Leu Lys Pro Gly Ser Ser Pro Arg Leu Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Val Pro Ile Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Phe Ser Leu Thr Val Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Ala Gly Ser Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 17 0 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 36 <211> 452 <212> PRT <213> homo sapiens <400> 36 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 Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser V al Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 37 <211> 214 <212> PRT <213> homo sapiens <400> 37 Gln Val Val Met Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Leu Lys Pro Asp Gln Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Val Pro Ile Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Phe Thr Val Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 38 <211> 446 <212> PRT <213> murine <400> 38 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 225 230 235 240 Ile Phe Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 39 < 211> 214 <212> PRT <213> murine <400> 39 Gln Ile Ala Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys V al Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Met 20 25 30 Tyr Trp Tyr Gln Leu Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Met Ala Ser Gly Val Pro Ile Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Phe Ser Leu Thr Val Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile 85 90 95 Thr Phe Gly Ala Gly Ser Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 40 <211> 710 <212> PRT <213> homo sapiens <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp 100 105 110 Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg 465 470 475 480 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe 485 490 495 Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 500 505 510 Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn 515 520 525 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser 530 535 540 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 545 550 555 560 Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp 565 570 575 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly 580 585 590 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu 595 600 605 Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr 610 615 620 Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln 625 630 635 640 Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys 645 650 655 Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr 660 665 670 Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr 675 680 685 Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly 690 695 700 Thr Lys Leu Glu Leu Lys 705 710 <210> 41 <211> 7842 <212> DNA <2 13> artificial sequence <220> <223> artificial sequence <400> 41 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780 atcagaagaa cttagatcat tata taatac agtagcaacc ctctattgtg tgcatcaaag 840 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag ag ttaggcag ggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920 atcgatacta gtattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 1980 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 2040 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 2100 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 2160 tgacgcaaat gggcggtagg cgtgtacggt gggaggttta tataagcaga gctcgtttag 2220 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagattct 2280 agagccgcca ccatggccct cccagtaacc gccctcctgc tcccccttgc tttgctgctg 2340 cacgccgcac ggcccgctag cgaagttcag cttgtcgaat ctgggggagg gttggttcag 2400 ccgggaggga gtctgcgcct ttcttgcgtg gcttcaggct ttaccttttc cagttttggg 2460 atgcattggg tacgacaagc acctgggaaa ggactgga gt gggtggcata tatatcaagc 2520 ggcagcggaa acttctacta cgttgacact gtaaaaggga gattcaccat ctcccgagac 2580 aacgctaaaa actcactcta tcttcaaatg aatagcctgc gagctgagga tacggcggtt 2640 tactactgcg cgcgatcaac atattaccac gggtccagag gcgcgatgga ctactggggg 2700 caagggactt tggttactgt gggtggcgga ggcagcggcg gtggtggttc cggaggcggc 2760 ggttctcaag tcgttatgac ccaaagcccc gcatttcttt ctgtgactcc aggcgagaag 2820 gtgacgataa cctgttcagc cagttccagt gtctccagta tgtattggta tcaactgaaa 2880 ccagatcagg caccgaagct tttgatatat gacacatcta aaatggcatc aggggtaccc 2940 ataaggttta gcgggtccgg ctcagggacc gattttacgt ttactgtctc atccgtcgag 3000 gcggaagatg cagcgaccta ttactgccag cagtggagta gttatccccc catcacgttt 3060 ggcggcggta cgaaagtgga gataaaggac tacaaagacg atgacgacaa gctcgagacc 3120 acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 3180 ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 3240 ttcgcctgtg atttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 3300 ctagtaacag tggcctttat tattttctgg gtgaggagta aga ggagcag gctcctgcac 3360 agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 3420 tatgccccac cacgcgactt cgcagcctat cgctccaaac ggggcagaaa gaaactcctg 3480 tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 3540 agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 3600 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 3660 ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 3720 ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 3780 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 3840 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 3900 caggccctgc cccctcgcta ataggaattc gtcgacaatc aacctctgga ttacaaaatt 3960 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 4020 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 4080 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 4140 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattg c caccacctgt 4200 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 4260 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgactcaa ttccgtggtc cttgctga ttccgtggtc cttgctga ttccgtggtg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 4440 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 4500 atctcccttt gggccgcctc cccgcctggt acctttaaga ccaatgactt acaaggcagc 4560 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca 4620 acgaaaataa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc 4680 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg 4740 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag 4800 acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca 4860 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc 4920 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 4980 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta 5040 gctatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 5100 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 5160 ggaggctttt ttggaggcct agacttttgc agagacggcc caaattcgta atcatggtca 5220 tagctg tttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 5280 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 5340 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5400 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5460 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5520 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5580 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5640 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5700 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5760 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 5820 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5880 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 5940 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6000 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 6060 acagtatttg g tatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 6120 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 6180 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6240 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6300 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6360 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6420 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6480 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6540 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6600 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6660 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6720 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6780 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6840 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6900 tccgtaagat gcttttc tgt gactggtgag tactcaacca agtcattctg agaatagtgt 6960 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 7020 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 7080 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7140 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7200 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7260 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7320 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7380 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc 7440 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 7500 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 7560 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac 7620 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat 7680 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 7740 cgccagctgg cgaaaggggg at gtgctgca aggcgattaa gttgggtaac gccagggttt 7800 tcccagtcac gacgttgtaa aacgacggcc agtgccaagc tg 7842 <210> 42 <211> 234 <212> PRT Thr <213> homo sapiu Val400> 42 Ser Thr Thr Ala Ser Gly Glu Thr Thr Ala Ser 1 5 Pro Le 10 15 Thr Ser Glu Pro Leu Ser Ser Lys Met Tyr Thr Thr Ser Ile Thr Ser 20 25 30 Asp Pro Lys Ala Asp Ser Thr Gly Asp Gln Thr Ser Ala Leu Pro Pro 35 40 45 Ser Thr Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser Ile Gly Ala 50 55 60 Ser Thr Gly Ser Pro Leu Pro Glu Pro Thr Thr Tyr Gln Glu Val Ser 65 70 75 80 Ile Lys Met Ser Ser Val Pro Gln Glu Thr Pro His Ala Thr Ser His 85 90 95 Pro Ala Val Pro Ile Thr Ala Asn Ser Leu Gly Ser His Thr Val Thr 100 105 110 Gly Gly Thr Ile Thr Thr Asn Ser Pro Glu Thr Ser Ser Arg Thr Ser 115 120 125 Gly Ala Pro Val Thr Thr Ala Ala Ser Ser Leu Glu Thr Ser Arg Gly 130 135 140 Thr Ser Gly Pro Leu Thr Met Ala Thr Val Ser Leu Glu Thr Ser 145 150 155 160 Lys Gly Thr Ser Gly Pro Val Thr Met Ala Thr Asp Ser Leu Glu 165 170 175 Thr Ser Thr Gly Thr Thr Gly Pro Val Thr Met Thr Thr Gly Ser 180 185 190 Leu Glu Pro Ser Ser Gly Ala Ser Gly Pro Gln Val Ser Ser Val Lys 195 200 205 Leu Ser Thr Met Met Ser Pro Thr Thr Ser Thr Asn Ala Ser Thr Val 210 215 220 Pro Phe Arg Asn Pro Asp Glu Asn Ser Arg 225 230 <210> 43 <211> 17 <212> PRT <213> murine <400> 43 Tyr Ile Ser Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val Lys 1 5 10 15 Gly <210> 44 <211> 121 <212> PRT <213> murine < 400> 44 Asp Val Gln Val Glu Ser Gly Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 1 5 10 15 Arg Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ala 35 40 45 Tyr Ile Ser S er Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu 65 70 75 80 Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 45 <211> 121 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 45 Glu Val Gln 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 Thr Phe Ser Ser Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <211> 7 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 46 Gly Ser Gly Ser Gly Ser Gly 1 5 <210> 47 <211> 10 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 47 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly 1 5 10 <210> 48 <211> 8 <212> PRT <213> homo sapiens <400> 48 Ile Asn Glu Gly Ser Pro Leu Trp 1 5 <210> 49 <211> 6 <212> PRT <213> homo sapiens <400> 49 Arg Arg Arg Gln Lys Arg 1 5 <210> 50 <211> 13 <212> PRT <213> homo sapiens <400> 50 Arg Arg Pro Thr Leu Thr Thr Phe Phe Gly Arg Arg Lys 1 5 10 <210> 51 <211> 15 <212 > PRT <213> homo sapiens <400> 51 Pro Pro Ser Thr Ser Ile Asn Glu Gly Ser Pro Leu Trp Thr Ser 1 5 10 15 <210> 52 <211> 15 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 52 Pro Pro Ser Thr Ser Val Asn Glu Gly Ser P ro Leu Gly Thr Ser 1 5 10 15 <210> 53 <211> 6 <212> PRT <213> homo sapiens <400> 53 Glu Gly Ser Pro Leu Trp 1 5 <210> 54 <211> 7 <212> PRT <213> homo sapiens <400> 54 Pro Pro Ser Thr Ser Ile Asn 1 5 <210> 55 <211> 2 <212> PRT <213> homo sapiens <400> 55 Thr Ser 1 <210> 56 <211> 5 <212> PRT <213> murine <400> 56 Ser Phe Gly Met His 1 5 <210> 57 <211> 17 <212> PRT <213> murine <400> 57 Tyr Ile Ser Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp Thr Val Lys 1 5 10 15 Gly <210> 58 <211> 13 <212> PRT <213> murine <400> 58 Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr 1 5 10 <210> 59 <211> 10 <212> PRT <213> murine <400> 59 Ser Ala Ser Ser Ser Val Ser Ser Met Tyr 1 5 10 <210> 60 <211> 7 <212> PRT <213> murine <400> 60 Asp Thr Ser Lys Met Ala Ser 1 5 <210> 61 <211> 10 <212> PRT <213> murine <400> 61 Gln Gln Trp Ser Ser Tyr Pro Pro Ile Thr 1 5 10 <210> 62 <211> 7 < 212> PR T <213> murine <400> 62 Gly Phe Thr Phe Ser Ser Phe 1 5 <210> 63 <211> 6 <212> PRT <213> murine <400> 63 Ser Ser Gly Ser Gly Asn 1 5 <210> 64 <211> 13 <212> PRT <213> murine <400> 64 Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr 1 5 10 <210> 65 <211> 10 <212> PRT <213> murine <400 > 65 Ser Ala Ser Ser Ser Val Ser Ser Met Tyr 1 5 10 <210> 66 <211> 7 <212> PRT <213> murine <400> 66 Asp Thr Ser Lys Met Ala Ser 1 5 <210> 67 <211 > 10 <212> PRT <213> murine <400> 67 Gln Gln Trp Ser Ser Tyr Pro Pro Ile Thr 1 5 10 <210> 68 <211> 10 <212> PRT <213> murine <400> 68 Gly Phe Thr Phe Ser Ser Phe Gly Met His 1 5 10 <210> 69 <211> 10 <212> PRT <213> murine <400> 69 Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr 1 5 10 <210> 70 <211> 13 <212> PRT <213> murine <400> 70 Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr 1 5 10 <210> 71 <211> 10 <212> PRT <213> murine <400> 71 SerAla Ser Ser Ser Val Ser Ser Met Tyr 1 5 10 <210> 72 <211> 7 <212> PRT <213> murine <400> 72 Asp Thr Ser Lys Met Ala Ser 1 5 <210> 73 <211> 10 < 212> PRT <213> murine <400> 73 Gln Gln Trp Ser Ser Tyr Pro Pro Ile Thr 1 5 10 <210> 74 <211> 6 <212> PRT <213> murine <400> 74 Ser Ser Phe Gly Met His 1 5 <210> 75 <211> 13 <212> PRT <213> murine <400> 75 Trp Val Ala Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr 1 5 10 <210> 76 <211> 14 <212> PRT <213> murine <400> 76 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp 1 5 10 <210> 77 <211> 6 <212> PRT <213> murine <400> 77 Ser Ser Met Tyr Trp Tyr 1 5 <210> 78 <211> 10 <212> PRT <213> murine <400> 78 Leu Leu Ile Tyr Asp Thr Ser Lys Met Ala 1 5 10 <210> 79 <211> 9 <212> PRT <213> murine <400> 79 Gln Gln Trp Ser Ser Tyr Pro Pro Ile 1 5 <210> 80 < 211> 8 <212> PRT <213> murine <400> 80 Gly Phe Thr Phe Ser Ser Phe Gly 1 5 <210> 81 <211> 8 <212> PRT <213> murine <400> 81 Ile Ser Ser Ser Gly Ser Gly Asn Phe 1 5 <210> 82 <211> 15 <212> PRT <213> murine <400> 82 Ala Arg Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr 1 5 10 15 <210> 83 <211 > 5 <212> PRT <213> murine <400> 83 Ser Ser Val Ser Ser 1 5 <210> 84 <211> 2 <212> PRT <213> murine <400> 84 Asp Thr 1 <210> 85 <211 > 10 <212> PRT <213> murine <400> 85Gln Gln Trp Ser Ser Tyr Pro Pro Ile Thr 1 5 10

Claims (54)

An anti-CD43 antibody or antigen-binding fragment thereof for use in a method of treating CD43 positive cancer, comprising:
administering to a patient having a CD43 positive cancer a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment;
wherein said anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 61 to 91 of wild-type CD43,
Said CD43 positive cancer is diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, Endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive nerve An anti-CD43 antibody or antigen-binding fragment thereof, selected from the group consisting of primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-CD43 antibody or antigen-binding fragment binds amino acids 71 to 78 of wild-type CD43. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-CD43 antibody or antigen-binding fragment binds amino acids 73 to 78 of wild-type CD43. 4. The method of any one of claims 1 to 3, wherein the anti-CD43 antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain,
VH domain is
VH CDR1 sequence of SEQ ID NO: 1;
VH CDR2 sequence of SEQ ID NO: 43; and
comprising the VH CDR3 sequence of SEQ ID NO: 3;
VL domains are:
VL CDR1 sequence of SEQ ID NO: 4;
VL CDR2 sequence of SEQ ID NO: 5; and
An anti-CD43 antibody or antigen-binding fragment thereof comprising the VL CDR3 sequence of SEQ ID NO: 6. 5. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 4, wherein the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 12. 6. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 4 or 5, wherein the anti-CD43 antibody is a murine antibody produced by a hybridoma cell line deposited under ICLC Accession No. ICLC PD No. 16001 (UMG1). . 6. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 4 or 5, wherein the anti-CD43 antibody is a chimeric antibody further comprising a human constant region domain. The anti-CD43 antibody or antigen-binding fragment thereof of claim 7 , wherein the human constant region domain is an IgG domain. 9. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody heavy chain sequence is SEQ ID NO: 34 and the antibody light chain sequence is SEQ ID NO: 35. 5. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-CD43 antibody or antigen-binding fragment comprises a human variable domain framework region. 11. The method of claim 10, wherein the VH domain has a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; wherein the VL domain has a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. An anti-CD43 antibody or antigen-binding fragment thereof. 12. The anti-CD43 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the anti-CD43 antibody is a monoclonal antibody. 13. The antibody or antigen-binding fragment of any one of claims 1-12, wherein the anti-CD43 antibody or antigen-binding fragment is F(ab), F(ab)'2, scFv, diabody, single domain antibody, tandab. (tandab), or a flexibody, an anti-CD43 antibody or antigen-binding fragment thereof. 14. The anti-CD43 antibody or antigen- thereof according to any one of claims 1 to 13, wherein the anti-CD43 antibody or antigen-binding fragment is capable of inducing antibody dependent cytotoxicity (ADCC) in the presence of effector cells. binding fragment. 15. The anti-CD43 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the anti-CD43 antibody or antigen-binding fragment is capable of depleting tumor-associated macrophages (TAM). 16. The anti-CD43 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug. 17. The anti-CD43 antibody of any one of claims 1 to 16, wherein the patient has diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, or mantle cell lymphoma. antigen-binding fragments. 17. The anti-CD43 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the patient has multiple myeloma. 17. The anti-CD43 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the patient has melanoma. 17. The anti-CD43 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the patient has testicular cancer. 21. The anti-CD43 antibody or antigen-binding fragment thereof according to claim 20, wherein the testicular cancer is selected from the group consisting of seminal carcinoma, embryonic carcinoma, yolk tumor, and teratoma. 17. The method according to any one of claims 1 to 16, wherein the patient has nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, male medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity immature teratoma), or leiomyosarcoma, an anti-CD43 antibody or antigen-binding fragment thereof. A bispecific antibody for use in a method of treating CD43 positive cancer, comprising:
administering to a patient having a CD43 positive cancer a therapeutically effective amount of the bispecific antibody;
The bispecific antibody has a first binding specificity for an epitope within amino acids 71 to 78 of wild-type CD43,
CD43 positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A bispecific antibody selected from the group consisting of primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. 24. The bispecific antibody of claim 23, wherein the bispecific antibody has a second binding specificity for CD3. A CAR-T cell for use in a method of treating CD43 positive cancer, comprising:
administering a therapeutically effective amount of CAR-T cells to a patient having a CD43 positive cancer;
CAR-T cells bind to an epitope within amino acids 71 to 78 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A CAR-T cell selected from the group consisting of primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. An anti-CD43 antibody or antigen-binding fragment thereof for use in a method for identifying a CD43 positive cancer, comprising:
detectably contacting a sample comprising CD43 positive cancer cells with an anti-CD43 antibody or antigen-binding fragment;
the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm An anti-CD43 antibody or antigen-binding fragment thereof, selected from the group consisting of primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. An anti-CD43 antibody or antigen-binding fragment thereof for use in a method of diagnosing and treating CD43 positive cancer, comprising:
detectably contacting the sample from the patient with an anti-CD43 antibody or antigen-binding fragment;
diagnosing the patient as a CD43 positive cancer if binding to the anti-CD43 antibody or antigen-binding fragment is detected;
and administering to the patient a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment,
the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm An anti-CD43 antibody or antigen-binding fragment thereof, selected from the group consisting of primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. A method for treating CD43 positive cancer, comprising:
administering to a patient having a CD43 positive cancer a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment;
the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 61 to 91 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A method selected from the group consisting of primitive neuroectodermal tumors, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. The method of claim 28 , wherein the anti-CD43 antibody or antigen-binding fragment binds amino acids 71 to 78 of wild-type CD43. The method of claim 28 , wherein the anti-CD43 antibody or antigen-binding fragment binds amino acids 73 to 78 of wild-type CD43. 31. The method of any one of claims 28-30, wherein the anti-CD43 antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain,
VH domains are:
VH CDR1 sequence of SEQ ID NO: 1;
VH CDR2 sequence of SEQ ID NO: 43; and
comprising the VH CDR3 sequence of SEQ ID NO: 3;
VL domains are:
VL CDR1 sequence of SEQ ID NO: 4;
VL CDR2 sequence of SEQ ID NO: 5; and
A method comprising the VL CDR3 sequence of SEQ ID NO: 6. 32. The method of claim 31, wherein the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 12. 33. The method of claim 31 or 32, wherein the anti-CD43 antibody is a murine antibody produced by a hybridoma cell line deposited under ICLC Accession No. ICLC PD No. 16001 (UMG1). 33. The method of claim 31 or 32, wherein the anti-CD43 antibody is a chimeric antibody further comprising a human constant region domain. 35. The method of claim 34, wherein the human constant region domain is an IgG domain. 36. The method of claim 35, wherein the antibody heavy chain sequence is SEQ ID NO: 34 and the antibody light chain sequence is SEQ ID NO: 35. The method of claim 31 , wherein the anti-CD43 antibody or antigen-binding fragment comprises a human variable domain framework region. 38. The method of claim 37, wherein the VH domain has a sequence selected from EQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; wherein the VL domain has a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. 39. The method of any one of claims 28-38, wherein the anti-CD43 antibody is a monoclonal antibody. 40. The method of any one of claims 28-39, wherein the anti-CD43 antibody or antigen-binding fragment is F(ab), F(ab)'2, scFv, diabody, single domain antibody, tandab. (tandab), or a flexibody. 41. The method of any one of claims 28-40, wherein the anti-CD43 antibody or antigen-binding fragment is capable of inducing antibody dependent cytotoxicity (ADCC) in the presence of effector cells. 42. The method of any one of claims 28-41, wherein the anti-CD43 antibody or antigen-binding fragment is capable of depleting tumor-associated macrophages (TAMs). 43. The method of any one of claims 28-42, wherein the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug. 44. The method of any one of claims 28-43, wherein the patient has diffuse large B cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, or mantle cell lymphoma. 44. The method of any one of claims 28-43, wherein the patient has multiple myeloma. 44. The method of any one of claims 28-43, wherein the patient has melanoma. 44. The method of any one of claims 28-43, wherein the patient has testicular cancer. 48. The method of claim 47, wherein the testicular cancer is selected from the group consisting of seminal carcinoma, embryonic carcinoma, yolk tumor, and teratoma. 44. The method according to any one of claims 28 to 43, wherein the patient has nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, male medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity immature teratoma), or leiomyosarcoma. A method for treating CD43 positive cancer, comprising:
administering to a patient having a CD43 positive cancer a therapeutically effective amount of the bispecific antibody;
The bispecific antibody has a first binding specificity for an epitope within amino acids 71 to 78 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A method selected from the group consisting of primitive neuroectodermal tumors, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. 51. The method of claim 50, wherein the bispecific antibody has a second binding specificity for CD3. A method for treating CD43 positive cancer, comprising:
administering a therapeutically effective amount of CAR-T cells to a patient having a CD43 positive cancer;
CAR-T cells bind to an epitope within amino acids 71 to 78 of wild-type CD43,
CD43 positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A method selected from the group consisting of primitive neuroectodermal tumors, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. A method for identifying CD43 positive cancer comprising:
detectably contacting a sample comprising CD43 positive cancer cells with an anti-CD43 antibody or antigen-binding fragment thereof;
the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A method selected from the group consisting of primitive neuroectodermal tumors, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma. A method for diagnosing and treating CD43 positive cancer, comprising:
detectably contacting the sample from the patient with an anti-CD43 antibody or antigen-binding fragment;
diagnosing the patient as a CD43 positive cancer if binding to the anti-CD43 antibody or antigen-binding fragment is detected;
and administering to the patient a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment;
the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71 to 78 of wild-type CD43,
CD43-positive cancers include diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic giant cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endoderm endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectoderm A method selected from the group consisting of primitive neuroectodermal tumors, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.

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