Monoclonal antibodies targeting unique cancer-associated epitopes of CD43

Abstract

The present invention relates to a monoclonal mouse antibody produced by a hybridoma cell deposited under ICLC accession number ICLC PD n° 16001. In addition, the present invention relates to an antibody comprising a heavy chain variable region comprising complementary determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementary determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the 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. In addition, the present invention relates to antibodies recognizing the same epitope.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Application No. 16 / 449,255, filed on June 21, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Biological Deposit

[0004] The hybridoma secreting mAb UMG1 was deposited under the Budapest Treaty on August 4, 2016 at the Centro Biotecnologie Avanzate (ABC), Interlab Cell Line Collection (ICLC) under the accession number ICLC PD n°16001. 3. Background technology

[0006] CD43 is a leukocyte marker that is normally restricted to hematopoietic lineage cells. CD43 is widely expressed in most cellular components of peripheral blood and bone marrow origin. The precursor form of CD43 migrates with an apparent molecular weight of 54 kD. In its mature form, CD43 is highly glycosylated with a molecular weight of 115 to 200 kD. CD4+ thymocytes and monocytes express the 115 kD form, while activated CD4+ and CD8+ T cells, B cells, neutrophils, and platelets express the 130 kD form. CD43 is involved in a variety of functions, such as cell adhesion, apoptosis, and migration (Ostberg et al., Immunology Today 19: 546-50, 1998).

[0007] A mouse anti-human CD43 monoclonal antibody, UN1, was first described 25 years ago. The UN1 monoclonal antibody was initially selected for its high reactivity to human immature thymocytes (Tassone et al., Tissue Antigens 44:73-82, 1994), and was later shown to bind not only to immature thymocytes, but also to various fetal tissues (Cecco et al., Tissue Antigens 51:528-535, 1998; Tassone et al., Int. J. Oncology 20:707-711, 2002) and various solid tumors, including breast cancer, colon cancer, gastric cancer and lung squamous cell carcinoma, but not to 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 UN1 epitopes in breast cancer cells correlates with the progression stage of the disease (Tassone et al., Anticancer Res. 22: 2333-40, 2002). The epitope recognized by UN1 is a carcinoembryonic antigen expressed in cancer tissues but not in most non-tumor adult tissues. This evidence makes UN1 monoclonal antibodies an attractive tool for tumor detection and immunotherapy (see Tuccillo et al., Mol. Cancer Ther. 13 (3), 2014).

[0008] Using immunoprecipitation and tandem mass spectrometry, it was found that the UN1 antibody recognizes an epitope on CD43 that includes a monosaccharide, GalNAc, 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).

[0009] However, despite extensive functional characterization of the UN1 antibody, its CDR sequence has never been determined. Hybridomas secreting the UN1 antibody have never been deposited in a biobank, and no UN1 hybridoma master or working cell bank has been established. Antibodies that bind to the same or similar epitopes as the UN1 antibody are needed for cancer therapy, especially for the treatment of T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma, and for cancer diagnostics. 4. Summary of the invention

[0011] Over the past 25 years, we have grown cells that were ultimately derived from the original UN1 hybridoma. A recent subclone secretes a monoclonal antibody, called UMG1, that retains some, but not all, of the binding characteristics of the original UN1 antibody and has a unique binding specificity that provides special advantages.

[0012] Briefly, UMG1 antibodies bind to a small fraction of lymphocytes in peripheral blood mononuclear cells (PBMCs) from healthy human donors (Example 1). Most of the UMG1-positive lymphocytes are CD45+CD3+CD4+CD8-CD127+CCR7+T lymphocytes (Example 2).

[0013] Like UN1, the UMG1 antibody binds to T-ALL cell lines that mainly belong to the EGIL T3 classification (Example 3). However, unlike the UN1 antibody, the UMG1 antibody does not bind to breast cancer cells (Example 3). In a first experiment, the UMG1 antibody did not show any binding to cancer cells in lung, colorectal and breast cancer tumors (Example 5), which is in contrast to previous observations for UN1 (see de Laurentiis et al., Molecular & Cellular Proteomics 10:1-12, 2011, Fig. 9 ). However, UMG1 does associate with cellular immune infiltrates in a variety of tumors, including lung, colorectal, and breast cancer tumors (Example 5). Although UMG1 does not associate with myeloid-derived cells in healthy donor PBMCs (Example 1), the UMG1 epitope is expressed in tumor-associated macrophages, and expression of the UMG1 epitope is elevated when macrophages are co-cultured and interact with cancer cells (Example 6).

[0014] UMG1 also binds to some B-cell derived malignancies, including the Waldenstrom's macroglobulinemia cell line (Example 3).

[0015] UMG1 binds to a small population of neutrophils from healthy donors (Example 9). UMG1 does not bind to activated T lymphocytes from healthy donors (Example 10).

[0016] Tissue microarrays of healthy human tissues showed a distinct distribution of UMG1 mAb binding, primarily confined to the thymus (mainly on cortical thymocytes), with rare scattered immune infiltrates in organs such as lymph nodes, intestine, and lung (Example 11).

[0017] Tissue microarray data showed that in addition to lymphoma, the UMG1 epitope is also expressed in melanoma and testicular cancer of various origins (Example 11). Expanded screening was performed on multiple tissue microarrays, and the binding of UMG1 antibodies to tumor cells and immune infiltration were pediatric tumors of different origins (Example 11).

[0018] A chimeric antibody (ch-UMG1) constructed by fusing the variable region of the UMG1 mouse antibody with the human IgG Fc region was able to induce antibody-dependent cell-mediated cytotoxicity (ADCC) against the T-ALL cell line HPB ALL and the T lymphoma cell line H9 in the presence of effector cells from human PBMCs (Example 17). The ch-UMG1 antibody was also able to induce ADCC against Waldenstrom's macroglobulinemia cells (Example 18). A humanized antibody (h-UMG1) constructed by transplanting the CDRs of the UMG1 heavy and light chains into a human framework was 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 were activated in the presence of H9 T lymphoma cells (Example 22), in which the CAR targeting moiety was a scFv with all 6 CDRs of the UMG1 antibody, predicting that UMG1-guided CAR-T therapy would be effective in treating T cell lymphoma.

[0019] The UMG1-CD3 bispecific antibody is able to bind to UMG1+ or CD3+ positive cells and redirect T cell cytotoxicity to target cancer cells (Examples 23 to 30).

[0020] The specificity of the UMG1 antibody makes it particularly suitable for treating a subset of tumors that express its target epitope, including lymphomas (such as non-Hodgkin lymphomas derived from peripheral B cells or peripheral T cells, including but not limited to diffuse large B-cell lymphoma, MALT lymphoma, T lymphoma, anaplastic large cell lymphoma, follicular lymphoma and mantle cell lymphoma), testicular cancers (such as seminoma, embryonal carcinoma, yolk sac tumor and teratoma), multiple myeloma, melanoma and solid tumors that aberrantly express the antigenic epitope, such as pediatric cancers or solid tumors in which depletion of tumor-associated macrophages will show therapeutic benefit.

[0021] Thus, in a first aspect, provided herein is an anti-CD43 antibody or antigen-binding fragment thereof for use in a method of treating a CD43-positive cancer, comprising: administering a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment to a patient having a CD43-positive cancer, wherein the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 61-91 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma.

[0022] In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds to amino acids 71-78 of wild-type CD43. In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds to amino acids 73-78 of wild-type CD43.

[0023] 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: a VHCDR1 sequence of SEQ ID NO: 1; a VH CDR2 sequence of SEQ ID NO: 43; and a VH CDR3 sequence of SEQ ID NO: 3; wherein the VL domain comprises: a VL CDR1 sequence of SEQ ID NO: 4; a VL CDR2 sequence of SEQ ID NO: 5; and a VL CDR3 sequence of SEQ ID NO: 6.

[0024] 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 with ICLC accession number 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.

[0025] 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 the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the VL domain has a sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0026] 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.

[0027] 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).

[0028] In some embodiments, the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug.

[0029] 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.

[0030] In some embodiments, the patient has multiple myeloma.

[0031] In some embodiments, the patient has melanoma.

[0032] In some embodiments, the patient has testicular cancer. In some embodiments, the testicular cancer is selected from the group consisting of seminoma, embryonal carcinoma, yolk sac tumor, and teratoma.

[0033] In some embodiments, the patient has Wilms tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, or leiomyosarcoma.

[0034] In another aspect, provided herein is a bispecific antibody for use in a method of treating a CD43-positive cancer, comprising: administering a therapeutically effective amount of a bispecific antibody to a patient having a CD43-positive cancer, wherein the bispecific antibody has a first binding specificity for an epitope within amino acids 71-78 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma. In some embodiments, the bispecific antibody has a second binding specificity for CD3.

[0035] In another aspect, provided herein is a CAR-T cell for use in a method of treating a CD43-positive cancer, comprising: administering a therapeutically effective amount of CAR-T cells to a patient having a CD43-positive cancer, wherein the CAR-T cells bind to an epitope within amino acids 71-78 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma.

[0036] In another aspect, provided herein is an anti-CD43 antibody or antigen-binding fragment thereof for use in a method of identifying a CD43-positive cancer, the method comprising: detectably contacting a sample comprising CD43-positive cancer cells with an anti-CD43 antibody or antigen-binding fragment, wherein the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71-78 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma.

[0037] In another aspect, provided herein is an anti-CD43 antibody or antigen-binding fragment thereof for use in a method for diagnosing and treating CD43-positive cancer, the method comprising: detectably contacting a sample from a patient with the anti-CD43 antibody or antigen-binding fragment, diagnosing the patient with CD43-positive cancer if binding to the anti-CD43 antibody or antigen-binding fragment is detected, and administering a therapeutically effective amount of the anti-CD43 antibody or antigen-binding fragment to the patient, wherein the anti-CD43 antibody or antigen-binding fragment binds to the amino acid residue of wild-type CD43. The invention relates to an epitope within the amino acid sequence of amino acids 71-78 of the first embodiment of the present invention, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma and leiomyosarcoma.

[0038] In another aspect, provided herein is a method for 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 an antigen-binding fragment thereof, wherein the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 61-91 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma.

[0039] In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds to amino acids 71-78 of wild-type CD43. In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds to amino acids 73-78 of wild-type CD43.

[0040] 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: a VH CDR1 sequence of SEQ ID NO: 1; a VHCDR2 sequence of SEQ ID NO: 43; and a VH CDR3 sequence of SEQ ID NO: 3; wherein the VL domain comprises: a VL CDR1 sequence of SEQ ID NO: 4; a VL CDR2 sequence of SEQ ID NO: 5; and a VL CDR3 sequence of SEQ ID NO: 6.

[0041] 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 with ICLC accession number 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.

[0042] 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 the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and the VL domain has a sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0043] 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 flexobody.

[0044] 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).

[0045] In some embodiments, the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug.

[0046] 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.

[0047] In some embodiments, the patient has multiple myeloma.

[0048] In some embodiments, the patient has melanoma.

[0049] In some embodiments, the patient has testicular cancer. In some embodiments, the testicular cancer is selected from the group consisting of seminoma, embryonal carcinoma, yolk sac tumor, and teratoma.

[0050] In some embodiments, the patient has Wilms tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, or leiomyosarcoma.

[0051] In another aspect, provided herein is a method for treating a CD43-positive cancer, comprising: administering a therapeutically effective amount of a bispecific antibody to a patient having a CD43-positive cancer, wherein the bispecific antibody has a first binding specificity for an epitope within amino acids 71-78 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma. In some embodiments, the bispecific antibody has a second binding specificity for CD3.

[0052] In another aspect, provided herein is a method for treating a CD43-positive cancer, comprising: administering a therapeutically effective amount of CAR-T cells to a patient having a CD43-positive cancer, wherein the CAR-T cells bind to an epitope within amino acids 71-78 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma.

[0053] In another aspect, provided herein is 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, wherein the anti-CD43 antibody or antigen-binding fragment binds to an epitope within amino acids 71-78 of wild-type CD43, and wherein the CD43-positive cancer is selected from the group consisting of diffuse large B-cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma.

[0054] In another aspect, provided herein is a method for diagnosing and treating CD43-positive cancer, comprising: detectably contacting a sample from a patient with an anti-CD43 antibody or antigen-binding fragment, diagnosing the patient with CD43-positive cancer if binding to the anti-CD43 antibody or antigen-binding fragment is detected, and administering a therapeutically effective amount of the anti-CD43 antibody or antigen-binding fragment to the patient, wherein the anti-CD43 antibody or antigen-binding fragment binds to an expression within amino acids 71-78 of wild-type CD43. The invention relates to a method for treating a patient with a CD43-positive cancer. The method comprises: treating a patient with a CD43-positive cancer. The method comprises: treating a patient with a CD43-positive cancer. The method comprises: treating a patient with a CD43-positive cancer. The method comprises: treating a patient with a CD43-positive cancer. 5. Description of the drawings

[0056] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0057] The features and advantages of the present disclosure may be better understood with reference to the following detailed description which describes exemplary embodiments, which sets forth illustrative scenarios in which the principles of the present disclosure are utilized:

[0058] Figure 1A and 1B The expression of the epitope recognized by the UMG1 antibody on peripheral blood mononuclear cells from a cohort of healthy donors is shown and compared with a commercial CD43 antibody. Figure 1AThe scatter plot shows the data obtained by flow cytometry. The x-axis represents the detected forward scatter (FSC) and the v-axis represents the side scatter (SSC). Each dot corresponds to a cell. Figure 1B The histogram depicts the phycoerythrin signal intensity on the x-axis. The y-axis relates the signal intensity to the maximum signal intensity of the unstained sample (i.e., 100%). The red curve represents the unstained control, the blue curve represents the scrambled IgG1-stained cells (i.e., negative control), the orange curve represents the cells stained with the monoclonal antibody UMG1, and the green curve represents the cells stained with the commercial anti-CD43 antibody.

[0059] Figures 2A-2D Four representative scatter plots of cell populations recognized by the UMG1 antibody produced by hybridoma cells deposited according to the present invention are shown. Figure 2A and 2C Two scatter plots belonging to lymphocytes are shown. Figure 2B and 2D Lymphocytes detected by the UMG1 antibody produced by the hybridoma cells deposited according to the present invention. Figure 2A and 2B In the figure, the x-axis represents the CD4 signal intensity, and the y-axis represents the CD8 signal intensity. Figure 2C and 2D In the figure, the x-axis represents the CD45ro signal intensity, and the y-axis represents the CCR7 signal intensity.

[0060] Figure 3A-3B Two histograms are shown. Figure 3A Shown is the expression of UMG1 detected by UMG1 antibody on the BCWM.1 cell line. Figure 3B UMG1 expression on MWCL.1 cell line is shown. Unfilled curves represent unstained controls, curves filled with horizontal stripes represent cells stained with secondary mAb, curves filled with vertical stripes represent cells stained with scrambled IgG plus secondary mAb, and curves filled with diagonal stripes represent cells stained by mAb UN1.

[0061] Figure 4 Tumor-associated macrophages (TAMs) recognized by the UMG1 antibody are shown. Arrows show TAMs infiltrating colorectal cancer samples.

[0062] Figure 5A-5BFigure 5 shows THP1 derived macrophages stained with: control IgG1, in the absence of tumor cells (first row), UMG1-CH (a chimeric antibody according to aspect 2 of the invention, in which the original murine Fc region is replaced by a fully human IgG1 Fc), in the absence of tumor cells (second row), and ch-UMG1, in the presence of the PANC1 pancreatic cancer cell line (third row, specifically shown in Figure 5B ). The first column represents DAPI staining, the second column represents antibodies plus Alexa Fluor488-labeled secondary antibodies, and the third column represents overlay images.

[0063] Figure 6A-6B is a bar graph showing the results of a degranulation assay to assess HPB-ALL ( Fig. 6A ) and H9 cell lines ( Figure 6B ) in antibody-dependent cell-mediated cytotoxicity (ADCC). The numbers on the x-axis represent different test samples, indicating: no target (1), effector plus target cells (E+T) (2), negative control (NC) 200μg / ml (3), ch-UMG1 10μg / ml (4), ch-UMG1 50μg / ml (5), ch-UMG1 100μg / ml (6), ch-UMG1 200μg / ml (7), positive control (PC) 200μg / ml (8). The y-axis represents the percentage of CD107a+NK cells affected by ADCC out of the total number of CD107a+NK cells tested for each sample.

[0064] Figure 7 is a bar graph showing the results of a degranulation assay to evaluate antibody-dependent cell-mediated cytotoxicity (ADCC) in the BCWM.1 cell line. The numbers on the x-axis represent different samples. The numbers on the x-axis represent different tested samples, indicating: no target (1), effector plus target cells (E+T) (2), negative control (NC) 200μg / ml (3), ch-UMG1 10μg / ml (4), ch-UMG1 50μg / ml (5), ch-UMG1 100μg / ml (6), ch-UMG1 200μg / ml (7), positive control (PC) 200μg / ml (8). The y-axis represents the percentage of CD107a+ NK cells affected by ADCC out of the total number of CD107a+ NK cells tested for each sample.

[0065] Figure 8It is a bar graph showing that in the presence of H9 cells, lymphocytes expressing CD3+ (CAR-T) are able to release significantly higher amounts of interferon gamma (IFNγ). The y-axis shows the concentration of IFNγ expressed in ng / ml. On the x-axis, the numbers shown represent the different cells tested: (1) represents untransduced T cells (negative control); (2) represents T cells transduced with control CAR (vehicle control); (3) represents T cells transduced with CAR-UMG1.

[0066] Fig. 9 is a bar graph showing that in the presence of H9 cells, CAR-T is able to release significantly higher amounts of interleukin 2 (IL-2). The y-axis shows the concentration of IL2 expressed in ng / ml. On the x-axis, the numbers shown represent the different cells tested: (1) represents untransduced T cells (negative control); (2) represents T cells transduced with control CAR (vehicle control); (3) represents T cells transduced with CAR-UMG1.

[0067] Fig.10 is a bar graph showing that CAR-T cells are able to induce selective killing of H9 cells. The y-axis represents the dead / live cell ratio. The x-axis reports: H9 alone (1), H9 in the presence of non-transduced T cells (2), H9 in the presence of T cells transduced with a control CAR (3), and H9 in the presence of T cells transduced with a UMG-1 CAR, also referred to as UMG1-CAR (4).

[0068] Fig.11 is a line graph representing the tumor volume curves of in vivo experiments comparing control IgG1 (rituximab) with a humanized form of UMG1-mAb (h-UMG1) and a non-fucosylated form of UMG1-mAb (ah-UMG1). In the figure, h-UMG1 is represented by a line with squares, ah-UMG1 is represented by a line with triangles, and control IgG1 is represented by a line with circles.

[0069] Fig. 12A and 12B Representative flow cytometry results of direct staining with h-UMG1-PE and three commercially available CD43 antibodies are shown. Fig. 12A Staining in the ALL-SIL human cell line is shown. Fig. 12B Staining in the KE-37 cell line is shown.

[0070] Fig.13A and 13B Competitive binding assays are shown. Fig.13A Representative results of competitive binding assays between h-UMG1, h-UMG1-PE and three commercial CD43 antibodies on the CEM cell line are shown. Fig. 13BRepresentative results of competitive binding assays between h-UMG1, h-UMG1-PE and three commercial CD43 antibodies on the HPB-ALL cell line are shown.

[0071] Figures 14A-14C Representative images of m-UMG1 staining in inflammatory infiltrates of three different human tumors are shown. Fig.14A Shown is m-UMG1 staining in colorectal adenocarcinoma. Fig. 14B Shown is m-UMG1 staining in lung adenocarcinoma. Fig. 14C Shown is m-UMG1 staining in breast cancer.

[0072] Figures 15A-15F Representative results for Example 12 are shown. Fig.15A The amino acid sequence of full-length CD43 is shown (SEQ ID NO: 17). Fig. 15B 15 is a diagram depicting the CD43 protein variants used to transfect HEK293T cells. FIGS. 15C and 15E show Western blot results of protein lysates of transfected HEK293T cells. Fig.15D and 15F is a bar graph showing the FACS results of transfected HEK293T cells.

[0073] Fig.16 Shown is the screening of h-UMG1 antibodies for antigen affinity on HPB-ALL and H9 cell lines, which are known to be positive for the UMG1 epitope.

[0074] Figures 17A-17B Shown are comparative flow cytometric profiles of h-UMG1 and UN1 in four different cell lines of the hematopoietic lineage. Fig.17A Shown are flow cytometric profiles of UN1 reported in hematopoietic cell lines as provided by (Tassone et al., Tissue Antigens 44:73-82, 1994). Fig. 17B Flow cytometry profiles of UMG1 in cell lines of the hematopoietic lineage as provided in Example 8 are shown.

[0075] Figures 18A-18B Representative FACS images of treatment with UMG1-CD3 bispecific antibody are shown to demonstrate the expression of cytokines in the cell line ALL-SIL ( Fig.18B ) and KE-37( Fig.18A ) were used to perform T cell cytotoxicity assays as provided in Example 23.

[0076] Fig.19Evaluation 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, which is a non-glycosylated CD43 protein, is shown. See Example 15a.

[0077] Fig. 20 Depicted are plasmid maps of constructs used to make various embodiments of the CAR-T provided herein.

[0078] Figures 21A-21B The efficacy of ah-UMG1 monoclonal antibody (defucosylated h-UMG1) and UMG1-CD3 bispecific antibody on T-ALL cells was shown. Fig.21A The efficacy of ah-UMG1 monoclonal antibody and UMG1-CD3 bispecific antibody on CEM cell line was shown. Fig. 21B The efficacy of ah-UMG1 mAb and UMG1-CD3 bispecific antibody on T-ALL primary blasts is shown.

[0079] Figures 22A-22C The efficacy of different concentrations of UMG1-CD3 bispecific antibody on T-ALL cell lines is shown. Fig.22A The efficacy of the UMG1-CD3 bispecific antibody on the CEM cell line was shown. Fig. 22B The efficacy of the UMG1-CD3 bispecific antibody on the KE37 cell line was shown. Fig. 22C The efficacy of the UMG1-CD3 bispecific antibody on the ALL-SIL cell line is shown.

[0080] Fig.23 Shown are cell apoptosis induced by different doses of UMG1-CD3 bispecific compared to untreated control (NC).

[0081] Fig.24 The role of CD8+ and CD4+ T-cells in inducing responses to UMG1-CD3 bispecific therapy is shown.

[0082] Fig.25 Proliferation of PBMCs in the absence or presence of increasing concentrations of UMG1-CD3 bispecific antibody is shown.

[0083] Figures 26A-26B shows the proliferation of PBMCs in the absence or presence of UMG1-CD3 bispecific antibody, Fig.26A shows the proliferation of PBMCs in the absence of the UMG1-CD3 bispecific antibody, Fig.26B Proliferation of PBMCs in the presence of UMG1-CD3 bispecific antibody is shown.

[0084] Figures 27A-27B shows the expression of T cell activation markers in the absence or presence of increasing concentrations of UMG1-CD3-bispecific antibody, Fig.27A The percentage of CD69-positive cells is shown. Fig.27B The percentages of CD25-positive cells are shown.

[0085] Figures 28A-28D Induction of IFNγ and TNFα in CD4+ and CD8+ T cells was shown. Fig.28A Induction of IFNγ in CD4+ T cells was shown. Fig.28B Induction of IFNγ in CD8+ T cells was shown. Fig.28C Induction of TNFα in CD4+ T cells was shown. Fig.28D Induction of TNFα in CD8+ T cells was shown.

[0086] Fig.29 Shown are the effects of UMG1-CD3 bispecific treatment on NFκB protein expression in PBMCs and CCRF-CEM cell lines.

[0087] Figures 30A-30F The in vitro efficacy of the UMG1-CD3 bispecific against multiple myeloma cell lines was demonstrated. Fig. 30A The inhibition of the UMG1 epitope on the Delta 47 cell line was shown. Fig. 30B The in vitro efficacy of the UMG1-CD3 bispecific against the Delta 47 cell line was demonstrated. Fig. 30C The inhibition of the UMG1 epitope on the H929 cell line was shown. Fig.30D The in vitro efficacy of the UMG1-CD3 bispecific against the H929 cell line was demonstrated. Fig.30E The inhibition of the UMG1 epitope on the KMS26 cell line was shown. Fig. 30C The in vitro efficacy of the UMG1-CD3 bispecific on the KMS26 cell line is shown.

[0088] Figures 31A-31C The in vitro efficacy of the UMG1-CD3 bispecific against the testicular cancer (seminoma) cell line TCAM2 was demonstrated. Fig.31A The expression of the UMG1 epitope on TCAM2 cell lines is shown. Fig.31B The in vitro efficacy of the UMG1-CD3 bispecific against the TCAM2 cell line is shown compared to a negative control (NC). Fig.31C The in vitro efficacy of the UMG1-CD3 bispecific on TCAM2 cell lines compared to negative control (NC) and ah-UMG1 monoclonal antibody is shown.

[0089] Fig.32The binding activity of different concentrations of the UMG1-CD3 bispecific on CEM, Jurkat and KE37 cell lines is shown.

[0090] Fig.33 Binding of h-UMG1 mAb to inactivated and activated neutrophils is shown.

[0091] Figures 34A-34D The FACS results of IgG isotype control and h-UMG1 mAb binding to activated T cells are shown. Fig.34A Binding of IgG isotype control to CD25 positive cells is shown. Fig.34B Binding of IgG isotype control to CD69 positive cells is shown. Fig.34C The binding of h-UMG1 monoclonal antibody to CD25 positive cells is shown. Fig.34D Binding of h-UMG1 mAb to CD69-positive cells is shown.

[0092] Fig.35 The results of epitope mapping of h-UMG1 mAb (humanized anti-CD43 mAb) binding to human CD43 peptide microarray are shown.

[0093] Fig.36 The epitope mapping results of h-UMG1 mAb (humanized anti-CD43 mAb) binding to PPSTSINEGSPLWTS (SEQ ID NO: 51) peptide microarray are shown.

[0094] Fig.37A -C shows the heat map, substitution matrix and amino acid map, representing the conserved amino acids bound by h-UMG1 mAb, Fig.37A A heat map is shown. Fig.37B The substitution matrix is ​​shown, Fig.37C Amino acid diagrams are shown.

[0095] Fig.38A -C shows the binding of UMG1 monoclonal antibody on human tissues, Fig.38A Figure 4 shows the binding of UMG1 on thymocytes, characterized by the increased presence of positive cells in the cortical thymus (inlets), Fig.38B UMG1 mAb membrane and cytoplasmic binding on human tonsils is shown. Fig.38C Shown is cytoplasmic binding observed on macrophages within lung tissue.

[0096] Fig.39A -B shows the binding of UMG1 monoclonal antibody to diffuse large B-cell lymphoma and T-cell lymphoma, Fig.39A showed binding of UMG1 to diffuse large B-cell lymphoma, Fig.39BBinding of UMG1 to T-cell lymphoma is shown. Representative images from LY2084 tissue microarrays.

[0097] Fig.40A -B shows the binding of UMG1 mAb to melanoma and seminoma tissues, Fig.40A shows the binding of UMG1 to melanoma tissue (ME2081), Fig.40B Binding of UMG1 to seminoma tissue (TE2081) is shown.

[0098] 6. Detailed description

[0099] 6.1. Definitions

[0100] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art.

[0101] The monoclonal antibody "UMG1" is a mouse anti-human antibody produced by a hybridoma cell line deposited under ICLC accession number ICLC PD No. 16001.

[0102] As used herein, unless otherwise limited, the term "antibody" has its broadest art-recognized meaning and includes all known forms, including, but not limited to, bivalent monospecific monoclonal antibodies, bivalent bispecific antibodies, trivalent trispecific antibodies, F(ab) fragments, F(ab)'2 fragments, scFv fragments, diabodies, single domain antibodies including camelid VHH single domain antibodies, Tandabs and flexobodies.

[0103] As used herein, the terms "treat" or "therapy" are used in their most broadly accepted clinical sense. These terms include, but are not limited to, alleviation of signs or symptoms of a disease; amelioration of signs or symptoms of a disease; relief of symptoms; reduction in the extent of a disease; stabilization of the disease (i.e., not worsening); delay or slowing of disease progression; improvement or remission of the disease state; remission (whether partial or complete), whether detectable or undetectable; cure; prolongation of survival, as compared to expected survival if not receiving treatment. Unless expressly stated otherwise, "treat" or "therapy" does not mean prophylaxis or prevention of a disease.

[0104] Reference to a "subject" or "individual" refers to any subject, particularly a mammalian subject, in need of diagnosis, prevention or treatment. Mammalian subjects include humans, domestic animals, livestock and zoo animals, competitive animals or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc. Unless otherwise specified, "patient" refers to a human "subject".

[0105] The term "sufficient amount" refers to an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate protein aggregation in a cell.

[0106] The term "therapeutically effective amount" refers to an amount effective for treating a disease. "Preventively effective amount" refers to an amount that can effectively slow down or prevent the onset of a disease.

[0107] In the present invention, "comprises," "comprising," "containing," "having," "having" and language variations thereof have the meanings ascribed to them by U.S. patent law, allowing for the presence of additional components other than those explicitly listed.

[0108] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Unless otherwise clearly indicated, the terms "including", "for example", etc. are intended to convey inclusion without limitation.

[0109] 34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110

[0110] Unless otherwise stated or obvious from the context, as used herein, the term "about" is understood to be within the normal tolerance range in the art.

[0111] 6.2. General Overview

[0112] The present invention provides novel humanized and murine CD43 antibodies, and binding molecules derived therefrom, that have distinct expression and binding properties compared to those of other commercially available CD43 antibodies previously disclosed.

[0113] 6.3.CD43 Binding Protein

[0114] 6.3.1. Mouse monoclonal UMG1 antibody

[0115] In a first aspect, the present invention relates to monoclonal mouse antibodies produced by hybridoma cells deposited under ICLC PD n°16001.

[0116] The hybridoma cells were deposited at Centro Biotecnologie Avanzate (ABC), Interlab Cell Line Collection (ICLC), Largo Rosanna, 10, 16132 Genoa, Italy, under accession number ICLC PD n° 16001 on August 4, 2016. The antibodies tested in the examples given below. As shown in the examples, the antibodies bind to a specific epitope on CD43 in the part of the protein that can be sialic acid glycosylated.

[0117] In this first aspect, the present invention further relates to an antibody comprising a heavy chain variable region comprising complementary determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementary determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the 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. These sequences are also given in SEQ ID NOs: 1-6.

[0118] In some embodiments, the antibody comprises all three heavy chain complementarity determining regions (CDRs) and all three light chain CDRs from an antibody.

[0119] The above CDR sequences are the CDR sequences of monoclonal mouse antibodies produced by hybridoma cells deposited as ICLC PD n°16001, and were determined by sequencing.

[0120] As used herein, the term "CDR" or "complementarity determining region" refers to non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by 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), where definitions include overlapping or subsets of amino acid residues when compared to each other. The amino acid residues comprising the CDRs defined in each of the above-mentioned references are listed for comparison. Preferably, the term "CDR" is a CDR defined by Kabat based on sequence comparison. CDRH1, CDRH2 and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 represent light chain CDRs.

[0121] Such monoclonal antibodies may have framework sequences from any species. Preferably, they may have a mouse or human framework.

[0122] As used herein, the term "framework (FR) amino acid residues" refers to those 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 the variable region but not part of the CDR (e.g., using the Kabat definition of CDR).

[0123] Methods for producing monoclonal antibodies having CDR sequences as described above are known in the art and include introducing a nucleic acid sequence encoding the CDR into an appropriate expression vector encoding the desired framework sequence. Other methods are described below.

[0124] In a second aspect, the invention relates to an antibody that recognizes the same epitope as the antibody according to the first aspect.

[0125] Typically, and as is generally known in the art, antibodies are proteins belonging to the protein family of immunoglobulins, and their variable regions include framework regions and complementary determining regions as defined above. Naturally, antibodies are produced by plasma cells in response to certain antigens. In general, 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 the heavy chain may be one of five mammalian Ig heavy chains: α, δ, ε, γ and μ. The type of heavy chain usually defines the class (isotype) of the antibody: IgA, IgD, IgE, IgG and IgM antibodies. Similarly, the constant region of the light chain may be one of two types of mammalian Ig light chains: κ and λ. The variable regions of the heavy and light chains are usually composed of a unique combination of many protein sequences, allowing binding to specific antigens.

[0126] According to the present invention, the term "antibody" also includes isolated antibodies.

[0127] Typically, 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 the antibody. Additionally, two constructs of one heavy chain and one light chain can be linked by the constant regions of their heavy chains, forming a "Y" shaped molecule, with the two arms depicting the antigen binding variable regions and the stem depicting the constant region.

[0128] The antibody according to the second aspect may be a complete antibody, which means that it typically comprises a heavy chain of three or four constant domains and a light chain of one constant domain and corresponding variable domains, wherein each domain may include other modifications, such as mutations, deletions or insertions, which do not change the overall domain structure.

[0129] In addition, the antibodies according to the second aspect of the invention may form homodimers or heterodimers or homo- or hetero-multimers, wherein "dimer" and "multimer" respectively indicate that two and at least three antibodies can be combined to form a complex. The prefix "homo" indicates that the complex can be formed by the same antibody molecules, and the prefix "hetero" indicates that the complex can be formed by different antibody molecules.

[0130] Generally, the term "antibody" is intended to include all of the above-mentioned immunoglobulin isotypes, i.e., the antibody may be an IgA, IgD, IgE, IgG or IgM antibody, including any subclass of these isotypes. Preferably, the antibody is an IgG antibody. Since antibodies can be recombinantly expressed and produced, the antibody may also contain two different heavy chain constant regions, such as one IgG1 and one IgG2 heavy chain, or heavy chains from different species. However, the heavy chains are preferably from the same species. In addition, the antibody may contain a λ or κ light chain.

[0131] An antibody that recognizes the same epitope as one of the antibodies of the first aspect of the invention may also be an antibody comprising a heavy chain variable region comprising complementary determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementary determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the 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.

[0132] Furthermore, an antibody that recognizes the same epitope as one of the antibodies of the first aspect of the invention may be an antibody in which the CDR has at least one conservative amino acid exchange compared to the above sequence, for example, with an amino acid that is similar in chemical structure, properties and / or function to the original amino acid.

[0133] An antibody that recognizes the same epitope as an antibody 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 below.

[0134] Typically, the antibodies according to the second aspect of the invention may have a sequence, particularly in its variable region, which is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g. at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to a monoclonal mouse antibody produced by the hybridoma cells deposited under ICLC PD n°16001.

[0135] In some embodiments, the mouse antibody comprises a variable heavy chain having an amino acid sequence with 60-100% sequence identity to SEQ ID NO: 7, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to DVQLVESGGGLVQPGSRKLSCVASGFTFSFGMHWVRQAPEGLEWVAYISSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYGHSGRGGQGTSVTVS (SEQ ID NO: 7). In some embodiments, the mouse antibody comprises an amino acid sequence with 60% or more sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence with 70% or more sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence with 80% or more sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO:7. In some embodiments, the mouse antibody comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO:7. In some embodiments, the mouse antibody comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO:7. In some embodiments, the mouse antibody comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO:7. In some embodiments, the mouse antibody comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO:7. In some embodiments, the mouse antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:7. In some embodiments, the mouse antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO:7.

[0136] In some embodiments, the mouse antibody comprises a variable light chain having an amino acid sequence with 60-100% sequence identity to SEQ ID NO: 12, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to QIALTQSPAIMSASPGEKVTMTCSASSSVSSMYWYQLKPGSSPRLLIYDTSKMASGVPIRFSGSGSGTSFSLTVSRVEAEDAATYYCQQWSSYPPITFGAGSKLELK (SEQ ID NO: 12). In some embodiments, the mouse antibody comprises an amino acid sequence with 60% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence with 70% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence with 80% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 12.

[0137] In some embodiments, the mouse antibody comprises a variable heavy chain and a variable light chain, wherein the amino acid sequence of the variable heavy chain has 60-100% sequence identity to SEQ ID NO:7, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDYWGQGTSVTVSS (SEQ ID NO:7), and the amino acid sequence of the variable light chain has 60-100% sequence identity to SEQ ID NO:7, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDYWGQGTSVTVSS (SEQ ID NO:7). NO: 12 has 60-100% sequence identity, for example, 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to QIALTQSPAIMSASPGEKVTMTCSASSSVSSMYWYQLKPGSSPRLLIYDTSKMASGVPIRFSGSGSGTSFSLTVSRVEAEDAATYYCQQWSSYPPITFGAGSKLELK (SEQ ID NO: 12).

[0138] 6.3.2. UMG1 Monospecific, Bispecific and Multispecific Antibodies

[0139] Generally, the antibody according to the present invention may be a monoclonal antibody, a bispecific antibody or a multispecific antibody. Such antibodies are known in the art.

[0140] As used in the context of the present invention, the term "monoclonal" should be understood in the broadest sense to describe antibodies produced by a single B lymphocyte clone or antibodies having identical or similar amino acid sequences.

[0141] The term "bispecific" as used herein can be understood in the broadest sense to describe an antibody that interacts with two different epitopes. A bispecific antibody can be derived from two monoclonal antibodies. Optionally, the two different epitopes may be located on the same antigen, but may also be located on two different antigens.

[0142] The term "multispecific" as used herein can be understood in the broadest sense to describe an antibody that interacts with three or more different types of epitopes. Optionally, these epitopes can be located on the same antigen or on two or more antigens.

[0143] Preferably, the antibody according to aspect 2 of the present invention is a monoclonal antibody.

[0144] Furthermore, the antibody according to aspect 2 of the present invention is preferably a bispecific or multispecific antibody.

[0145] Methods for preparing antibodies are well known to those skilled in the art. Preferably, antibodies are produced by making hybridoma cells. Methods for producing hybridoma cells and methods for producing antibodies with the aid of hybridoma cells are well known to those skilled in the art. Typically, mice are injected with the desired antigen and killed a few days later to isolate splenocytes that secrete antibodies against the desired antigen. Generally, these antibody-secreting splenocytes are fused with immortalized non-secreting myeloma cells to form hybridoma cells. These hybridoma cells are then typically screened and hybridomas that produce the desired antibodies are selected. The selected hybridomas can then be cultured in vivo or in vitro, and the desired antibodies can be isolated.

[0146] Bifunctional or bispecific antibodies may have antigen binding sites with different specificities. Those skilled in the art are familiar with various forms of bispecific antibodies and their preparation. For example, these include BSIgG, which is an IgG molecule comprising two different heavy chains and two different light chains, which is secreted by so-called "hybrid hybridomas", and heterologous antibody conjugates produced by chemical coupling of antibodies or antibody fragments of different specificities (Segal DM et al., Current Opin. Immunol. 1999, 11: 558-562; Van Spriel AB et al., Immunology Today 2000, 21: 391-397, each of which is incorporated herein by reference in its entirety).

[0147] Preparation: Bispecific antibodies can be produced to deliver cells, cytotoxins or drugs to specific sites. An important use may be to deliver host cytotoxic cells (e.g., NK or cytotoxic T cells) to specific cell targets (PJ Lachmann, Clin. Exp. Immunol. 1990, 79: 315, which is incorporated herein by reference in its entirety). Another important use may be to deliver cytotoxic proteins to specific cell targets (V. Raso, T. Griffin, Cancer Res. 1981, 41: 2073; S. Honda et al., Cytotechnology, 1990, 4: 59, each of which is incorporated herein by reference in its entirety). Another important use may be to deliver anticancer non-protein drugs to specific cell 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 herein by reference in its entirety). Such bispecific antibodies can be prepared by chemical cross-linking (M. Brennan et al., 1985, Science 229: 81; the entire text of which is incorporated by reference), disulfide exchange or the production of hybridomas (tetraploids). Quadromas can be constructed by fusing hybridomas that secrete two different types of antibodies against two different antigens (Milstein and Cuello, Nature, 1983, 305: 537-539, the entire text of which is incorporated by reference herein).

[0148] The term "epitope" as used in the context of the present invention may be understood in the broadest sense as a portion of the CD43 molecule that can be recognized and bound by an antibody produced by a hybridoma cell deposited under ICLCPD n° 16001 at one or more of the antigen binding regions of the antibody. The portion of the antibody that binds to an epitope is called a paratope. In many cases, an epitope has conformational properties that specifically generate a paratope binding site.

[0149] Epitopes usually consist of chemically active surface groupings of molecules (such as amino acids or sugar side chains) and usually have specific three dimensional structural properties as well as specific charge properties.

[0150] In addition, it is known and appreciated by those skilled in the art that the interaction between an epitope and an antibody can generally be based on the primary structure of the antigen, i.e., a continuous sequence of amino acids. Typically, this interaction may also be based on the secondary structure, tertiary structure, or quaternary structure of the epitope and post-translational modifications, such as glycosylation. The interaction between an epitope and an antibody may also be based on the three-dimensional structure of the antigen and the resulting surface features of the antigen, which may involve discontinuous portions of the amino acid sequence, which contain distantly located amino acids that interact with the antibody.

[0151] An antibody recognizes the "same epitope" as the antibody of the first aspect when the two antibodies recognize the same or spatially overlapping epitopes. Generally speaking, the most extensive and rapid method for determining whether two epitopes recognize the same or spatially overlapping epitopes is a competition assay, which can usually be configured in a variety of different formats using labeled antigens or labeled antibodies. For example, the antigen is immobilized on a 96-well plate, and then the ability of the unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label.

[0152] An antibody that recognizes the "same epitope" as the antibody according to the first aspect typically refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody typically blocks binding of the antibody to its antigen by 50% or more in a competition assay.

[0153] Generally, the epitope recognized and bound by the antibody produced by the hybridoma cell deposited with ICLC PD n°16001 can be identified by any suitable epitope mapping method known in the art in combination with the antibody produced by the hybridoma cell deposited with ICLC PD n°16001.

[0154] An example of such a method includes screening peptides of varying lengths derived from CD43 for binding to antibodies produced by hybridoma cells deposited as ICLC PD n°16001, wherein the smallest fragment that can specifically bind to the antibody generally contains the epitope sequence recognized by the antibody. In general, CD43 peptides can be produced synthetically or by proteolytic digestion of CD43. Those skilled in the art are familiar with methods for identifying peptides that bind to antibodies, such as mass spectrometry. In another example, NMR spectroscopy can be used to identify residues that interact with the antibodies of the invention. For example, it has been consistently found that 15 N and 2 H-labeled CD43 peptides can be mixed with unlabeled antibodies, and those amino acids in the labeled peptide that interact with the unlabeled antibody can be detected as their positions in the NMR spectrum change. Typically, the difference between the two spectra can identify the amino acids in CD43 that are involved in the antibody interaction. Preferably, mass spectrometry is used to identify peptides that bind to the antibody.

[0155] Exemplarily, the epitope recognized and bound by the antibody produced by the hybridoma cells deposited under ICLC PD n°16001 can also be identified by a method comprising amplifying various DNA fragments of CD43 DNA by polymerase chain reaction (PCR), integrating these fragments to form expression vectors comprising their connection to histidine fusion proteins, and after protein expression, detecting the epitope, for example by Western blotting.

[0156] In another example, to determine the site on CD43 recognized and bound by the antibody produced by the hybridoma cells deposited as ICLC PD n° 16001, an expression vector of a CD43 clone having a deletion mutation can be introduced by the PCR method to prepare a mutant series, such as an E. coli mutant series, which expresses proteins having various deletion sites in CD43. These E. coli mutants can be cultured and induced to express. Western blot analysis can be performed using cell lysates as antigens.

[0157] Other methods for identifying the epitope recognized and bound by the antibodies produced by the hybridoma cells deposited under ICLC PD n° 16001 may include detection by immunoassay, such as enzyme-linked immunosorbent assay (ELISA).

[0158] The term "affinity" used in the context of the present invention can be understood in the broadest sense as the intensity of the interaction between an epitope and an epitope binding site of an antibody. Methods for determining the absolute value of antibody affinity, i.e., the affinity constant, are well known to those skilled in the art. However, relative values ​​of antibody affinity can also be determined generally, i.e., the affinities of two antibodies are compared without determining their absolute values. Methods for comparing antibody affinities 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 then labeled with immunofluorescence secondary antibodies. Typically, after detection by flow cytometry, the intensity of the antibody signal can be compared.

[0159] Screening methods: Methods for identifying antibodies according to the second aspect that recognize the same epitope as the antibodies according to the first aspect are well known to those skilled in the art. For example, antibodies according to the second aspect can be identified by phage display based on antibody libraries.

[0160] Therefore, the antibodies of the present invention that recognize the same epitope may also be human antibodies.

[0161] 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.

[0162] Chimeric antibodies are antibodies in which at least one region of an immunoglobulin from one species is fused to another region of an immunoglobulin from another species by genetic engineering to reduce its immunogenicity (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397).

[0163] 6.3.3. Humanized UMG1 Antibody

[0164] 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 of the antibody according to the first aspect.

[0165] Generally, humanized antibodies are chimeric antibodies of a specific type. For example, humanized antibodies can be produced by transferring DNA of human antibodies to DNA encoding mouse antibody frameworks or by transferring DNA of mouse antibodies to DNA encoding human antibody frameworks. Preferably, DNA of human antibodies is transferred to DNA encoding mouse antibody frameworks. In general, the transfer of DNA includes transferring one or more DNA sequences to DNA encoding the target antibody framework. Alternatively, variable and constant regions and heavy and light chains can be partially or completely humanized. Preferably, the heavy chain variable region and light chain variable region of mouse antibodies are humanized. More preferably, the heavy chain variable region and light chain variable region of mouse antibodies are humanized by changing the DNA sequence encoding 1 to 50, preferably 1 to 30, more preferably 1 to 20 amino acids. The transferred DNA may generally contain DNA regions of six hypervariable loops that determine antigen specificity, also referred to as complementarity determination regions (CDRs), or DNA regions that do not contain CDRs, or both. Preferably, humanization includes transferring DNA that does not contain CDRs.

[0166] Typically, the resulting DNA construct can then be used to express and produce antibodies that are generally less or non-immunogenic than the non-human parent antibody. This includes the production of modified antibodies, such as aglycosylated antibodies or defucosylated antibodies. These methods are well known in the art. Therefore, the antibodies of the invention that recognize the same epitope can also be non-glycosylated antibodies or non-fucosylated antibodies.

[0167] 6.3.4. Engineered humanized antibodies

[0168] The present invention also provides engineered humanized antibodies that recognize CD43. The h-UMG1 antibody may comprise one or more variable heavy chain regions or light chain regions provided in SEQ ID NOs: 8-11 and SEQ ID NOs: 13-16, respectively. One skilled in the art can generate various embodiments by making one or more conservative substitutions to the amino acid residues provided by the present invention. "Conservative substitution" or "conservative amino acid substitution" refers to the replacement of an amino acid with a chemically or functionally similar amino acid.

[0169] In some embodiments, the antibody is IgG1, IgG2, IgG4 or IgM. In some embodiments, the antigen binding protein is a Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a scFv fragment, a scFv Fc fragment and / or a single domain antibody.

[0170] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 8, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVSYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSS (SEQ ID NO: 8). In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8.

[0171] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 9, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVSYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSS (SEQ ID NO: 9). In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9.

[0172] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 10, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSS (SEQ ID NO: 10). In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 10.

[0173] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 11, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to QVQLVESGGGVVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSS (SEQ ID NO: 11). In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 11.

[0174] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 13, for example, 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to EIVLTQSPATLSLSPGERATLSCSASSSVSSMYWYQQKPGLAPRLLIYDTSKMASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSYPPITFGQGTRLEIK (SEQ ID NO: 13).

[0175] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 13.

[0176] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 14, for example, 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to EIALTQSPATLSLSPGERATLSCSASSSVSSMYWYQLKPGLAPRLLIYDTSKMASGIPIRFSGSGSGTDFTLTVSRVEPEDFAVYYCQQWSSYPPITFGQGTRLEIK (SEQ ID NO: 14).

[0177] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 14.

[0178] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 15, for example, 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to QVVMTQSPAFLSVTPGEKVTITCSASSSVSSMYWYQQKPDQAPKLLIYDTSKMASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSYPPITFGGGTKVEIK (SEQ ID NO: 15).

[0179] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 70% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 80% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 95% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 97% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 99% or more sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 15.

[0180] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 16, for example, 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to QVVMTQSPAFLSVTPGEKVTITCSASSSVSSMYWYQLKPDQAPKLLIYDTSKMASGVPIRFSGSGSGTDFTFTVSSVEAEDAATYYCQQWSSYPPITFGGGTKVEIK (SEQ ID NO: 16).

[0181] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:8 and SEQ ID NO:13.

[0182] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 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 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 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 100% sequence identity to SEQ ID NO:8 and SEQ ID NO:14.

[0183] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 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 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 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 that has 100% sequence identity to SEQ ID NO:8 and SEQ ID NO:15.

[0184] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence that has 100% sequence identity to SEQ ID NO:8 and SEQ ID NO:16.

[0185] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence that has 100% sequence identity to SEQ ID NO:9 and SEQ ID NO:13.

[0186] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence that has 100% sequence identity to SEQ ID NO:9 and SEQ ID NO:14.

[0187] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:9 and SEQ ID NO:15.

[0188] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence that has 100% sequence identity to SEQ ID NO:9 and SEQ ID NO:16.

[0189] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence that has 100% sequence identity to SEQ ID NO:10 and SEQ ID NO:13.

[0190] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:10 and SEQ ID NO:14.

[0191] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15. In some embodiments, the h-UMG1 antibody comprises an amino acid sequence that has 100% sequence identity to SEQ ID NO:10 and SEQ ID NO:15.

[0192] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:10 and SEQ ID NO:16.

[0193] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:11 and SEQ ID NO:13.

[0194] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:11 and SEQ ID NO:14.

[0195] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:9 and SEQ ID NO:15.

[0196] In some embodiments, the h-UMG1 antibody comprises an amino acid sequence having 60% or more 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 70% or more 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 80% or more 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 85% or more 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 90% or more 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 95% or more 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 100% 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 60% or greater 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 70% or greater 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 80% or greater 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 85% or greater 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 90% or greater 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 95% or greater 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 97% or greater 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 99% or greater 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 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 100% sequence identity to SEQ ID NO:11 and SEQ ID NO:16.

[0197] In another preferred embodiment, the monoclonal antibody of the antibody according to aspect 2 is capable of inducing antibody-dependent cellular cytotoxicity (ADCC) against the following cells: EGIL T3 subgroup of T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoblastic lymphoma cells and Waldenstrom's macroglobulinemia (WM) cells.

[0198] Lymphocytes belong to the group of white blood cells and are mediators of humoral and cell-mediated immunity. There are two groups of lymphocytes, B cells and T cells.

[0199] Just like many other cell types, B cells and T cells can develop abnormally into B cell and T cell tumors. Because there are many different stages in the development of B cells and T cells, there are various types of tumors. Both B cells and T cells originate from lymphoid precursor cells.

[0200] In the case of B cells, this lymphoid progenitor cell development proceeds through many stages of B cell development, each containing a certain definable cell type, until a plasma cell is formed. One of these stages includes the so-called "IgM-secreting B cells", which eventually develop into antibody-producing plasma cells. Tumors that originate from "IgM-secreting B cells" are called "Waldenstrom's macroglobulinemia" (WM). WM is a rare, indolent, incurable disease. It is characterized by the accumulation of clonal IgM-secreting lymphoplasmacytic cells in the bone marrow.

[0201] T cells develop from lymphoid progenitors into mature T cells only at a few developmental stages. Tumors can specifically evolve from mature T cells or lymphoid progenitors, the latter leading to B-cell or T-cell acute lymphoblastic leukemia (B-ALL) and (T-ALL), respectively. T-cell phenotype T-ALL accounts for about 20% of all acute lymphoblastic leukemia cases and occurs more often 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 based on the prevalent localization to specific sites, such as the bone marrow in T-ALL or the secondary lymphoid organs in T-LBL. The European Group for the Study of Immunological Characteristics of Leukemia (EGIL) divided T-ALL into four subgroups based on its immunophenotype (Bene MC, Leukemia 1995; 9: 1783):

[0202] 1) EGIL T1 (pro-), characterized by cytoplasmic positivity for CD3 (cCD3) and surface expression of CD7;

[0203] 2) EGIL T2 (pre-), characterized by ccCD3, CD7 positivity and CD2 or CD5 positivity;

[0204] 3) EGIL T3 (cortical), characterized by cCD3, CD1a positivity and the presence or absence of surface CD3 (sCD3), and

[0205] 4) EGIL T4 (mature leukemia), characterized by cCD3 and sCD3 positivity and CD1a negativity.

[0206] As used herein, the term "antibody-dependent cellular cytotoxicity (ADCC)" is the killing of cells bound and labeled by an antibody by cytotoxic effector cells, such as natural killer (NK) cells.

[0207] To examine whether an antibody is able to induce ADCC, the following assay can be used. Degranulation assays are performed by incubating peripheral blood mononuclear cells (PBMCs) from healthy donors (including effector cells) with target cells expressing the epitope at different antibody concentrations. 4 Target cells were seeded in 96-well round-bottom plates and cultured for 30 min at 37°C with 5% CO2 in the presence of different concentrations of antibodies (0, 10, 50, 100, and 200 μg / ml) or control IgG1. Subsequently, 0.4×10 6PBMCs (fixed effector cells (E): target cells (T) = 10: 1) were added to each well along with 20 μl / ml phycoerythrin (PE)-coupled anti-CD107a monoclonal antibody (mAb) (BD), and the cells were then incubated at 37°C for 3 hours under 5% CO2. After 1 hour, 6 μg / ml monensin was added to each well (GolgiStop, BD). At the end of the incubation period, cells were stained with anti-CD56 coupled to allophycocyanin (APC) and anti-CD3 coupled to perichlorophyllin protein complex (PerCp) and analyzed on an ATTUNE NxT flow cytometer (THERMO Scientific). NK cells (CD3- / CD56+) that induced target cell lysis (CD107a+) were measured by detecting CD3- / CD56+ / CD107a+ cells. Therefore, with the increase of antibody concentration, the increase of CD3- / CD56+ / CD107a+ cells confirms the potential of antibody to induce ADCC. The obtained data allow the design of immune targeting methods, which are urgent and unmet clinical needs of, for example, T cell acute lymphoblastic leukemia / lymphoblastic lymphoma. Other methods well known to those skilled in the art for detecting whether an antibody can induce ADCC can also be used.

[0208] 6.3.5. UMG1-binding molecules

[0209] In a third aspect, the invention provides a binding molecule derived from the UMG1 antibody according to aspect 1 or aspect 2.

[0210] According to the invention, the binding molecule is a molecule derived from the monoclonal mouse UMG1 antibody produced by the hybridoma cells deposited as ICLC PD n° 16001. Preferably, the binding molecule is an immunoglobulin-containing molecule, ie it comprises at least one immunoglobulin (Ig) domain.

[0211] In a preferred embodiment, the binding molecule of the present invention is selected from single-chain antibodies. In a more preferred embodiment, the binding molecule is selected from the group consisting of single-chain variable fragments (scFv), polymers of scFv (e.g., diabodies, triabodies or tetrabodies), antibody fragments, preferably Fab, Tandab and flexible bodies.

[0212] The structure of antibodies, especially the functions of their CDRs are generally well known in the art (Carter PJ. Potent antibody therapeutics by design. Nature Rev. Immunol. 6: 343-357, 2006, which is incorporated herein by reference in its entirety). Single-chain Fv (scFv) and multimers thereof, Tandab, diabodies and flexible bodies are generally standard antibody formats known in the art, for example, from WO1988 / 001649A1, WO1993 / 011161A1, WO1999 / 057150A2 and EP1293514B1, each of which is incorporated herein by reference in its entirety.

[0213] In scFv, the two antigen-binding variable regions of the light and heavy chains (VH Fv and VL Fv) of an antibody are usually artificially connected by a linker peptide, designated as a single-chain variable fragment or single-chain antibody (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 herein by reference in its entirety). The antigen binding site may be composed of the variable domains of the light and heavy chains of a monoclonal antibody. Several studies have shown that scFv fragments may indeed have complete intrinsic antigen binding affinity with one binding site of a whole antibody.

[0214] In the context of the present invention, diabodies are scFvs with two binding specificities and can be monospecific and bivalent or bispecific and bivalent.

[0215] Tandab and flexobody are other antibody formats, for example, which are defined in US2007031436 and EP1293514B1, respectively, which are incorporated herein by reference in their entirety.

[0216] Antibody fragments containing protein idiotypes can be generated by techniques known in the art. For example, these fragments include, but are not limited to, F(ab')2 fragments that can be produced by digesting antibody molecules with pepsin; Fab' fragments that can be produced by reducing the disulfide bridges of F(ab')2 fragments; Fab fragments that can be produced by treating antibody molecules with papain and a reducing agent; and Fv fragments.

[0217] 6.3.6. Antibody-drug conjugates (ADCs)

[0218] The antibody or binding molecule of the present invention can be further linked to an active substance, preferably a toxin, a nanoparticle, a cytokine or a radionucleotide. 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, WO 1990 / 012592 A1, WO 2007 / 030642 A2, WO 2004 / 067038 A1, WO 2004 / 003183 A1, US 2005 / 0074426 A1, WO 1994 / 004189 A1; each of which is incorporated herein 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]; Wolska-Washer et al., “Safety and Tolerability of Antibody-Drug Conjugates in Cancer,” Drug Saf. 2019 Feb;42(2):295-314; 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 December; 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 disclosures of each of which are incorporated herein by reference. .

[0219] In various embodiments, the binding molecule is coupled to a therapeutic agent (i.e., a drug) to form a binding molecule-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (such as radioisotopes), immunomodulators (such as cytokines, chemokines, or checkpoint inhibitors), and toxins (such as cytotoxic agents). In certain embodiments, as described in detail in Section 6.7.3 below, the therapeutic agent is connected to the binding molecule via a linker peptide.

[0220] Methods for preparing antibody-drug conjugates (ADCs) that can be suitable for conjugating drugs to binding molecules disclosed herein are described in, for example, U.S. Pat. No. 8,624,003 (one-pot method), U.S. Pat. No. 8,163,888 (one-step method), U.S. Pat. No. 5,208,020 (two-step method), U.S. Pat. No. 8,337,856, U.S. Pat. No. 5,773,001, U.S. Pat. No. 7,829,531, U.S. Pat. No. 5,208,020, U.S. Pat. No. 7,745,394, WO 2017 / 136623, WO 2017 / 015502, WO 2017 / 015496, WO 2017 / 015495, WO 2004 / 010957, WO 2005 / 077090, WO 2005 / 082023, WO 2006 / 065533, WO 2007 / 030642, WO 2007 / 103288, WO 2013 / 173337, WO 2015 / 057699, WO 2015 / 095755, WO 2015 / 123679, WO 2015 / 157286, WO 2017 / 165851, WO 2009 / 073445, WO 2010 / 068759, WO 2010 / 138719, WO 2012 / 171020, WO 2014 / 008375, WO 2014 / 093394, WO 2014 / 093640,WO 2014 / 160360,WO 2015 / 054659,WO 2015 / 195925,WO 2017 / 160754, Storz (MAbs. 2015 Nov-Dec; 7(6): 989-1009), Lambert et al (AdvTher, 201734: 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., 201114:790-6), Hudak et al. (Angew Chem Int. EdEngl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 20127: 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. .

[0221] 6.3.7. Chimeric Antigen Receptor (CAR)

[0222] The present invention also relates to a chimeric antigen receptor (CAR) comprising the binding molecule of aspect 3 linked to an intracellular domain, preferably comprising one or more signal transduction domains.

[0223] Preferably, the present invention relates to a chimeric antigen receptor (CAR) comprising an scFv of a preferred embodiment of the binding molecule of aspect 3, which is connected to an intracellular region comprising the CD3ζ chain, a signal transduction region of a T cell receptor, and two co-stimulatory domains CD28 and 4-1BB.

[0224] When expressed in T cells or NK cells, CAR according to the present invention is a relevant tool for targeting malignant cells with epitopes recognized and bound by monoclonal antibodies of aspect 1 or aspect 2. The term "chimeric antigen receptor" (CAR) used herein refers to a synthetic receptor comprising a targeting portion associated with one or more signal transduction domains in a single fusion molecule. In general, the binding portion of CAR includes scFv, but may also include other binding entities. Binding portions based on receptor or ligand domains have also been successfully applied. The signal transduction domain of CAR can be from the cytoplasmic region of CD3ζ or Fc receptor γ chain, but can also be from other cytoplasmic regions. The first generation of CAR has been shown to successfully redirect the cytotoxicity of T cells. Signal transduction domains from costimulatory molecules, as well as transmembrane and hinge domains have been added to second and third generation CARs, thereby successfully conducting some therapeutic trials in humans, in which T cells can be redirected to malignant cells expressing CD19 (Porter DL et al., N Eng J Med, 2011).

[0225] 6.3.7.1. CAR-T Implementation Methods

[0226] Those skilled in the art will appreciate that the CAR-T provided by the present invention can be designed for specific applications provided by the present invention (D. Xu et al., Onctarget. 2018 Mar 2; 9(17)), the entire text of which is incorporated herein by reference.

[0227] 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 Exp Med. (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 (Pulè et al., (2005) Mol Ther. 12(5); Geiger et al., Blood (2001) 98; Wilkie et al., (2008) J In some embodiments, the CAR is a fourth generation TRUCKS CAR (Chmielewski et al., Cancer Res (2011) 71.); In some embodiments, the CAR is an armed CAR (Pegram et al., (2012) Blood 119; Curran et al., (2015) Mol Ther. 2015 April; 23 (4)); In some embodiments, the CAR is an engineered co-stimulatory CAR (Zhao et al. (2015) Cancer Cell 28); In some embodiments, the CAR is a SynNotch / sequential AND gated CAR (Roybal et al., (2016) Cell 164); In some embodiments, the CAR is a cis- and trans-co-stimulatory CAR (Stephan et al., (2007) Nat Med 13 (12)); In some embodiments, the CAR is a dual-targeting CAR (Wilkie et al., (2012) J Clin Immunol.32(5)); in various embodiments, CAR is a combined CAR / AND gate-selected CAR (Kloss et al., (2013) Nat Biotechnol 31(1)); in various embodiments, CAR is a TanCAR-selected CAR (Ahmed et al., (2013) Mol Ther Nucleic Acids.2:e105); in various embodiments, CAR is a Go-CART-selected CAR (Foster et al., (2014)); the disclosures of which are incorporated herein by reference in their entirety.

[0228] In a specific embodiment, the CAR is a pCAR, as described in U.S. Pre-Grant Publication No. US 2019 / 0002521, which is incorporated herein by reference in its entirety.

[0229] 6.3.7.2. CAR constructs with primary intracellular signaling domain (CAR-UMG1)

[0230] In some embodiments, the CAR construct comprises a primary intracellular signal transduction domain. When, for example, the extracellular domain fused to the antigen binding domain binds to a cognate antigen, the primary intracellular signaling domain produces an intracellular signal. The primary intracellular signal transduction domain is derived from a primary stimulatory molecule, for example, it comprises an intracellular sequence of a primary stimulatory molecule. The primary intracellular signal transduction domain includes enough primary stimulatory molecule sequences to produce an intracellular signal, for example, when the antigen binding domain fused thereto binds to a cognate antigen.

[0231] A primary stimulatory molecule is a molecule that, upon binding to a cognate ligand, mediates an immune effector response, e.g., in a cell in which it is expressed. Typically, it generates an intracellular signal that is dependent on binding to a cognate ligand comprising an antigen. The TCR / CD3 complex is a typical primary stimulatory molecule; it generates an intracellular signal upon binding to a cognate ligand, e.g., a peptide-loaded MHC molecule. Typically, e.g., in the case of a TCR / CD3 primary stimulatory molecule, the intracellular signal generated by the primary intracellular signaling domain is dependent on binding of the primary stimulatory molecule to an antigen.

[0232] Primary stimuli may mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of the cytoskeletal structure.

[0233] For example, in the presence of costimulation, stimulation can lead to optimization of the immune effector function of CART cells, such as an increase. For example, in the case of CART cells, stimulation can mediate T cell responses, such as proliferation, activation, differentiation, etc.

[0234] In some embodiments, the primary intracellular signal transduction domain comprises a signal transduction motif, e.g., an immunoreceptor tyrosine-based activation motif or ITAM. The primary intracellular signal transduction domain may comprise an ITAM containing a cytoplasmic signal transduction sequence from, for example, TCR ζ (CD3 zeta, CDζ), common FcR γ (FCER1G), Fc γ Rlla, FcR β (Fc ε Rib), CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcsRI, DAP10, DAP 12, and CD66d.

[0235] The main intracellular signal transduction domain comprises a functional fragment or analog of a main stimulatory molecule (e.g., CD3 zeta, CD3ζ). The main intracellular signal transduction domain may include an entire intracellular region or a fragment of an intracellular region, which is sufficient to produce an intracellular signal when the antigen binding domain to which it is fused binds to a cognate antigen. In some examples, the main intracellular signal transduction domain has at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity with the entire intracellular region of a naturally occurring primary stimulatory molecule or a fragment of an intracellular region sufficient to produce an intracellular signal, and the naturally occurring primary stimulatory molecule is, for example, a human, or other mammals, such as non-human species, such as rodents, monkeys, apes or rat cells. Primary stimulatory molecules.

[0236] In some embodiments, the primary intracellular signaling domain is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues with corresponding residues of the entire intracellular region of a naturally occurring human primary stimulatory molecule, such as a naturally occurring human primary stimulatory molecule disclosed herein, or a fragment of the intracellular region sufficient to produce an intracellular signal.

[0237] 6.3.7.3. CAR constructs with co-stimulatory signaling domains (CAR-UMG1)

[0238] In some embodiments, the CAR construct includes a co-stimulatory signal transduction domain, and when the extracellular domain (e.g., antigen binding domain) fused thereto binds to a cognate ligand, the co-stimulatory signal transduction domain produces an intracellular signal. The co-stimulatory signal transduction domain is derived from a co-stimulatory molecule. The co-stimulatory signal transduction domain includes enough major co-stimulatory molecule sequences to produce an intracellular signal, for example, when the extracellular domain (e.g., antigen binding domain) fused thereto binds to a cognate ligand.

[0239] The co-stimulatory domain can be a domain that optimizes the performance (e.g., persistence or immune effector function) of a T cell comprising a CAR comprising the co-stimulatory domain.

[0240] Co-stimulatory molecules are cell surface molecules other than antigen receptors or their counter-ligands, which are required to promote immune effector responses. In some cases, they are necessary for efficient or enhanced immune responses. Typically, co-stimulatory molecules produce intracellular signals that depend on binding to cognate ligands, and in some embodiments, cognate ligands are not antigens, such as antigens recognized by the antigen binding domains of CART cells. Typically, signal transduction from primary stimulatory molecules and co-stimulatory molecules contributes to immune effector responses, and in some cases, both signals are necessary for efficient or enhanced immune effector responses.

[0241] The costimulatory domain includes a functional fragment or analog of a costimulatory molecule (such as ICOS, CD28 or 4-1BB). It may include a fragment of the entire intracellular region or the intracellular region, for example, when the antigen-binding domain to which it is fused is combined with a cognate antigen, it is sufficient to produce an intracellular signal. In some embodiments, the costimulatory domain has at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity with the entire intracellular region or the intracellular region fragment sufficient to produce an intracellular signal, and the intracellular signal domain is from a naturally occurring costimulatory molecule, such as a human, or other mammals, such as non-human species, such as rodents, monkeys, apes or mouse intracellular costimulatory molecules.

[0242] Exemplary co-stimulatory domains include, but are not limited to, those selected from the group consisting of CD27, CD27, CD28, 4-1BB (CD137), Qx40, CD30, CD40, ICQS (CD278), ICAM-1, LFA-1 (CD11a / CD18), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD8, CDS, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactil e), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, ClOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IP0-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, and PAG / Cbp.

[0243] In some embodiments, the costimulatory signaling domain is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the corresponding residues of the entire intracellular region of a naturally occurring human costimulatory molecule, such as a naturally occurring human costimulatory molecule disclosed herein, or a fragment of the intracellular region sufficient to produce an intracellular signal.

[0244] 6.3.7.4. Immune effector cells containing chimeric antigen receptors (CARs)

[0245] In the sixth aspect, the present invention provides a CD3+ lymphocyte, a NK lymphocyte, a cytokine-induced killer (CIK) cell, a γ-δ lymphocyte, a NKT cell or another immune effector cell, which comprises the chimeric antigen-ch-UMG1 according to aspect 4 or the expression vector according to aspect 5.

[0246] Generally, CD3 is a complex of four signaling chains that associate with the α:β heterodimer of the T cell receptor in a functional T cell receptor complex. T cell receptor signaling generally requires the CD3 complex. Generally, the CD3+ lymphocyte group contains only thymocytes and T cells. CD3+ cells can be detected, for example, by flow cytometry.

[0247] 6.3.8. Bispecific T cell engagers (BiTEs)

[0248] The two main approaches to T cell redirection involve genetic modification with chimeric antigen receptors (CARs) or the use of recombinant proteins called bispecific T cell engagers (BiTEs).

[0249] The present invention provides various embodiments of BiTE-UMG1 constructs (Huehls AM et al., "Bispecific T-cell engagers for cancer immunotherapy", Immunol Cell Biol. 2015 Mar; 93(3):290-6; 93(3):290-6; Zhukovsky EA et al., "Bispecific antibodies and CARs: generalized immunotherapeutics harnessing Tcell redirection", Curr Opin Immunol. 2016 Jun; 40:24-35), the entire disclosures of which are incorporated herein by reference.

[0250] Typically, BiTEs are composed of two single-chain variable fragments (scFv) connected in series via a flexible linker. One scFv binds to a T cell-specific molecule (usually CD3), while the second scFv binds to a tumor-associated antigen. This structure and specificity enable BiTEs to physically link T cells to tumor cells, ultimately stimulating T cell activation, tumor killing, and cytokine production.

[0251] In some embodiments, the BiTE-UMG1 construct targets a blood 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.

[0252] 6.4. Pharmaceutical compositions of CD43 binding proteins

[0253] In a seventh aspect, the present invention provides a pharmaceutical composition comprising 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, γ-δ lymphocytes, NKT cells or other immune effector cells as described in aspect 6.

[0254] As used herein, the term "pharmaceutical composition" is used interchangeably with the term "drug."

[0255] In some embodiments of the pharmaceutical composition, it is an antibody or an antigen-binding fragment 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.

[0256] In various embodiments, the pharmaceutical compositions are described in detail in U.S. Pat. No. 8,961,964, U.S. Pat. No. 8,945,865, U.S. Pat. No. 8,420,081, U.S. Pat. No. 6,685,940, U.S. Pat. No. 6,171,586, U.S. Pat. No. 8,821,865, U.S. Pat. No. 9,216,219, U.S. Application No. 10 / 813,483, WO 2014 / 066468, WO 2011 / 104381, and WO 2016 / 180941, each of which is incorporated herein by reference in its entirety.

[0257] 6.5. Preparation method

[0258] The UMG1 binding molecules (antibodies, proteins, antigens, etc.) provided by the present invention can be prepared using standard methods known in the art.

[0259] For example, UMG1 binding molecules can be prepared by expressing using standard cell-free translation, transient transfection, and stable transfection methods currently used for antibody preparation. In a particular embodiment, Expi293 cells (ThermoFisher) can be used to produce binding molecules using protocols and reagents from ThermoFisher, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethyleneimine, as described in detail by Fang et al. (Biological Procedures Online, 2017, 19: 11), the entire contents of which are incorporated herein. The expressed protein can be easily purified using standard methods known in the art (e.g., CH1 affinity resins, such as CaptureSelect CH1 resins) and protocols provided by ThermoFisher. Further purification can be accomplished using ion exchange chromatography, as routinely used in the art.

[0260] 6.6. Administration

[0261] The UMG1 pharmaceutical composition provided by the present invention can be administered by any suitable administration route, including but not limited to parenteral administration, including subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, nasal and pulmonary administration routes.

[0262] 6.7. Combination therapy

[0263] The present invention also provides combination therapy. In some embodiments, the pharmaceutical composition provided herein is used in combination with another therapeutic therapy. The therapeutic therapy can be surgical therapy, radiotherapy, holistic therapy, cell therapy, tissue regeneration or other pharmaceutical compositions known to be used to treat cell proliferation diseases or cancer.

[0264] In some embodiments, when the pharmaceutical compositions provided herein are used in therapeutic combinations, the therapeutically effective doses are different. Methods for experimentally determining the therapeutically effective doses of drugs and other agents used in a combined treatment regimen include using metronomic dosing, i.e., by providing more frequent, lower doses to minimize toxic side effects.

[0265] Combination therapy regimens include treatment regimens in which administration of a compound described herein is initiated before, during, or after treatment with the above-described second agent and continued to any time during treatment with the second agent or after termination of treatment with the second agent. Such regimens also include treatments in which a compound described herein and a second agent used in combination are administered simultaneously or at different times and / or at reduced or increased intervals during treatment.

[0266] Combination therapy also includes cyclical treatments that start and stop at different times to assist in the clinical management of the patient. For example, the compounds in the combination therapy described herein are administered once a week at the beginning of treatment, reduced to once every two weeks, and further reduced as appropriate.

[0267] 6.8. Preparation

[0268] The present invention also provides various UMG1 pharmaceutical preparations, which contain an effective amount of UMG1 antigen, antibody or binding molecule or protein.

[0269] In some embodiments, the pharmaceutical composition is formulated in any conventional manner using one or more physiologically acceptable carriers, which contain excipients and adjuvants that facilitate processing of the active compound into a preparation that can be used in medicine. The correct formulation depends on the chosen route of administration. Any pharmaceutically acceptable techniques, carriers and excipients are optionally used as appropriate. Pharmaceutical compositions comprising UMG1 antibodies or UMG1 binding molecules are manufactured in a conventional manner, for example, by way of example only, by conventional mixing, dissolving, granulating, preparing gels, crushing, emulsifying, encapsulating, embedding or compressing processes.

[0270] UMG1 pharmaceutical compositions may optionally include other medical or pharmaceutical agents, carriers, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts for regulating osmotic pressure, buffers and / or other therapeutically valuable substances. Methods for preparing compositions comprising compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form solids, semisolids or liquids.

[0271] Solid formulations of the composition include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories.

[0272] Liquid formulation compositions include solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles or nanoparticles, which contain the compounds disclosed herein. Semisolid compositions include, but are not limited to, gels, suspensions and creams. The pharmaceutical compositions described herein are in the form of liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc.

[0273] 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 each total composition contains 0.0000001-10% (by weight). In addition, the antibodies, binding molecules or CD3+ lymphocytes described herein are preferably used in a carrier. The choice of carrier may depend 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. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to make the composition isotonic. Examples of carriers include, but are not limited to, physiological saline, Ringer's solution, and glucose solution. Acceptable excipients, carriers or stabilizers are preferably nontoxic at the dosages and concentrations used, and include buffers such as citrate, phosphate and other organic acids; salt-forming counterions such as sodium and potassium; low molecular weight (>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; and other The composition may also include at least one other active compound, such as a chemotherapeutic agent.

[0274] Preferably, the antibodies, binding molecules, CD3+ lymphocytes and / or active compounds 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 is administered.

[0275] 6.9. Polynucleotides encoding CD43 binding proteins

[0276] In an eighth aspect, the present invention provides a nucleic acid or polynucleotide encoding the UMG1 antibody according to aspect 1 or 2 or the UMG1 binding molecule according to aspect 3.

[0277] Also provided herein are polynucleotides encoding antibodies, which are optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA unstable elements. The method of generating an optimized nucleic acid encoding an antibody or its fragment (e.g., a light chain, a heavy chain, a VH domain, or a VL domain) for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA can be implemented by adjusting the optimization method accordingly, for example, U.S. Patent Nos. 5965726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498; each of which is incorporated by reference in its entirety. For example, potential splicing sites and unstable elements (e.g., elements rich in A / T or A / U) in RNA can mutate without changing the amino acid encoded by the nucleic acid sequence to increase the stability of RNA for recombinant expression. These changes take advantage of the degeneracy of the genetic code, for example, using an alternative codon for the same amino acid. In some embodiments, it may be desirable to change one or more codons to encode conservative mutations, such as similar amino acids having chemical structures and properties and / or functions similar to the original amino acids. Such methods may increase the expression of antibodies or fragments thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more relative to the expression of antibodies encoded by unoptimized polynucleotides.

[0278] In certain embodiments, an optimized polynucleotide sequence encoding an antibody or fragment thereof described herein (e.g., a VL domain and / or a VH domain) can be hybridized with an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an antibody or fragment thereof described herein (e.g., a VL domain and / or a VH domain). In a specific embodiment, an optimized nucleotide sequence encoding an antibody or fragment described herein hybridizes with an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an antibody or fragment thereof described herein under high stringency conditions. In a specific embodiment, an optimized nucleotide sequence encoding an antibody or fragment thereof described herein hybridizes with an antisense polynucleotide of a non-optimized nucleotide sequence encoding an antibody or fragment thereof described herein under high, medium, or low stringency hybridization conditions. Information about hybridization conditions has been described, see U.S. Patent Application Publication No. US2005 / 0048549 (e.g., paragraphs 72-73).

[0279] The polynucleotides of the present invention can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotides can be determined. The nucleotide sequences encoding the antibodies described herein and the modified forms of these antibodies can be determined using methods well known in the art, that is, the nucleotide codons encoding specific amino acids are known to be assembled in a manner to produce nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as Kutmeier G et al., (1994), BioTechniques 17: 242-6; which is incorporated by reference in its entirety), in short, including the synthesis of overlapping oligonucleotides containing portions of the sequences encoding the antibodies, annealing and connecting these oligonucleotides, and then amplifying the connected oligonucleotides by PCR.

[0280] Alternatively, the polynucleotides encoding the antibodies described herein can be generated from nucleic acids from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, genomic DNA obtained from hybridoma cells producing an antibody of interest can be used to perform PCR amplification using synthetic primers that hybridize with the 3' end and 5' end of a known sequence. This PCR amplification method can be used to obtain nucleic acids comprising sequences encoding antibody light chains and / or heavy chains. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding variable light chain regions and / or variable heavy chain regions of an antibody. Amplified nucleic acids can be cloned into vectors to express in host cells and used for further cloning, such as producing chimeric antibodies and humanized antibodies.

[0281] If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library produced by any tissue or cell expressing the antibody (e.g., a hybridoma cell selected to express an antibody described herein), or nucleic acid isolated from any tissue or cell expressing the antibody (e.g., a hybridoma cell selected to express an antibody described herein), preferably a cDNA library produced by poly A+ RNA) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe for a specific gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method known in the art.

[0282] DNA encoding the antibodies of the invention described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHOGS System TM (Lonza) CHO cells), or myeloma cells that otherwise do not produce immunoglobulin, to synthesize antibodies in recombinant host cells.

[0283] To produce complete antibodies, PCR primers including VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify the VH or VL sequences in scFv clones or other clones. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as a human γ4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as a human κ or λ constant region. In certain embodiments, the vector for expressing the VH or VL domain includes a promoter, a secretion signal, a variable region cloning site, a constant domain, and a selection marker, such as neomycin. The VH and VL domains can also be cloned into a vector expressing the necessary constant region. Then, using techniques known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line to produce a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0284] For example, the DNA may also be modified by replacing the murine sequences with coding sequences for human heavy and light chain constant domains, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0285] Site-directed or high-density mutagenesis or other mutagenesis methods of the variable region can be used to optimize the monoclonal antibody for specificity, affinity, etc. In particular, affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783; each strategy is incorporated by reference in its entirety) are known in the art and can be used to generate high affinity human antibodies.

[0286] 6.10. Hybridoma cells producing UMG1 monoclonal antibodies

[0287] In a ninth aspect, the present invention provides a hybridoma cell which produces a monoclonal antibody of the antibody according to aspect 1 or 2.

[0288] 6.11. Hybridoma Compositions

[0289] The present invention also provides a hybridoma composition, which is deposited as ICLC PD n°16001.

[0290] 6.12. Methods for Producing UMG1 Monoclonal Antibodies

[0291] In an eleventh aspect, the present invention provides a method for preparing a monoclonal antibody according to aspect 1 or 2, the method comprising isolating the antibody from a hybridoma cell deposited as ICLC PD n°16001.

[0292] 6.13. Isolation of cells using UMG1 antibodies and / or binding molecules

[0293] In the twelfth aspect, the present invention provides a method for identifying or isolating T-cell acute lymphoblastic leukemia cells, T lymphoma cells, Waldenstrom's macroglobulinemia cells or tumor-associated macrophages, comprising contacting a cell sample containing the cells with a monoclonal antibody according to aspect 1 or 2 or a binding molecule according to aspect 3.

[0294] In general, macrophages are the most representative non-malignant cells in the tumor microenvironment. Tumor-associated macrophages (TAMs) are believed to have pro-tumor inflammatory and immunosuppressive phenotypes that favor chemotherapy resistance, angiogenesis, cell motility, and intra / extravasation. Therefore, targeting TAMs may represent a new therapeutic approach and an unexplored clinical option that can improve the efficacy of current anticancer treatments.

[0295] The skilled person is generally familiar with methods based on antibodies or binding molecules for identifying or isolating specific cells, such as T-cell acute lymphoblastic leukemia cells, T lymphoma cells, Waldenstrom's macroglobulinemia cells or tumor-associated macrophages, such as methods based on flow cytometry fluorescence cell sorting, magnetic cell separation or single cell sorting, for example by a cell sorter.

[0296] 6.14. Methods for Producing Immune Effector Cells

[0297] In the thirteenth aspect, the present invention provides a method for producing CD3+ lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γ-δ lymphocytes, NKT cells or other immune effector cells expressing a chimeric antigen receptor of a chimeric antigen receptor according to aspect 4, comprising introducing an expression vector of the expression vector according to aspect 5 into the CD3+ lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γ-δ lymphocytes, NKT cells or other immune effector cells.

[0298] 6.15. Expression vector compositions

[0299] 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.

[0300] Typically, an expression vector is a plasmid used to introduce a desired nucleic acid sequence (e.g., a gene) into a target cell, which results in transcription and translation of a protein (i.e., a chimeric antigen receptor, antibody, or binding molecule) encoded by the nucleic acid sequence. Therefore, an expression vector typically includes regulatory sequences, such as promoter and enhancer regions, and polyadenylation sites, in order to guide efficient transcription of the nucleic acid sequence on the expression vector. The expression vector may also include additional necessary or useful regions, such as selectable markers for selection in eukaryotic or prokaryotic cells, purification tags for purifying the resulting protein, multiple cloning sites, or replication origins.

[0301] Typically, the expression vector can be a viral vector or a non-viral vector. Typically, various viral vectors can be used, such as retroviral vectors, such as lentiviral or adenoviral vectors, or plasmids. 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.

[0302] 6.16. Treatment methods

[0303] In another aspect, methods of treatment are provided, the methods comprising administering to a patient a binding molecule or antibody as described herein in an amount effective to treat the patient.

[0304] In some embodiments, the method comprises administering to the patient an amount of a binding molecule or antibody described herein effective to treat the patient with a CAR or CAR-T.

[0305] In some embodiments, the method comprises administering to the patient an amount of a binding molecule or antibody described herein effective to treat the patient with a BiTE.

[0306] In some embodiments, the method comprises administering to the patient an amount of a binding molecule or antibody described herein effective to treat the patient with the antibody-drug conjugate.

[0307] 6.16.1. Instructions

[0308] In some embodiments, the antibodies or binding molecules of the present invention can be used to treat proliferative diseases or cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a blood cancer, including but not limited to T cell malignancies, T cell leukemias, T cell lymphomas, T cell acute lymphoblastic leukemias, multiple myeloma, B cell malignancies, myeloid malignancies, acute myeloid leukemias, and chronic myeloid leukemias.

[0309] In some embodiments, the cancer or proliferative disease can be cancer from the bladder, blood, blood immune cells (e.g., T cells or B cells, monocytes, etc.), bone, bone marrow, brain, breast, colon, colorectal, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, pancreas, skin, stomach, testicles, tongue, or uterus.

[0310] In some embodiments, the cancer or tumor treated with the antibodies or binding molecules of the invention can be a malignant tumor; non-malignant; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; hairy stromal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid; branchial alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; aerobic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosis Sexual cancer; adrenocortical carcinoma; endometrioid carcinoma; adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; cervical adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; ovarian stromal tumor, malignant; malignant thecoma; malignant granulosa cell tumor; androgenic tumor, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; malignant lipid cell tumor; malignant paraganglioma; extramammary paraganglioma, malignant; pheochromocytoma; tongue cancer; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; giant Malignant melanoma in nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mixed Müllerian tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; ovarian goiter, malignant; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic malignancy; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Malignant pineal gland; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Schwannoma, malignant; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Parasarcoma; Malignant lymphoma, small lymphocytic;Diffuse large cell lymphoma; Follicular lymphoma; Mycosis fungoides; Other non-Hodgkin lymphomas; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative intestinal disease; Leukemia; Lymphoid leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblastic leukemia; Myeloid sarcoma; Hairy cell leukemia and / or Waldenstrom's macroglobulinemia.

[0311] In the thirteenth aspect, the present invention provides a method for producing CD3+ lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γ-δ lymphocytes, NKT cells or other immune effector cells expressing a chimeric antigen receptor of a chimeric antigen receptor according to aspect 4, comprising introducing an expression vector of the expression vector according to aspect 5 into the CD3+ lymphocytes, NK lymphocytes, cytokine-induced killer (CIK) cells, γ-δ lymphocytes, NKT cells or other immune effector cells.

[0312] In some embodiments, the cancer treated by the antibodies disclosed herein is a non-Hodgkin lymphoma derived from peripheral B cells or peripheral T cells, including but not limited to diffuse large B cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0313] In some embodiments, the cancer treated by the antibodies disclosed herein is multiple myeloma.

[0314] In some embodiments, the cancer treated by the antibodies disclosed herein is melanoma.

[0315] In some embodiments, the cancer treated by the antibodies disclosed herein is testicular cancer, including but not limited to seminoma, embryonal carcinoma, yolk sac tumor, and teratoma.

[0316] In some embodiments, the cancer treated by the antibodies disclosed herein is a pediatric malignancy, such as Wilms' tumor, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, or leiomyosarcoma.

[0317] 6.17. Example

[0318] The following examples are intended to illustrate the present invention, but should not be construed as limiting the present invention. These examples include technical features, and it should be understood that the present invention also relates to any combination of the technical features presented in the examples.

[0319] 6.17.1. Example 1: UMG1 binding specificity - UMG1 antibody binds to lymphocytes but not to myeloid cells in PBMCs

[0320] The binding of UMG1 to peripheral blood mononuclear cells (PBMC) from healthy donors was examined.

[0321] Methods: Peripheral blood mononuclear cells (PBMCs) from different healthy donors were obtained by Ficoll gradient separation. Subsequently, cells were seeded in 5 ml tubes and stained with 1 μg / ml of UMG1 antibody or 1 μg / ml of negative control "scrambled" mouse IgG1 antibody in 100 μl of binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)) and incubated at 4°C for 30 minutes. Cells were then washed twice in binding solution and stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 30 minutes in the dark at 4°C. Subsequently, cells were washed twice in binding solution. Cells were analyzed on an ATTUNE NxT flow cytometer (THERMOScientific). One tube per donor was not stained and one tube per donor was stained with FITC-conjugated secondary antibody only as a negative control.

[0322] Results: The UMG1 antibody was able to recognize lymphocyte subsets with various prevalences (range: 0-15%) in different human donors. The UMG1 antibody did not show any reactivity with any other cell population within PBMCs, including myeloid-derived cells, indicating that myeloid-derived cells in PBMCs of healthy subjects are negative for the expression of the UMG1 epitope (see Figure 1A and Figure 1B ).

[0323] In contrast, when we used a commercial anti-CD43 antibody (S7 from Becton Dickinson) to probe the same PBMCs for CD43 expression, all lymphocytes and myeloid cells were positive (see Figure 1B ).

[0324] Therefore, the epitope on CD43 recognized by the UMG1 antibody exhibits a specific, restricted expression pattern in PBMC cells, which is different from the expression pattern of the epitope recognized by the commercial anti-CD43 antibody (S7).

[0325] 6.17.2. Example 2: UMG1 Binding Specificity - PBMC T Lymphocyte Subpopulations Bound by UMG1 Antibodies

[0326] This example further characterized the lymphocyte subpopulations detected by the UMG1 antibody by immunomagnetic sorting of the corresponding lymphocytes.

[0327] Methods: Briefly, 15 μg of UMG1 antibody was mixed with the manufacturer's components (EasySep TM "Do it yourself" selection kit (STEMCELL Technologies)) to obtain a solution that can be used for immunomagnetic separation. After FcR blocking, this solution was added to PBMCs from 3 different donors, in which at least 10% of the lymphocytes were detected by the antibody, and incubated at room temperature (rt) for 15 min. Magnetic nanoparticles were added to the solution and the cells were incubated for another 10 min at RT. The solution was then placed in a magnet to remove unbound cells.

[0328] Results: Almost all cells detected by UMG1 antibody were CD45 + CD3 + CD4 + CD8 - CD127 + CCR7 + T lymphocytes (see Figures 2A-2D and Table 1).

[0329]

[0330]

[0331] 6.17.3. Example 3: UMG1 Binding Specificity—Only T-ALL and Waldenstrom's Macroglobulinemia Cancer Cell Lines Express the UMG1 Epitope

[0332] In this experiment, we evaluated the expression of UMG1 epitopes in various hematopoietic and non-hematopoietic cancer cell lines.

[0333] Methods: Briefly, cells were seeded in 5 mL tubes and stained with 1 μg / ml monoclonal antibody UMG1 or 1 μg / ml scrambled mouse IgG1 antibody in 100 μl binding solution (phosphate buffered saline (PBS) + 0.5% fetal bovine serum (FBS)) and incubated at 4°C for 30 minutes. Cells were then washed twice in binding solution and stained with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 30 minutes in the dark at 4°C. 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 unstained and one tube for each cell line was stained with FITC-conjugated secondary antibody only.

[0334] Results: Patients with EGIL T3 classification and Waldenstrom's macroglobulinemia ( Figure 3A-3B) T-ALL cell lines were positive for UMG1 epitope expression, while other cell lines tested were negative for UMG1 epitope (see Table 2). UMG1 antibody recognized T-ALL and Waldenstrom's macroglobulinemia cell lines, but not other hematopoietic and non-hematopoietic tumors.

[0335]

[0336]

[0337] 6.17.4. Example 4: UMG1 binding specificity - UMG1 binds to T-ALL human cell lines with a binding pattern that is different from that of commercial CD43 antibodies

[0338] This example demonstrates the unique binding properties of the UMG1 antibody compared to commercially available CD43 antibodies in two different T-ALL human cell lines, ALL-SIL and KE-37.

[0339] Methods: Commercially available CD43 antibodies: clone, CD431G10 (Becton Dickinson), CD43 MEM-59 (Invitrogen), and CD43 L-10 (Invitrogen) were compared with the UMG1 antibody.

[0340] Cells were collected from various human cell lines (100,000 cells / tube). Cells were washed by adding 2 ml of cold staining buffer and centrifuging the cells at 1200 rpm for 5 minutes at room temperature, and the supernatant was discarded. Primary antibody UMG1 was added at a concentration (1 μg / ml in a final volume of 100 μL of cells. Cells were gently mixed by pulse vortexing. Next, cells were incubated for 15 minutes at 2-8°C, protected from light. Cells were washed twice, 2 mL of staining buffer was added, and cells were centrifuged at 1200 rpm for 5 minutes at room temperature, and the supernatant was discarded. Fluorescently labeled secondary antibodies were diluted in a final volume of 100 μL of cells according to the manufacturer's instructions and incubated at 2-8°C for at least 15 minutes, protected from light. Cells were washed twice as described above, then resuspended in 500 μL PBS 1X and analyzed by flow cytometry.

[0341] Results: Through FACS analysis, we observed that UMG1 antibody, CD431G10 (BectonDickinson), CD43MEM-59 (Invitrogen), and CD43 L-10 (Invitrogen) had different expression densities and intensities in ALL-SIL and KE-37 cell lines. Fig. 12A and 12BThese observations suggest that the UMG1 antibody binds to a different site on CD43 than the three different commercial antibodies against CD43.

[0342] 6.17.5. Example 5: UMG1 binding specificity - UMG1 response with tumor immune infiltration

[0343] 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.

[0344] Methods: Paraffin-embedded tissue samples from three different human cancers were sectioned, deparaffinized, and then analyzed by immunohistochemistry for the expression of the UMG1 epitope using the following method.

[0345] The samples were deparaffinized in a heating block at 65°C for 30 minutes. Next, the sections were immersed in (1) xylene for 10 minutes, (2) xylene for 5 minutes, and then rehydrated through graded alcohols: 90% ethanol for 2 minutes; 70% ethanol for 2 minutes. The slides were rinsed in tap water, followed by a final rinse in deionized water.

[0346] Antigen exposure was performed using Novocastra Epitope Retrieval Solution, pH 9 (Leica Biosystems) in a 98°C thermostatic bath for 30 min. Endogenous peroxidase was neutralized with peroxidase blocker for 10 min. Peroxidase blocker: 3 / 4%, (v / v) H2O2. Next, the samples were washed twice in PBS for 5 min each. After washing, the samples were incubated with protein blocker for 8 min. Protein blocker: 0.4% casein in phosphate-buffered saline. After blocking, the slides were washed twice in PBS for 5 min each.

[0347] Sections were stained with primary antibody UMG1 ("m-UMG1") at a dilution of 1:300 at 4°C overnight. Next, the stained sections were washed twice in PBS for 5 minutes each. After washing, the samples were incubated with rabbit anti-mouse IgG for 30 minutes and then washed twice in PBS for 5 minutes each. After washing, the samples were incubated with Novolink polymer, anti-rabbit poly-HRP IgG for 30 minutes and then washed twice in PBS for 5 minutes each.

[0348] Staining on sections was revealed by AEC (3-amino-9-ethylcarbazole) substrate chromogen (Dako) followed by washing in tap water. Sections were counterstained with hematoxylin for 5 min and then washed again in tap water. Sections were mounted using Ultramount aqueous permanent 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).

[0349] Results: We observed UMG1 staining in the immune infiltrate of various solid tumors. More specifically, we found significant reactivity in tumor-associated macrophages in lung, colorectal, and breast cancer tissues. Fig.14A (colorectal adenocarcinoma (grade 2, G2), 14B (lung adenocarcinoma), and 14C (breast, triple-negative ductal invasive breast cancer (G2, basal-like)).

[0350] Notably, UMG1 failed to directly stain cancer cells, unlike other previously described CD43 antibodies such as UN1 (see UN1 staining in De Laurentiis, A. et al., Molecular Cellular Proteomics, 2011, Fig. 9 ).

[0351] These results suggest that the binding pattern of the UMG1 antibody to CD43 is distinct from other characterized CD43 antibodies, especially UN1, which has been previously shown to bind to CD43 in cancer cells.

[0352] 6.17.6. Example 6: UMG1 binding specificity - UMG1 epitope is expressed in tumor-associated macrophages. When macrophages are co-cultured and interact with cancer cells, UMG1 epitope expression increases

[0353] In this example, specimens from different types of cancer were evaluated for UMG1 epitope expression by immunohistochemistry, and it was found that the specific CD43 epitope bound by UMG1 was highly expressed by tumor-associated macrophages (TAMs).

[0354] Methods: Different types of cancer were stained as described in the Examples herein.

[0355] Results: Immunohistochemistry (Table 3, Figure 4 and Figures 14A-14C ) evaluated specimens from different types of cancer and found that UMG1+ macrophages were highly infiltrating components of most tumors, with specificity and high infiltration levels in pancreatic and ovarian cancers, although myeloid-derived cells in peripheral blood mononuclear cells of healthy subjects lacked the UMG1 epitope.

[0356]

[0357] To better understand the significance of the UMG1 epitope in macrophages, in a second experiment, changes in the expression of the UMG1 epitope were evaluated in a macrophage differentiation model with or without co-cultured cancer cells. For this, we used THP-1 monocytic leukemia cells; as shown in Example 3, these cells do not express the UMG1 epitope.

[0358] Methods: Differentiated human non-polarized M0 macrophages (THP-1M) were obtained by culturing in complete appropriate medium in the presence of 50ng / ml phorbol 12-myristate 13-acetate (PMA) for 48h. The medium was then replaced with fresh medium without PMA. Next, PANC1 pancreatic cancer cell line cells were added to the selected wells at a 1:1 ratio and incubated for 48h.

[0359] The cells were then prepared for immunofluorescence analysis. Briefly, after fixation, THP-1M cells were stained with a chimeric antibody derived from UMG1, chUMG1 (described further in Example 16) or a human IgG1 control and incubated overnight at 4°C. FITC anti-human monoclonal secondary antibody was then added to the cells for 2 hours. After washing, the cells were stained with DAPI ( Antifade mounting medium from Vectorlabs was added to cells and coverslips prior to analysis.

[0360] Results: Figure 5A As shown, THP-1 derived macrophages stained with control IgG1 were completely negative, while macrophages stained with ch-UMG1 were weakly (faintly) positive. Interestingly, in the presence of PANC1 cells, THP1 derived macrophages showed strong (bright) expression of UMG1. The figure shows a specific interaction between THP-1 derived macrophages (white arrows) and PANC1 cells (red arrows) ( Figure 5A , left side).

[0361] These findings indicate that UMG1-specific epitopes are significantly upregulated (i.e., elevated) when macrophages are co-cultured and interact with cancer cells in a reconstituted tumor microenvironment. This elevated expression implies that the UMG1 epitope is a suitable target for therapeutic approaches to eliminate tumor-associated macrophages. In addition to this relevant potential as a therapeutic tool, UMG1 may also prove useful for detection, prognostic role analysis, and predictive studies.

[0362] 6.17.7. Example 7: UMG1 Binding Specificity - Competitive Binding Assays Demonstrate that the UMG1 Binding Site on CD43 is Unique Compared to Commercially Available CD43 Antibodies

[0363] To determine whether the binding site of UMG1 is the same or different from that of commercially available CD43 antibodies, competitive binding assays between (i) h-UMG1 (a humanized form of the UMG1 antibody, further described in Example 19 below) and h-UMG1 conjugated to phycoerythrin (h-UMG1-PE) and (ii) h-UMG1 and three commercially available CD43 antibodies were performed on two different cell lines, CEM and HPB-ALL.

[0364] Methods: Competition binding assays were performed using the following antibodies and analyzed by FACS analysis: unconjugated h-UMG1, h-UMG1-PE, and commercially available CD43 antibodies: MEM-59PE (Invitrogen), L-10 PE (Invitrogen), and 1G10PE (Becton Dickinson). Briefly, CEM and HPB-ALL cells were incubated for 20 min in the dark on ice with h-UMG1 unconjugated, increasing concentrations (0.016 μg / ml, 0.08 μg / ml, 0.4 μg / ml, 1 μg / ml, 2 μg / ml), in the presence of 1 μg / ml of one of the CD43 clones or h-UMG1-PE (positive control).

[0365] Approximately 500,000 cells were collected and stained for each test. Cells were analyzed and measured using FACS Canto (Becton Dickinson) and DIVA software (BD FACSDiva TM Software) was used for analysis. For each measurement, 10,000 events were gated using DIVA software. Each experiment was performed in triplicate.

[0366] Results: As expected, in both CEM and HPB-ALL cell lines, unconjugated h-UMG1 competed for h-UMG1-PE binding. That is, by increasing the concentration of unstained h-UMG1, the number of stained cells labeled with h-UMG1-PE decreased. Fig.13A and 13B (Circled line).

[0367] In contrast, unconjugated h-UMG1 did not compete for the binding of other commercial CD43 antibodies (MEM-59 (Invitrogen), L-10 (Invitrogen), and 1G10 (Becton Dickinson). In fact, the number of stained cells labeled with anti-CD43 was not reduced by increasing the concentration of unconjugated h-UMG1 antibody. See, Fig.13A and 13B (Line with upward triangle, line with downward triangle, line with square).

[0368] These results suggest that the binding site of the h-UMG1 antibody is different from that of the three commercially available CD43 antibodies.

[0369] 6.17.8. Example 8: UMG1 Binding Specificity - Flow Cytometry Profiles of h-UMG1 in Hematopoietic Cell Lines Compared to Historically Published Data for UN1

[0370] As mentioned above, UN1 has been reported in the literature to bind directly to a variety of cancer cell lines, whereas this was not the case for UMG1 in the experiments reported above in Examples 3 and 5. Instead, UMG1 bound to tumor-associated macrophages infiltrating solid tumors.

[0371] Since hybridomas secreting UN1 antibodies have never been deposited in a biobank, nor have UN1 hybridoma master or working cell banks been established, it is not possible to perform side-by-side experimental comparisons of the original UN1 antibody and UMG1, we further explored the similarities and differences between UN1 and UMG1 binding by repeating the experiments first reported in Tassone et al., Tissue Antigens 44:73-82, 1994. In this reference, UN1 binding to different hematopoietic cell lines, such as JURKAT, MOLT-4, CEM, and HPB-ALL lines, was assessed by flow cytometry expression.

[0372] Method: Cells of human cell lines were collected at approximately 100,000 cells / tube. Cells were then washed by adding 2 mL of cold staining buffer and centrifuging the cells at 1200 rpm for 5 minutes at room temperature and the supernatant was discarded.

[0373] In a final volume of 100 μL of cells, add h-UMG1 primary antibody at a concentration of 1 μg / ml. Next, mix the cells by pulse vortexing and incubate at 2-8°C for 15 minutes, protected from light. Then, wash the excess primary antibody twice by adding 2 mL of staining buffer to the pellet and centrifuge cells at 1200 rpm for 5 minutes at room temperature, and discard the supernatant. Add fluorescently labeled secondary antibodies at the recommended dilution in a final volume of 100 μL of cells and incubate at least 15 minutes at 2-8°C, protected from light.

[0374] Excess secondary antibody was then washed from the cells twice by adding 2 mL of staining buffer, centrifuging the cells at 1200 rpm for 5 minutes at room temperature, and discarding the supernatant. The washed pelleted cells were resuspended in 500 μL of PBS 1X and analyzed by flow cytometry.

[0375] result: Fig.17A Shown are historical flow cytometric profiles of UN1 performed in 1994 by the Tassone laboratory (Tassone et al., Tissue Antigens 44:73-82, 1994) in JURKAT, MOLT-4, CEM, and HPB-ALL cell lines. Fig. 17B Shown are the results of flow cytometry profiles of h-UMG1 antibody in JURKAT, MOLT-4, CEM and HPB-ALL cell lines.

[0376] Comparison showed that both UMG1 and UN1 did not bind to JURKAT cells but bound to MOLT-4, CEM and HPB-ALL cell lines.

[0377] Notably, the flow cytometry profiles of UMG1 in the CEM cell line showed an approximately 1 log shift compared to UN1. The difference in the curves for UN1 and UMG1 suggests a difference in binding affinity to CEM cells.

[0378] 6.17.9. Example 9: UMG1 Binding Specificity - h-UMG1 Monoclonal Antibody Binding on Activated Neutrophils

[0379] In this example, UMG1 epitope expression was assessed on inactivated and activated neutrophils isolated from peripheral blood of healthy human donors.

[0380] Methods: Whole blood from two healthy donors was collected in heparin anticoagulated vacuum cans, diluted 1:1 with PBS, and separated by Ficoll-Paque density gradient centrifugation (600 rcf, 15 min, RT, no brake). RBC (red blood cell pellet) was suspended in 1xPBS, followed by the addition of 6% dextran solution. Dextran was precipitated for 30 min at room temperature in the dark. 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 s and lysis was stopped using 1x PBS. The samples were centrifuged for 10 min, 400 rcf, no brake, the supernatant was discarded, and the granulocyte pellet was resuspended in culture medium. For each healthy donor granulocyte sample, half was cultured only in growth medium, while the other half was stimulated with PMA (5 ng / ml) and ionomycin (250 ng / ml) for 15 min at 37°C, 5% CO2. Samples were collected, washed in staining solution, and Fc blocked. Cells were incubated with CD11b Pe-Cy7 (pan-granulocyte marker), CD55 (neutrophil activation marker), and UMG1-APC or APC-conjugated IgG1 human isotype control and acquired in flow cytometry to assess h-UMG1 expression on inactivated and activated granulocytes.

[0381] Results: The binding of h-UMG1 mAb on inactivated and activated neutrophil populations was similar to that of the isotype control (IgG1). Only a minority of neutrophils expressed the specific CD43 epitope targeted by h-UMG1 mAb ( Fig.33 ). CD43 is commonly expressed on neutrophils, a finding that reinforces the uniqueness of the UMG1 monoclonal antibody target.

[0382] 6.17.10. Example 10: UMG1 binding specificity - h-UMG1 monoclonal antibody binding to activated T lymphocytes

[0383] In this example, UMG1 epitope expression was assessed on activated T lymphocytes from peripheral blood of healthy donors.

[0384] Methods: Whole blood from healthy donors (HD) was collected in EDTA anticoagulated vacutainers. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation. Lymphocytes from 3 HD patients were activated with PMA (25 ng / ml) and ionomycin (1 μg / ml) for 24 h at 37°C, 5% CO2. After activation, cells were collected, 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 cytometric analysis was performed to evaluate h-UMG1 expression on CD25+ T cells and CD69+ T cells.

[0385] Results: No significant difference in h-UMG1 mAb binding was observed compared to the IgG isotype control group in activated T lymphocytes ( Figures 34A-34D ). Therefore, h-UMG1 monoclonal antibody does not target activated T lymphocytes.

[0386] 6.17.11. Example 11: UMG1 Binding Specificity - Immunohistochemical Analysis of mUMG1 Binding in Healthy and Tumor Tissues

[0387] Methods: Tissue microarrays (TMAs) used for immunohistochemical analysis included: dog female normal organs (DGF281); rat normal organs (RAT901a); mouse normal organs (MO541c); rhesus monkey normal organs (RhFDA1a); cynomolgus monkey normal organs (CyFDA1c); multiple organ cancers and normal tissues (MC5003c); malignant melanoma and skin tissues (ME2081); lymphoma investigation tissues (Ly2084); breast cancer tissues (BR1505d); testicular disease (TE2081); embryonic tumor test (T001a); human digestive system (GI1441); human brain tumor (GL2082); human pediatric malignancies (PC701), produced by Biomax, USA, and FDA standard paraffin tissue array human normal organs produced by BioChain (Catalog No.: T8234701). Descriptions of the selected TMAs are provided below.

[0388] FDA Standard Tissue Array (Human Tissue, T8234701-5): Normal human tissue microarray provided on five slides, containing 30 different human normal tissue types and 3 donors / tissue types.

[0389] Multi-organ cancer and normal tissue (MC5003c): High-density multi-organ tumor with normal tissue microarray, containing 20 types of organs, each organ was obtained from 25 individuals (20 tumors and 5 normal tissues), single core per case.

[0390] Malignant Melanoma (ME2081): Malignant melanoma and skin tissue microarray, including 88 cases of malignant melanoma, 16 skin tissues, and duplicate cores for each case.

[0391] Lymphoma Investigation Tissue (Ly2084): Lymphoma Investigation Tissue Microarray (slide 4 of 520 Lymphoma Investigation slides) containing 104 malignancies (64 B-cell lymphomas, 24 mucosa-associated lymphoma tissues, 6 T-cell lymphomas, 4 Hodgkin lymphomas, 4 anaplastic large cell lymphomas, 1 mantle cell lymphoma and 1 Burkitt lymphoma) with duplicate cores for each case.

[0392] Testicular Disease (TE2081): Testicular tumor tissue array, including 46 seminoma, 8 yolk sac tumor, 16 embryonal carcinoma, 5 teratoma, 3 tuberculoma, 6 atrophy, 15 adjacent normal tissue and 5 normal tissue, with duplicate cores for each case.

[0393] Human pediatric malignancies (PC701): Normal tissues of pediatric malignancies, including 21 cases of Wilms' tumor, 13 cases of neuroblastoma, 7 cases of endodermal sinus carcinoma, 4 cases of retinoblastoma, 3 cases of hepatoblastoma, 2 cases of medulloblastoma, 4 cases of lymphoma, 1 case each of choroid plexus papilloma, glioblastoma, adrenocortical carcinoma, embryonal rhabdomyosarcoma, ependymoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, immature teratoma, and leiomyosarcoma, plus 7 normal tissues, single nucleus for each case.

[0394] For immunostaining, tissue sections were dewaxed and rehydrated. Antigen unmasking was performed using Novocastra Epitope Extraction Solution, pH 9 EDTA buffer at 98°C for 30 minutes in a thermostatic bath (FALC Instruments SrL, Treviglio (BG) Italy, model WB-MD 5).

[0395] TMA was incubated with UMG1 monoclonal primary antibody (m-UMG1 monoclonal antibody was used in this application at a dilution of 1:300) overnight at 4°C. Immunostaining was visualized using a matrix chromogen by polymer detection method (Novolink polymer detection system Novocastra-Leica Biosystems Newcastle, product number: RE7280-K) or AEC (3-amino-9-ethylcarbazole, Dako, reference number K3464). Slides were counterstained with Harris hematoxylin (Novocastra, Leica Biosystems).

[0396] All sections were analyzed under a Zeiss Axio Scope A1 light microscope (Zeiss, Germany), and micrographs were collected using an Axiocam503 color digital camera and ZEN2 imaging software (Zeiss, Germany).

[0397] UMG1 expression assessment: Slides were scanned using an Aperio CS2 Leica. Protein expression levels were scored manually by determining signal intensity using a 4-grade scale (negative (0), weak (1), moderate (2), and strong (3)). See Micke P et al., 2014, Int J Cancer, 135: 2206-2214, incorporated herein by reference in its entirety.

[0398] Results: In normal human tissues, in the thymus ( Fig.38A ) and tonsillar lymph nodes ( Fig.38B Positive binding of UMG1 mAb was observed on 100% of the 12 cells. Membrane staining was moderately / intensely positive (score 2-3) on more than 50% of lymphocytes and a small fraction of elements with monocyte / macrophage morphology. Rare binding was observed on scattered immune cells in other organs, but in these cells, expression was more compatible with cytoplasmic expression without membrane reinforcement (see Fig.38C and lung tissue).

[0399] In the lymphoma spectrum, cytoplasmic and membrane staining is frequently observed and highlighted with varying intensities ( Figures 39A-39B , Table 6). The membrane expression of the UMG1 monoclonal antibody target epitope on malignant cells at multiple observation points was also clear, although the intensity and distribution varied.

[0400] Regarding malignant melanoma, UMG1 monoclonal antibody targets a region expressed on the malignant cell membrane and TAMs ( Fig.40A , Table 6). In addition, based on preliminary data on tumor-associated macrophage epitope expression, some samples stained positive for intratumoral leukocyte infiltration.

[0401] Among testicular tumors, seminomas have a higher prevalence of tumor clones ( Fig.40B , Table 6), followed by embryonal carcinoma and yolk sac tumor. Peritumoral healthy tissue and normal healthy testis were negative.

[0402]

[0403]

[0404] 1 Number of samples that can be evaluated (i.e. considered to be correctly stained)

[0405] 2Number of samples labeled with UMG1 antibody on tumor cell membrane

[0406] 3 Number of samples in which UMG1 antibody labeled tumor cells exceeding 10%

[0407] 4 Number of samples with tumor cells labeled with UMG1 antibody and intensity scores of 2+ or 3+

[0408] 5 The number of samples with more than 10% immune cells infiltrating the tumor microenvironment (labeled with UMG1 antibody)

[0409] 6 Not applicable or not evaluated

[0410]

[0411] 1 Number of samples that can be evaluated (i.e. considered to be correctly stained)

[0412] 2 Number of samples labeled with UMG1 antibody on tumor cell membrane

[0413] 3 Number of samples in which UMG1 antibody labeled tumor cells exceeding 10%

[0414] 4 Number of samples with tumor cells labeled with UMG1 antibody and intensity scores of 2+ or 3+

[0415] 5 The number of samples with more than 10% immune cells infiltrating the tumor microenvironment (labeled with UMG1 antibody)

[0416] 6 Not applicable or not evaluated

[0417] Another group of cancers that showed expression of the UMG1 mAb epitope in both tumor cells and immune infiltration were pediatric tumors of varying origin (see Table 7).

[0418] In addition, in certain sites (MC5003c tissue microarray) representing multiple solid tumors of different origins, UMG1 epitope expression on cancer cells or immune cell infiltrates (tumor-associated macrophages (TAMs) and other immune cell infiltrates) was observed. UMG1 mAb binding in these samples had different distribution levels and intensities (data not shown).

[0419] 6.17.12. Example 12: UMG1 binding specificity - epitope binding site on CD43

[0420] Binding of various CD43 protein variants to h-UMG1 antibodies was examined by Western blotting and FACS analysis to determine the binding site of h-UMG1 on CD43 in HEK293T wild-type cells that do not express CD43.

[0421] CD43 protein variants: Fig.15A The sequences of the tested CD43 protein clones are provided in Table 4 and the sequence listing as shown in SEQ ID NOs: 17-24. Wild-type CD43, denoted "CD43#1", was generated using the entire 400 amino acid region. To engineer CD43 protein variants, the N-terminal domain was sequentially truncated. The first CD43 truncated variant "CD43#2" was generated using aa 31 to 400 of the full-length CD43. The second CD43 variant is shown as "CD43#3" and was generated using aa 41 to 400 of the full-length CD43. The third CD43 variant, "CD43#4", was generated using aa 61 to 400 of the full-length CD43. The fourth CD43 variant is referred to as "CD43#5" and consists of aa 91-400 of the full-length CD43. The fifth CD43 variant is labeled "CD43#6" and is deleted from aa 64 to 78.

[0422] In addition, single amino acid deletion variants were tested. The sixth CD43 variant, "CD43#7," had an amino acid deletion at aa 69, which is thought to be the GalNac site. The seventh CD43 variant, labeled "CD43#8," had an amino acid substitution at aa 69, T to N or "T69N."

[0423]

[0424]

[0425] Constructs: CD43 protein constructs were expressed using pLenti-CMV-(insert)-Histag-GFP-2A-Puro expression vector from Applied Biological Materials (ABM) Inc. service (Vancouver, Canada). His-tag and / or GFP detection served as positive controls for successful transfection and / or protein expression.

[0426] 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% CO2 in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin (Thermo Fisher Scientific, MA, USA). 72 h after transfection, cells were analyzed by Western blotting or flow cytometry (FACS).

[0427] Western blot: Western analysis was performed using the UMG1 antibody to determine whether the UMG1 antibody can bind and detect CD43 wild-type and CD43 protein variants of the expected kDa size. His-tag antibody was used as a positive control.

[0428] Briefly, NP40 lysis buffer was supplemented with Halt TM Whole-cell protein extracts were obtained with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, MA, USA). Bradford assay (Bio-Rad Laboratories, Berkeley, CA, USA) was used to estimate protein concentration. Cell lysates were loaded at a concentration of 60 μg per lane and subjected to NuPAGE. TM 3-8% Tris-acetate protein gel (Invitrogen, ThermoScientific, MA, USA) was used to separate the proteins. Turbo TM The cells were electrotransferred for 30 min using the BioRad Biotechnology transfer system (Bio-Rad Laboratories, Berkeley, CA, USA) and immunoblotted with anti-actin antibody purchased from Cell Signaling (data not shown), anti-His tag antibody purchased from abm (Vancouver, Canada) (#G020), and h-UMG1 primary antibody (all diluted 1:500). Goat anti-mouse and rabbit anti-human HRP-conjugated antibodies (Invitrogen) were used as secondary antibodies (diluted 1:3000). Immunoreactive bands were detected using SuperSignal TM West Pico PLUS chemiluminescent substrate (Thermo Scientific, MA, USA) was used for visualization by enhanced chemiluminescence detection.

[0429] Flow cytometry (FACS) analysis: FACS was performed to determine whether the antibodies could detect CD43 wild-type and CD43 variants expressed in HEK293T cell line cells.

[0430] FACS analysis was performed according to standard procedures, and the percentage of h-UMG1-positive cells among GFP-positive cells was detected using 1 μg / ml h-UMG1-PE-conjugated antibody. TM X-20, BD) and analyzed by DIVA software (BD FACSDiva TM At least 20,000 events were gated for each measurement.

[0431] Results: Western analysis showed that the UMG1 epitope binding site is located between aa 61 and 91 (as in wild-type CD43). Fig.15A The boxed sequence in , which shows the putative binding site for the h-UMG1 antibody. In addition, these studies showed that the UMG1 antibody binds specifically to UMG1 and not to the CD43His-spiked protein that lacks the specific extracellular region recognized by UMG1. Fig. 15C and 15E .

[0432] The FACS observations confirmed the results observed in Western blots, namely that the UMG1 epitope binding site is located between aa 61 to 91 of CD43 wild-type, as it could not be detected in GFP-expressing cells of CD43#5 and CD43#6 protein variants. Fig.15D and 15F .also, Fig.15E and 15F Western blot and FACS studies in the Figure 4 showed that the h-UMG1 antibody in HEK293T cells transgenic for CD43 also recognized the CD43 aa region from aa 61 to 91 if Treonine69 was deleted (CD43#7) or replaced with non-O-glycosylated amino acids (CD43#8). These results indicate binding to an epitope that should not have any sugar groups. As expected, wild-type HEK293T cells that were not transfected with CD43 did not show any reactivity with the UMG1 antibody.

[0433] 6.17.13. Example 13: Linear epitope mapping of UMG1 binding specificity-h-UMG1 mAb-CD43 epitope

[0434] Methods: Microarray content: The human CD43 (SEQ ID NO: 17) sequence was extended with a neutral GSGSGSG (SEQ ID NO: 46) linker at the C- and N-termini to avoid peptide truncation. The extended antigen sequence was translated into a 15 amino acid linear peptide with a peptide-peptide overlap of 14 amino acids. The resulting CD43 peptide microarray contained 400 different peptides, printed in duplicate (800 peptide spots) and framed by an additional HA (YPYDVPDYAG (SEQ ID NO: 47), 82 peptide spots) control peptide. Synthesis and analysis of the microarray was performed by Pepperpprint GmbH, Heidelberg.

[0435] CD43 peptide microarrays were prestained with secondary antibodies (goat anti-human IgG (H+L) DyLight680 (0.2 μg / ml)) and control antibodies (mouse monoclonal anti-HA (12CA5) DyLight800 (0.5 μg / ml)). After preswelling for 15 min in wash buffer (PBS, pH 7.4, containing 0.05% Tween 20) and incubation for 30 min in blocking buffer (Rockland blocking buffer MB-070), CD43 peptide microarrays were initially incubated with secondary antibodies and control antibodies in incubation buffer (wash buffer containing 10% blocking buffer) for 45 min at room temperature (RT) to investigate background interactions with linear CD43 peptides, which could interfere with the primary analysis.

[0436] The other CD43 peptide microarray copies were then incubated with humanized monoclonal anti-CD43 antibodies (h-UMG1 monoclonal antibodies) at 10 μg / ml and 100 μg / ml concentrations in incubation buffer (incubated for 16 hours at 4°C and 140 rpm), and then stained with secondary antibodies and control antibodies. The LI-COR Odyssey imaging system was used to read at a scan intensity of 7 / 7 (red = 700nm / green = 800nm), a scan offset of 0.65mm, and a resolution of 21 μm. The additional HA control peptides that constitute the peptide microarray were stained simultaneously with the control antibodies as internal quality controls to confirm the analytical quality and integrity of the peptide microarray.

[0437] At scan intensity 7 / 7 (red / green), we did not observe any background interaction of the secondary and control antibodies with the linear CD43 peptide, even though brightness and contrast were significantly increased. The analyzer performs data quantification. Control antibody staining produces the expected well-defined pattern of HA control spots for the peptide microarray (green), validating the integrity of the overall microarray and the quality of the analysis.

[0438] Quantification of spot intensities and peptide annotations was based on 16-bit grayscale TIFF files, which exhibit a higher dynamic range than 24-bit color TIFF files. Microarray image analysis was performed using The software algorithm decomposes the fluorescence intensity of each spot into raw signal, foreground signal, and background signal, and calculates the average median foreground intensity and the spot-to-spot deviation of duplicate spots. Based on the average median foreground intensity, an intensity map is generated, and the interactions in the peptide map are highlighted by intensity color coding, with high spot intensity in red and low spot intensity in white. We allow a maximum spot-to-spot deviation of 40%, otherwise the corresponding intensity value is zero.

[0439] We further plotted the average spot intensity of the humanized monoclonal antibody detection of the antigen sequence from the N-terminus to the C-terminus of CD43 to visualize the overall spot intensity and signal-to-noise ratio. The intensity plot was correlated with the peptide and intensity mapping and visual inspection of the microarray scan to determine the epitope of the humanized monoclonal anti-CD43 antibody. If it was unclear whether a certain amino acid contributed to the antibody binding, the corresponding letter was written in gray. The baseline of the intensity plot was leveled for a better overview of the data.

[0440] Results: We observed a weak antibody response to an epitope-like pattern formed by a neighboring peptide with the consensus motif INEGSPLW (SEQ ID NO:48; aa 71-78 on human CD43); in addition, we observed two stronger interactions with peptides with the highly basic consensus RRRQKR (SEQ ID NO:49) and RRPTLTTFFGRRRK (SEQ ID NO:50) due to nonspecific ionic binding of the antibody. Fig.35 ). The median signal-to-noise ratio can be best determined by simultaneously staining for an HA control peptide.

[0441] 6.17.14. Example 14: UMG1 binding specificity - linear epitope mapping of h-UMG1 monoclonal antibodies - epitope substitution scanning

[0442] Methods: Microarray Contents: Wild-type peptide PPSTNIEGSPLWTS (SEQ ID NO: 51) The epitope substitution scan was based on the exchange of 20 main amino acids for all amino acid positions. The resulting peptide microarray contained 300 different wild-type peptide variants, printed in triplicate (900 peptide spots), 9 spots of the custom control peptide PPSTSVNEGSPGTS (SEQ ID NO: 52) and an additional HA control peptide framework (YPYDVPDYAG, 82 spots (SEQ ID NO: 47)). The synthesis and analysis of the microarrays were performed by PEPperPRINT GmbH, Heidelberg.

[0443] Pre-staining of peptide microarrays with secondary and control antibodies After pre-swelling in wash buffer for 15 min and incubation in blocking buffer for 30 min, peptide microarrays were initially incubated with secondary and control antibodies for 45 min at room temperature to analyze background interactions with wild-type peptide variants that could interfere with the primary analysis.

[0444] The other peptide microarray copies were then incubated with humanized monoclonal anti-CD43 antibodies at concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml (data not shown), and 250 μg / ml in incubation buffer, and then stained with secondary antibodies and control antibodies. After washing, the read was performed at a scanning intensity of 7 / 7 (red / green). The additional HA control peptide that constituted the peptide microarray was stained at the same time as an internal quality control to confirm the analytical quality and integrity of the peptide microarray.

[0445] At a scan intensity of 7 / 7 (red / green), even with significantly increased brightness and contrast, we did not observe any background interactions of the secondary and control antibodies with 300 wild-type peptide variants or other custom peptides (see adjusted scans). Data quantification was performed by the Analyzer. Control antibody staining produced the expected well-defined HA control spot pattern used to construct the peptide microarray, verifying the integrity of the overall microarray and the quality of the analysis.

[0446] Quantification of spot intensities and peptide annotations was based on 16-bit grayscale TIFF files, which exhibit a higher dynamic range than 24-bit color TIFF files. Microarray image analysis was performed using The software algorithm decomposes the fluorescence intensity of each spot into raw signal, foreground signal, and background signal, and calculates the average median foreground intensity and the spot-to-spot deviation of three spot replicates. Based on the average median foreground intensity, an intensity map is generated, and the interactions in the peptide map are highlighted by intensity color coding, with red representing high intensity and white representing low intensity. Very weak signals with blurred spot morphology are zeroed.

[0447] We further plotted the average spot intensity of the analysis against the microarray content in rows from the upper left to the lower right across the bottom of the chip with the humanized monoclonal antibody to visualize the overall spot intensity and signal-to-noise ratio. The intensity plots were correlated with the peptide and intensity maps and visual inspection of the microarray scans to identify variants of the wild-type peptide that interacted with the humanized monoclonal anti-CD43 antibody.

[0448] To gain insight into the epitope substitution scan of the wild-type peptide, we further generated heat maps of the microarray scans as well as substitution matrices and amino acid plots reflecting the amino acid preference at a given position. The dataset was analyzed to identify conserved and variable amino acid positions of the wild-type peptide.

[0449] The substitution matrix highlights the preference for a given amino acid by color coding (red for preferred amino acids, blue for less preferred amino acids) and is calculated by dividing the spot intensity for a given peptide by the average spot intensity of all 20 peptides substituted at the same position. The amino acid map is calculated by dividing the spot intensity for a given peptide by the spot intensity for the wild-type peptide. Thus, the position of the amino acid reflects the intensity ratio compared to the native wild-type peptide amino acid.

[0450] Results: Substitution scanning of the wild-type peptide PPSTNIEGSPLWTS (SEQ ID NO: 51) showed a weak but typical epitope substitution pattern with conserved (few or single spots in a row) and variable (continuous rows of spots) amino acid positions (see Fig.36 ). A low signal-to-noise ratio was observed and was well defined in the simultaneous control staining of the HA control peptide framework.

[0451] Heat map, substitution matrix and amino acid map of humanized monoclonal anti-CD43 antibodies analyzed for substitution scanning of wild-type peptide PPSTNIEGSPLWTS (SEQ ID NO:51, aa 66-80 of human CD43) highlighting the conserved core motif EGSPLW (SEQ ID NO:53, aa 73-78 of human CD43) framed by the N- and C-terminal variable fragments PPSTIN (SEQ ID NO:54, aa 66-72 of human CD43) and TS (SEQ ID NO:55, aa 79-80 of human CD43) (see Figures 37A-37C This finding is consistent with the epitope INEGSPLW suggested in previous epitope mapping of the full-length human CD43 protein.

[0452] The amino acids at positions 76 (P) and 77 (L) of human CD43 are essential for antibody binding and are completely intolerant of any amino acid substitutions. Amino acid positions 73 (E) and 78 (W) are highly conserved and can only tolerate conservative substitutions of D and F, respectively. Substitution of W at position 78 by F even leads to a significant increase in antibody binding. The sequence at amino acid position 74 (G) is less conserved and is easily substituted by M, acidic amino acids D and E, and aromatic amino acids W, F, and Y.

[0453] All other amino acid positions, including amino acid position 75 (S), showed variable characteristics. Among the 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 showed resistance to substitution by the basic amino acids K and H (but surprisingly, R was not included).

[0454] 6.17.15. Example 15a: UMG1 binding specificity - binding to the aglycosylated extracellular portion of CD43 compared to other CD43 antibodies

[0455] This example shows the binding affinity measurement between humanized UMG1 (h-UMG1) (H3-L4) and the extracellular part of CD43 (aa 20-253). The results are reported as the dissociation constant, KD.

[0456] Methods: Antibodies dissolved in water were manually printed onto bare gold-coated (47 nm thickness) PlexArray nanocapture sensor chips (Plexera Bioscience, Seattle, WA, USA) at 40% humidity. The affinity of the analyte (CD43) was measured at different concentrations. Each concentration was printed in duplicate, with each spot containing 0.2 uL of sample solution. The chip was incubated overnight at 4 °C, 80% humidity, and rinsed with 10× PBST for 10 min, 1× PBST for 10 min, and deionized water twice for 10 min. The chip was then blocked with 5% (w / v) skim milk in water overnight, rinsed with 10× PBST for 10 min, 1× PBST for 10 min, and deionized water twice for 10 min, and then dried under a stream of nitrogen before use. SPRi measurements were performed using a PlexAray HT (Plexera Bioscience, Seattle, WA, USA). Collimated light (660 nm) was passed through a coupling prism, reflected from the SPR-active gold surface, and received by a CCD camera. Various concentrations of analyte (extracellular portion of human recombinant CD43 (from aa 20-253); SEQ ID NO: 42) produced in an E. coli vector of CD43 were used in the experiment ( Fig.19 Different concentrations of analytes in the flow cell are indicated by different colored lines). Buffer and sample were injected into a 30 μL flow cell mounted on a coupled Prim by a non-pulsating piston pump. Each measurement cycle contained four steps: washing with PBST running buffer at a constant rate of 2 uL / s to obtain a stable baseline; injecting the sample at a rate of 5 uL / s for binding; washing the surface with PBST at a rate of 2 uL / s for 300 s and regenerating with 0.5% (v / v) H3PO4 at 2 μl / s for 300 s. All measurements were performed at 25 °C. The signal change (unit: AU) after binding and washing was recorded as the analytical value.

[0457] The selected protein grafted area in the SPR image was analyzed and the average reflectivity of the selected area was plotted over time. The real-time binding signal was recorded and analyzed by the data analysis module (DAM, PlexeraBioscience, Seattle, WA, USA). Kinetic analysis was performed using Biacore 4.1 software (Biacore, Inc.).

[0458] Results: SPR binding results showed the K between the extracellular part of CD43 and h-UMG1 D The value is 99.4nm( Fig.19 ). The results showed that it had a strong binding affinity to the target.

[0459] Furthermore, binding to the aglycosylated extracellular portion of CD43 (produced in E. coli without mammalian glycosylation) distinguishes UMG1 from other anti-CD43 antibodies (e.g., UN1 and MEM-59) that bind only to glycosylated or neuraminidase-sensitive epitopes (de Laurentiis A et al., Mol Cell Proteomics. 2011 May;10(5)).

[0460] 6.17.16. Example 15b: UMG1 binding specificity - Comparison of UMG1 binding characteristics with UN1 historical data

[0461] As shown in the above examples, UMG1 and its chimeric and humanized derivatives chUMG1 and h-UMG1, respectively, have several different binding properties compared to other anti-CD43 antibodies, including historically reported data on UN1. Table 5 compares the properties of UMG1 with historically reported data on UN1.

[0462]

[0463]

[0464]

[0465] 6.17.17. Example 16: Construction of ch-UMG1-chimeric antibody with UMG1 binding specificity

[0466] A chimeric antibody with UMG1 binding specificity (ch-UMG1) was constructed by fusing murine UMG1 VH (SEQ ID NO: 34) to a human VH constant region and murine UMG1 VL (SEQ ID NO: 35) to a human light chain constant region using standard techniques.

[0467] 6.17.18. Example 17: ch-UMG1-ch-UMG1 induces antibody-dependent cell-mediated cytotoxicity (ADCC) in T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma

[0468] To determine the potential activity of the monoclonal antibody ch-UMG1 as an immunotherapeutic tool, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was tested against two cell lines that have been shown in Example 3 to express the UMG1 epitope.

[0469] PBMCs from healthy donors (effector cells) were co-cultured with the T-ALL cell line HPB ALL or the T lymphoma cell line H9 (target cells) in the presence of different concentrations of ch-UMG1 as shown below.

[0470] Method: 4×10 4 Target cells were seeded in 96-well round-bottom plates and cultured for 30 min at 37°C with 5% CO2 in the presence of different concentrations of monoclonal antibody ch-UMG1 (0, 10, 50, 100, 200 μg / ml) or the highest dose (200 μg / ml) of chimeric negative or positive control (NC and PC, 200 μg / ml, respectively) IgG1. 6 PBMC (fixed E: T = 10: 1) were added to each well along with 20 μl / ml PE-conjugated anti-CD107a monoclonal antibody (Becton Dickinson), and the cells were then incubated for 3 h at 37°C under 5% CO2. After 1 hour, 6 μg / ml monensin 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).

[0471] Results: It was found that CD3- / CD56+ / CD107a+ cells increased significantly with the concentration of ch-UMG1 antibody, confirming the potential of ch-UMG1 antibody as an ADCC inducer ( Figure 6A-6B ).

[0472] The chimeric mAb ch-UMG1 is an effective immunotherapeutic tool for the treatment of T-cell ALL / LY. These data help design immune-targeted approaches, which are an urgent and unmet clinical need for T-cell ALL / LY.

[0473] 6.17.19. Example 18: ch-UMG1-ch-UMG1 induces antibody-dependent cell-mediated cytotoxicity (ADCC) of Waldenstrom's macroglobulinemia cells

[0474] 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 cells was evaluated.

[0475] Methods: We performed degranulation assays by co-culturing purified NK cells from healthy donors (effector cells) and BCWM.1 cell line (target cells) in the presence of different concentrations of ch-UMG1 antibody or negative / positive controls. We chose mAb cetuximab as negative control and mAb rituximab as positive control.

[0476] Specifically, 10 5 Target cells were seeded in 96-well round-bottom plates and cultured for 30 min at 37°C with 5% CO2 in the presence of different concentrations of ch-UMG1 antibody (0, 10, 50, 100, 200 μg / ml), 200 μg / ml cetuximab, or 200 μg / ml rituximab. 5 NK cells (fixed E: T = 1: 1) were added to each well along with 20 μl / ml PE-coupled anti-CD107a monoclonal antibody (BD), and the cells were then incubated for 2 hours at 37°C 5% CO2. After 1 hour, 6 μg / ml monensin was added to each well (GolgiStop, BD). At the end of the incubation period, the cells were stained with APC-coupled anti-CD56 and PerCp-coupled anti-CD3 and analyzed on an ATTUNE NxT flow cytometer (THERMOScientific).

[0477] Results: It was found that CD3- / CD56+ / CD107a+ cells increased significantly with the concentration of ch-UMG1 antibody, achieving exactly the same effect as rituximab. These results confirmed the potential of ch-UMG1 antibody as an ADCC inducer ( Figure 7 ). The chimeric monoclonal antibody ch-UMG1 is an effective immunotherapeutic tool for the treatment of Waldenstrom's macroglobulinemia.

[0478] 6.17.20. Example 19: Construction of h-UMG1-humanized UMG1 monoclonal antibody

[0479] Humanized UMG1 antibodies were constructed using a combination of human heavy chains SEQ ID NO: 8 to SEQ ID NO: 11 and human light chains SEQ ID NO: 13 to SEQ ID NO: 16 provided herein.

[0480] The expression and purification of the antibodies were as follows: The corresponding cDNA of the antibody was cloned into a vector system using conventional (non-PCR-based) cloning techniques. Gene synthesis vector plasmid. Plasmid DNA was prepared under low endotoxin conditions based on anion exchange chromatography. DNA concentration was determined by measuring absorption at a wavelength of 260 nm. Sanger sequencing verified the correctness of the sequence (up to two sequencing reactions per plasmid depending on the size of the cDNA).

[0481] Suspension-adapted CHO K1 cells (originally obtained from ATCC and adapted to serum-free growth in suspension culture) were used for production. For the aglycosylated antibody (ah-UMG1), GlymaxX technology (ProBioGen) was used and transiently expressed in CHO cells (Evitria). Seeds were grown in a chemically defined, animal component-free, serum-free medium. Cells were transfected with a custom proprietary transfection reagent and cultured in animal component-free, serum-free medium after transfection.

[0482] The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter). TM SuRe TM purification TM Purity was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7um 7.8x300mm) and DPBS as running buffer at 0.8ml / min.

[0483] Endotoxin content was measured using a Charles River Endosafe PTS system. Titers were measured using a ForteBio Protein A biosensor (kinetic analysis) and calculated based on a human IgG1 standard.

[0484] The affinity of putative h-UMG1 antibodies (constructed as described in Example 19) was tested on HPB-ALL and H9 cell lines known to be positive for the UMG1 epitope.

[0485] Methods: Four humanized heavy chain (H 1-4) and four humanized light chain (L 1-4) variants were generated by identifying the mouse complementarity determining regions (CDRs) and transplanting the CDRs into the human antibody framework by replacing selected residues in the nearest human germline sequence in the framework region, with the goal of protecting potential structurally important residues of the mouse counterpart. 16 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). All heavy chain variants adopted the IgG1 isotype.

[0486] In addition, 8 hybrid CHL (1-4) and H (1-4) CL variants were generated. These 8 hybrid variants include 4 with mouse heavy chain and human light chain, the selection range is L1-4 (SEQ ID NO: 13-16), and 4 with mouse light chain and human heavy chain, the selection range is H1-4 (SEQ ID NO: 8-11).

[0487] The recombinant gene was placed into an Evitria vector plasmid and transfected into CHO K1 cells (using eviFect, Evitria). Cells were grown post-transfection in animal component-free, serum-free medium (eviMake2, Evitria). The supernatant was collected by centrifugation and subsequently sterile filtered (0.2 μm filter).

[0488] 6.17.21. Example 20: h-UMG1 - Screening for h-UMG1 Antibodies Binding to HPB-ALL and H9 Cell Lines

[0489] Selection: Each humanized antibody was screened for affinity to target cells (estimated by mean fluorescence intensity, MFI) on 2 different cell lines (HPB-ALL and H9) and compared with the binding of chimeric (ch-UMG1) and hybrid monoclonal antibodies by flow cytometry (Attune NxT, ThermoScientific). Each screening was performed twice for a total of 4 replicates. All experiments were performed under the same conditions: the final concentration of all monoclonal antibodies was 1 μg / ml; rituximab (Roche) has been used as an IgG1 negative control; FITC mouse anti-human IgG (BD Biosciences) was used as a monoclonal secondary antibody.

[0490] Results: All evaluated antibodies were able to bind the target with at least the same affinity as the chimeric monoclonal antibody (ch-UMG1). Fig.16 One humanized antibody (H3-L4) achieved the highest MFI in the screen and was selected for further development and named UMG1. Fig.16 .

[0491] 6.17.22. Example 21: h-UMG1 - HPB-ALL tumor reduction in the NSG mouse model by using humanized UMG1 (h-UMG1) and aglycosylated h-UMG1 (ah-UMG1)

[0492] This example reports tumor volume curves from an in vivo experiment comparing a control IgG1 with a humanized form of the UMG1 monoclonal antibody (h-UMG1) and an aglycosylated form of the UMG1 monoclonal antibody (ah-UMG1).

[0493] Methods: 15 NOD-SCID-g-chain-null (NSG) mice were subcutaneously transplanted with 5x10 6 The mice were then randomly divided into groups and injected intraperitoneally with 15 mg / kg of control IgG1, h-UMG1, or h-UMG1 weekly starting from day 1 until death or tumor volume >2000 mm 3 or unacceptable toxicity. Tumor volume was assessed every other day, and the mean tumor volume of each treatment group at each time point was Fig.11 shown.

[0494] Results: Starting from day 29, the disease burden was significantly reduced in the h-UMG1 (line with squares) and ah-UMG1 (line with triangles) antibody-treated groups compared with the IgG1 control group (line with circles). Fig.11 These results suggest that both antibodies have potent antitumor activity.

[0495] 6.17.23. Example 22: CAR-UMG1-UMG1 targeted chimeric antigen receptor T cells (CAR-UMG1) induces T cell activation in the presence of H9 cells

[0496] To further enhance the potential of UMG1 antibodies as immunotherapeutic tools, third-generation CARs were developed.

[0497] Methods: The third-generation CAR was designed by coupling an extracellular domain consisting of a scFv derived from the UMG1 antibody sequence (SEQ ID NO: 7 for the heavy chain and SEQ ID NO: 12 for the light chain) with an intracellular region consisting of the CD3ζ chain (the signaling domain of the TCR) and two co-stimulatory domains, CD28 and 4-1BB, to mimic physiological T cell activation. Fig. 20 A diagram of the CAR construct is provided in (circular diagram) and the complete sequence of the CAR construct is provided in SEQ ID NO:41.

[0498] The construct was cloned as a CAR cassette in a lentiviral vector (Qin DY et al., Anticancer Drugs. 2016 September; 27(8): 711-22). Subsequently, the viral particles were used to transduce CD3+ lymphocytes from healthy donors at an infection multiplier (MOI) of 5, and the transduction efficiency was assessed by flow cytometry (about 38%). The ability of these CAR-T cells to release IFNγ and IL-2 in the presence of target cells and their selective cytotoxicity were tested.

[0499] Results: Figure 8 and Fig. 9 As shown in Figure 2, CAR-UMG1 released significantly higher amounts of interferon-γ (IFN-γ) and interleukin 2 (IL-2) only in the presence of H9 T-cell lymphoma cells. In addition, only CAR-UMG1 was able to induce selective killing of H9 cells (see Figure 2). Fig.10 ). These results demonstrate the ability of CAR-UMG1 to recognize H9 cells and induce T cell activation.

[0500] The chimeric antigen receptor CAR-UMG1 induced significant cytotoxicity against cells expressing the UMG1 epitope.

[0501] 6.17.24. Example 23: UMG1-CD3 bispecific antibody

[0502] To test the specificity of the UMG1-CD3 bispecific antibody and its ability to redirect T cell cytotoxicity to UMG1-positive cells, analyses were performed on the KE37 cell line, which expresses the UMG1 CD43 epitope but is CD3 negative (UMG1+, CD3-), and the ALL-SIL cell line, which is negative for both the UMG1 antigen and CD3 (UMG1-, CD3-).

[0503] Methods: UMG1-CD3 bispecific antibodies were generated using a UMG1-CD3 construct comprising SEQ ID NO: 40. Redirected T cell cytotoxicity was detected by flow cytometry using human peripheral blood mononuclear cells (PBMCs), KE37 cell line (UMG1+, CD3-), and ALL-SIL cell line (UMG1-, CD3-).

[0504] 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 after 72 hours of treatment was measured by flow cytometry as loss of target cell membrane integrity, which was reflected by nuclear uptake of 7AAD.

[0505] Figures 18A-18BRepresentative FACS images of experiments using 1 μg / ml UMG1-CD3 bispecific antibody and an E:T cell ratio of 20:1 are shown in .

[0506] Results: Compared with untreated cells (expressed as NT), KE37 cells treated with UMG1-CD3 bispecific antibody (see Fig.18A ) and ALL-SIL (see Fig.18B ) Increased killing was observed in both cell lines.

[0507] In addition, the KE37 cell line expressing the UMG1 antigen showed a high cell death rate, approximately 86% of the tested cell population, while the ALL-SIL cell line not expressing the UMG1 antigen showed a low cell death rate, approximately 22% of the tested cell population. These results suggest that T cell killing can be directed to UMG1+ cells using the UMG1-CD3 bispecific antibody. Figures 18A-18B .

[0508] 6.17.25. Example 24: UMG1-CD3 bispecific binding activity

[0509] This example tests the binding of the UMG1-CD3 bispecific antibody on the KE37 cell line (UMG1+, CD3-), CCRF-CEM cells (UMG1-, CD3+), and Jurkat cell line (UMG1-, CD3+).

[0510] Methods: Binding activity was detected by flow cytometry using KE37 cell line (UMG1+, CD3-), CCRF-CEM cells (UMG1-, CD3+) and Jurkat cells (UMG1-, CD3+).

[0511] T-ALL cell lines were incubated with increasing concentrations of UMG1-CD3 bispecific antibodies for 20 minutes. After two 1xPBS pH 7.4 (Gibco, 10010-015) washes (5 minutes, RT, 1300rpm), AF647 anti-human IgG (PE) was used to stain T-ALL cells for 20 minutes. After two 1x PBS pH7.4 (Gibco, 10010-015) washes (5 minutes, RT, 1300rpm), binding activity was assessed by flow cytometry as the percentage of PE-positive cells in treated cells compared to negative controls.

[0512] Results: Fig.32As shown, UMG1-CD3 bispecific binding reached a maximum level on KE37 at a concentration of 0.001 μg / ml, while a more gradual binding was observed on CCRF-CEM cells reaching 90% binding at a concentration of 10 μg / ml. In contrast, very low UMG1-CD3 bispecific binding was observed on Jurkat cells at all concentrations under our experimental conditions. The results indicate that the UMG1-CD3 bispecific antibody can bind to either UMG1+ or CD3+ positive cells. The results also indicate that the bispecific affinity of UMG1-CD3 for UMG1 is higher than that for CD3.

[0513] 6.17.26. Example 25: UMG1-CD3 bispecific antibody-mediated T cell cytotoxicity against T-ALL cells

[0514] This example tests the efficacy of the UMG1-CD3 bispecific antibody on T-ALL cell lines and patient-derived primary T-ALL cells.

[0515] Methods: UMG1-CD3 bispecific antibodies were generated using a UMG1-CD3 construct comprising SEQ ID NO: 40. Redirected T cell cytotoxicity was examined by flow cytometry using human peripheral blood mononuclear cells (PBMCs) and a panel of T-ALL cell lines and T-ALL primary blasts.

[0516] Increasing concentrations of UMG1-CD3 bispecific antibody were incubated with far red (in vitro) labeled target cells as well as effector cells at different PBMC E: T cell ratios of 1: 1, 5: 1, 10: 1 or 20: 1. After 48 hours of incubation, the cell mixtures were assessed by flow cytometry for loss of target cell membrane integrity, as reflected by nuclear uptake of 7AAD (BD Pharmingen).

[0517] Results: Compared with untreated cells, increased killing was observed mainly in UMG1-positive cells after treatment with the UMG1-CD3 bispecific antibody ( Figures 21A-21B ; Figures 22A-22C In addition, CCRF-CEM expressing UMG1 antigen (see Fig.21A and Fig.22A ), KE37 cell line (see Fig. 22B ) and EGILIII T-ALL blasts (see Fig. 21B ) showed a high cell death rate (80% to 95%) among the cell populations tested, while the ALL-SIL cell line that does not express the UMG1 antigen showed a low cell death rate (10%) among the cell populations tested (see Fig. 22C ).

[0518] 6.17.27. Example 26: UMG1-CD3 Bispecific - Confirmation of UMG1-CD3 Bispecific Antibody-Mediated T Cell Cytotoxicity against T-ALL Cells

[0519] T-ALL cell lines were co-cultured with total human PBMCs or PBMCs depleted of CD8+ T cells or CD4+ T cells using immunomagnetic beads.

[0520] Methods: Total human peripheral blood mononuclear cells or CD8+T cells or CD4+T cells immunomagnetically depleted (Miltenyi magnetic beads) peripheral blood mononuclear cells and CCRF-CEM cell lines were examined by flow cytometry for redirected T cell cytotoxicity.

[0521] UMG1-CD3 bispecific antibody (1 μg / ml) was incubated with far red (in vitro) labeled target cells and effector cells at a PBMC E: T cell ratio of 10: 1. After 48 hours of incubation, the cell mixture was assessed by flow cytometry for loss of target cell membrane integrity, as reflected by nuclear uptake of 7AAD (BD Pharmingen).

[0522] Results: Reduced target cell cytotoxicity was observed in lymphocyte-depleted samples compared with total PBMCs, confirming the activity of T cell-mediated UMG1-CD3 bispecific cytotoxicity against T-ALL cells (see Fig.24 ).

[0523] 6.17.28. Example 27: UMG1-CD3 bispecific antibody induces apoptosis in T-ALL cell lines

[0524] This example demonstrates the ability of the UMG1-CD3 bispecific to induce apoptosis in T-ALL cell lines.

[0525] Methods: Apoptosis was assessed by flow cytometry using human peripheral blood mononuclear cells (PBMCs) and CCRF-CEM cells.

[0526] Increasing concentrations of UMG1-CD3 bispecific antibody were incubated with far red (in vitro) labeled target cells and effector cells at a PBMC E:T cell ratio of 10: 1. Apoptosis was assessed by flow cytometry to measure the percentage of target cells positive for annexin after 24 hours of treatment.

[0527] Results: An increase in the rate of apoptosis was observed after treatment with the UMG1-CD3 bispecific antibody in a dose-dependent manner (see Fig.23 ).

[0528] 6.17.29. Example 28: UMG1-CD3 bispecific antibody induces PBMC activation

[0529] In this example, the ability of the UMG1-CD3 bispecific to induce PBMC activation by engaging CD3-positive T cells was tested.

[0530] Methods: Redirected T cell cytotoxicity was assessed by flow cytometry using human peripheral blood mononuclear cells (PBMCs) from different healthy donors.

[0531] Increasing concentrations of UMG1-CD3 bispecific antibodies were incubated with CFSE (Invitrogen) labeled PBMCs. Cell proliferation after 96 h of treatment was assessed by Cell-Titer Glo (Promega) and flow cytometry. PBMC activation was assessed by flow cytometric analysis of CD69 and CD25 positivity on CD4 and CD8 cells 24 h after treatment with UMG1-CD3 bispecific antibodies or negative control. Protein expression of phospho-NFκB-p65 (A-8, Santacruz) was assessed by Western blot analysis after 96 h of UMG1-CD3 bispecific antibody treatment with PBMCs or CCRF-CEM alone. Cytokine release was assessed after 4 h of incubation of PBMCs with Brefeldin A (Santacruz) as intracellular IFNγ and TNFα (BD Pharmingen) positivity in CD4 and CD8 cells 24 h after UMG1-CD3 bispecific antibody treatment.

[0532] Results: UMG1-CD3 bispecific treatment increased PBMC proliferation ( Fig.25 and Figures 26A-26B ). In the presence of UMG1-CD3-bispecific, upregulation of early and late T cell activation markers CD25 and CD69 on T cells was observed compared to the negative control (NC). Figures 27A-27B UMG1-CD3 bispecific-induced activation resulted in increased IFNγ and TNFα release and increased T cell proliferation ( Figures 28A-28D ). The NFκB pathway was activated by UMG1-CD3 bispecific treatment only in PBMCs but not in the CCRF-CEM cell line ( Fig.29 ). It is well known that the NFκB pathway is constitutively activated in the CCRF-CEM cell line.

[0533] 6.17.30. Example 29: In vitro efficacy of UMG1-CD3 bispecific against multiple myeloma cell lines

[0534] The efficacy of the UMG1-CD3 bispecific was evaluated on three different myeloma cell lines.

[0535] Methods: Human peripheral blood mononuclear cells (PBMCs) and multiple myeloma (MM) cell lines were used to examine the cytotoxicity of redirected T cells by flow cytometry. Two UMG1-positive MM cell lines (H929, Kms26) and one negative for the UMG1 target antigen epitope (Delta 47) were tested.

[0536] UMG1-CD3 bispecific antibody (1 μg / ml) was incubated with far red (in vitro) labeled target cells and effector cells at a PBMC E: T cell ratio of 10: 1. After 48 hours of treatment, the cell mixture was assessed by flow cytometry for loss of target cell membrane integrity, as reflected by nuclear uptake of 7AAD (BD Pharmingen).

[0537] Results: Compared with untreated cells (NC, negative control), increased killing was observed mainly in UMG1-positive cells after treatment with the UMG1-CD3 bispecific antibody. In addition, H929 and Kms26 UMG1-positive cells showed 80% to 95% cell death ( Figures 30A-30D ), while the delta47 cell line that does not express the UMG1 antigen showed 10% cell death ( Figures 30E-30F ).

[0538] 6.17.31. Example 30: In vitro efficacy of UMG1-CD3 bispecific against testicular cancer (seminoma) cell lines

[0539] The efficacy of the UMG1-CD3 bispecific was evaluated on a testicular cancer (seminoma) cell line.

[0540] Methods: Human peripheral blood mononuclear cells (PBMCs) and seminoma cell lines were used to measure the cytotoxicity of redirected T cells by flow cytometry. In particular, TCAM2 seminoma UMG1 epitope-positive cell lines were tested.

[0541] UMG1-CD3 bispecific antibody (1 μg / ml) was incubated with far-red (in vitro) labeled target cells and effector cells at a PBMC E:T cell ratio of 10:1. After 48 hours of treatment, the cell mixture was evaluated by flow cytometry for loss of target cell membrane integrity, as reflected by nuclear uptake of 7AAD (BD Pharmingen). A second experiment was performed under similar conditions, but a group of cells treated with Ah-UMG1 mAb (15 mg / kg) was added and the cell mixture was evaluated by flow cytometry for loss of target cell membrane integrity, as reflected by nuclear uptake of 7AAD (BD Pharmingen) after 24 hours of treatment.

[0542] Results: Compared with untreated cells, significant killing of TCAM2 cells was observed after 48 h of treatment with UMG1-CD3 bispecific antibody ( Figures 31A-31B Compared with ah-UMG1 monoclonal antibody, UMG1-CD3 bispecific antibody showed stronger killing effect after 24 hours of treatment ( Fig.31C ).

[0543] 6.18. Sequence

[0544] UMG1 heavy chain CDR1 [SEQ ID NO: 1]:

[0545] Gly Phe Thr Phe Ser Ser Ser Phe Gly Met His

[0546] UMG1 heavy chain CDR2 [SEQ ID NO: 2]:

[0547] Tyr Ile Ser Ser Gly Ser Gly Asn Phe Tyr Tyr Val Asp ThrVal Lvs

[0548] UMG1 heavy chain CDR3 [SEQ ID NO: 3]:

[0549] Ser Thr Tyr Tyr His Gly Ser Arg Gly Ala Met Asp Tyr UMG1 light chain CDR1 [SEQ ID NO: 4]:

[0550] Ser Ala Ser Ser Ser Va1 Ser Ser Met Tyr Trp Tyr

[0551] UMG1 light chain CDR2 [SEQ ID No: 5]:

[0552] Asp Thr Ser Lys Met Ala Ser

[0553] UMG1 light chain CDR3 [SEQ ID NO: 6]:

[0554] Gln G1n Trp Ser Ser Tyr Pro Pro Ile Thr

[0555] UMG1 VH (murine) (clone IGHV5-17*02) [SEQ ID NO: 7]:

[0556] ●

[0557] ○CDR is underlined and italicized

[0558] ○ Germline mutations are in bold

[0559] ■ V is A in germline

[0560] Humanized VH1 (clone IGHV3-48*01) [SEQ ID NO: 8]:

[0561] ●

[0562] ○CDR is underlined and italicized

[0563] ○ Mutations in the original mouse sequence are in bold

[0564] Humanized VH2 (clone IGHV3-48*01 with germline inversion) [SEQ ID NO: 9]:

[0565]

[0566] ○CDR is underlined and italicized

[0567] ○ Mutations in the original mouse sequence are in bold

[0568] Humanized VH3 (clone IGHV1-48*01 with germline inversion and conserved residues flanking CDRs) [SEQ ID NO: 10]:

[0569]

[0570] ○CDR is underlined and italicized

[0571] ○ Mutations in the original mouse sequence are in bold

[0572] Humanized VH4 (clone IGHV3-30*02 with germline inversion) [SEQ ID NO: 11]:

[0573]

[0574] ○CDR is underlined and italicized

[0575] ○ Mutations in the original mouse sequence are in bold UMG1 VL (murine, kappa) (clone IG IGKV4-55*01) [SEQ ID NO: 12]:

[0576]

[0577] ○CDR is underlined and italicized

[0578] ○ Germline mutations are in bold

[0579] ■A is V in the germline

[0580] ■L is Q in the germline

[0581] ■In the germline, I is V

[0582] ■F is Y in the germline

[0583] ■ V is I in germline

[0584] ■ V is M in germline

[0585] Humanized VL1 (clone IGKV3D-20*01) [SEQ ID NO: 13]:

[0586]

[0587] ○CDR is underlined and italicized

[0588] ○ Mutations in the original mouse sequence are in bold Humanized VL2 (IGKV3D-20*01 with germline reversal) [SEQ ID NO: 14]:

[0589]

[0590] ○CDR is underlined and italicized

[0591] ○ Mutations in the original mouse sequence are in bold

[0592] Humanized VL3 (clone IGKV6D-41*01) [SEQ ID NO: 15]:

[0593]

[0594] ○CDR is underlined and italicized

[0595] ○ Mutations in the original mouse sequence are in bold

[0596] Humanized VL4 (clone IGKV6D-41*01, partial germline reversion) [SEQ ID NO: 16]:

[0597]

[0598] ○CDR is underlined and italicized

[0599] ○ Mutations in the original mouse sequence are in bold

[0600] CD43 Clone #1 (wild-type CD43 band 400 aa) [SEQ ID NO: 17]:

[0601] ●MATLLLLLGVLVVSPDALGSTTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0602] CD43 Clone #2 (truncated CD43) (aa 31 to 400) [SEQ ID NO: 18]:

[0603] ●EPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0604] CD43 Clone #3 Truncated CD43 (aa 41 to 400) [SEQ ID NO: 19]:

[0605] ●QTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0606] CD43 clone #4 carries a short CD43 (aa 61 to 400) [SEQ ID NO: 20]:

[0607] ●QTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0608] CD43 Clone #5 Truncated CD43 (aa 91 to 400) [SEQ ID NO:21]:

[0609] EPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0610] CD43 Clone #6, deletion from aa 64 to 78 [SEQ ID NO:22]:

[0611] ●MATLLLLLGVLVVSPDALGSTTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0612] CD43 clone #7, deletion of aa 69 (O-glycosylation site of GalNac) [SEQ ID NO:23]:

[0613] ●MATLLLLLGVLVVSPDALGSTTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSALPPSSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0614] CD43 Clone #8 amino acid substitution T69N [SEQ ID NO:24]:

[0615] ●MATLLLLLGVLVVSPDALGSTTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSALPPSNSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0616] Human nucleotide CD43 full length [SEQ ID NO:25]:

[0617] ATGGCCACGC TTCTCCTTCT CCTTGGGGTG CTGGTGGTAA GCCCAGACGC TCTGGGGAGCACAACAGCAG TGCAGACACC CACCTCCGGA GAGCCTTTGG TCTCTACTAG CGAGCCCCTG

[0618] AGCTCAAAGA TGTACACCAC TTCAATAACA AGTGACCCTA AGGCCGACAG CACTGGGGACCAGACCTCAG CCCTACCTCC CTCAACTTCC ATCAATGAGG GATCCCCTCT TTGGACTTCC

[0619] ATTGGTGCCA GCACTGGTTC CCCTTTACCT GAGCCAACAA CCTACCAGGA AGTTTCCATCAAGATGTCAT CAGTGCCCCA GGAAACCCCT CATGCAACCA GTCATCCTGC TGTTCCCATA

[0620] ACAGCAAACT CTCTAGGATC CCACACCGTG ACAGGTGGAA CCATAACAAC GAACTCTCCAGAAACCTCCA GTAGGACCAG TGGAGCCCCT GTTACCACGG CAGCTAGCTC TCTGGAGACC

[0621] TCCAGAGGCA CCTCTGGACC CCCTCTTACC ATGGCAACTG TCTCTCTGGA GACTTCCAAAGGCACCTCTG GACCCCCTGT TACCATGGCA ACTGACTCTC TGGAGACCTC CACTGGGACC

[0622] ACTGGACCCC CTGTTACCAT GACAACTGGC TCTCTGGAGC CCTCCAGCGG GGCCAGTGGACCCCAGGTCT CTAGCGTAAA ACTATCTACA ATGATGTCTC CAACGACCTC CACCAACGCA

[0623] AGCACTGTGC CCTTCCGGAA CCCAGATGAG AACTCACGAG GCATGCTGCC AGTGGCTGTGCTTGTGGCCC TGCTGGCGGT CATAGTCCTC GTGGCTCTGC TCCTGCTGTG GCGCCGGCGG

[0624] CAGAAGCGGC GGACTGGGGC CCTCGTGCTG AGCAGAGGCG GCAAGCGTAA CGGGGTGGTGGACGCCTGGG CTGGGCCAGC CCAGGTCCCT GAGGAGGGGG CCGTGACAGT GACCGTGGGA

[0625] GGGTCCGGGG GCGACAAGGG CTCTGGGTTC CCCGATGGGG AGGGGTCTAG CCGTCGGCCCACGCTCACCA CTTTCTTTGG CAGACGGAAG TCTCGCCAGG GCTCCCTGGC GATGGAGGAG

[0626] CTGAAGTCTG GGTCAGGCCC CAGCCTCAAA GGGGAGGAGG AGCCACTGGT GGCCAGTGAGGATGGGGCTG TGGACGCCCC AGCTCCTGAT GAGCCCGAAG GGGGAGACGG GGCTGCCCCT

[0627] TAA

[0628] Human protein CD43 full length [SEQ ID NO:26]:

[0629] ●MATLLLLLGVLVVSPDALGSTTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLPVAVLVALLAVIVLVALLLLWRRRQKRRTGALVLSRGGKRNGVVDAWAGPAQVPEEGAVTVTVGGSGGDKGSGFPDGEGSSRRPTLTTFFGRRKSRQGSLAMEELKSGSGPSLKGEEEPLVASEDGAVDAPAPDEPEGGDGAAP

[0630] UMG1 chimeric heavy chain, nucleic acid (clone NUC 7200_evi-5 UMG.1.CH-h1.HC) [SEQ ID NO:27]:

[0631]

[0632] UMG1 chimeric light chain, nucleic acid (clone NUC 7201_evi-5 UMG.1.CH-hk.LC) [SEQ ID NO: 28]

[0633] ●GCGGCCGCCATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTCAGATCGCCCTGACCCAGAGTCCTGCAATTATGTCAGCCTCCCCGGGCGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCTGTCAGTTCAATGTACTGGTATCAGCTGAAGCCCGGCTCCTCCCCCAGGCTGCTGATCTACGACACAAGCAAAATGGCATCTGGCGTGCCCATTCGGTTCAGCGGCTCTGGAAGTGGGACTTCATTTTCCCTGACCGTGTCCAGAGTCGAGGCTGAAGATGCCGCTACATACTATTGTCAGCAGTGGTCTAGTTATCCCCCTATCACTTTCG GTGCAGGCAGCAAGCTCGAGCTGAAACGTACGGTCGCGGCGCCTTCTGTGTTCATTTTCCCCCCCATCTGATGAACAGCTGAAATCTGGCACTGCTTCTGTGGTCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCCAAAGTCCAGTGGAAAGTGGACAATGCTCTGCAGAGTGGGAATTC CCAGGAATCTGTCACTGAGCAGGACTCTAAGGATAGCACATACTCCCTGTCCTCTACTCTGACACTGAGCAAGGCTGATTACGAGAAACACAAAGTGTACGCCTGTGAAGTCACACATCAGGGGCTGTCTAGTCCTGTGACCAAATCCTTCAATAGGGGAGAGTGCTGATAGTAAAAGCTT Humanized heavy chain (VH3), nucleic acid (clone NUC 7683_evi-5 UMG.HUM3-h1.HC), nucleic acid [SEQ ID NO: 29]

[0634]

[0635] Humanized light chain (VL4), nucleic acid (clone NUC 7700_evi-5 UMG.HUM4-hk.LC) [SEQ ID NO: 30]

[0636] ●GCGGCCGCCATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTCAGGTGGTCATGACCCAGTCTCCTGCTTTCCTGTCCGTGACACCGGGCGAGAAGGTCACCATCACATGCTCCGCATCCAGCTCTGTCAGTTCAATGTACTGGTATCAGCTGAAGCCAGACCAGGCACCCAAACTGCTGATCTACGATACATCTAAAATGGCCAGTGGCGTCCCCATTAGGTTCTCGGGATCGGGGAGCGGAACTGACTTCACTTTTACCGTGTCGAGCGTCGAGGCCGAAGATGCCGCTACCTACTATTGTCAGCAGTGGTCTAGTTATCCCCCTATCACATTTG GCGGAGGGACTAAGGTGGAGATTAAGCGTACGGTCGCGGCGCCTTCTGTGTTCATTTTCCCCCCCATCTGATGAACAGCTGAAATCTGGCACTGCTTCTGTGGTCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCCAAAGTCCAGTGGAAAGTGGACAATGCTCTGCAGAGTGGGAATTC CCAGGAATCTGTCACTGAGCAGGACTCTAAGGATAGCACATACTCCCTGTCCTCTACTCTGACACTGAGCAAGGCTGATTACGAGAAACACAAAGTGTACGCCTGTGAAGTCACACATCAGGGGCTGTCTAGTCCTGTGACCAAATCCTTCAATAGGGGAGAGTGCTGATAGTAAAAGCTT Mouse heavy chain, nucleic acid (clone NUC29709_evi-5 UMG.VH-m1.HC) [SEQ ID NO:31]

[0637]

[0638] Mouse light chain, nucleic acid (clone NUC 29710_evi-5 UMG.VL-mk.LC) [SEQ ID NO:32]

[0639] ●GCGGCCGCCATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTCAGATCGCCCTGACCCAGAGTCCTGCAATTATGTCAGCCTCCCCGGGCGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCTGTCAGTTCAATGTACTGGTATCAGCTGAAGCCCGGCTCCTCCCCCAGGCTGCTGATCTACGACACAAGCAAAATGGCATCTGGCGTGCCCATTCGGTTCAGCGGCTCTGGAAGTGGGACTTCATTTTCCCTGACCGTGTCCAGAGTCGAGGCTGAAGATGCCGCTACATACTATTGTCAGCAGTGGTCTAGTTATCCCCCTATCACTT TCGGTGCAGGCAGCAAGCTCGAGCTGAAACGGGCTGACGCGGCGCCTACAGTCTCCATTTTTCCACCTAGTAGCGAACAGCTGACATCCGGGGGGGCTTCCGTCGTCTGCTTTCTGAACAACTTTTACCCCAAGGACATCAACGTGAAGTGGAAAATTGATGGCTCCGAGAGGCAGAACG GAGTCCTGAATTCTTGGACCGACCAGGATTCTAAGGACAGTACATATTCAATGTCCAGCACCCTGACACTGACTAAAGATGAGTACGAACGGCACAATAGCTATAACCTGCGAGGCAACCCATAAAACAAGCACAAGCCCAATCGTCAAATCCTTCAACCGTAATGAGTGTTGATAAGCTT

[0640] Bispecific human heavy chain, nucleic acid (NUC 32827_evi-5 UMG.VH3-h1.HC-CD3.scFv) [SEQ ID NO: 33]

[0641]

[0642] Chimeric heavy chain, (clone PRO 7200_evi-5 UMG.1.CH-h1.HC) [SEQ ID NO: 34]

[0643] ●DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0644] Chimeric light chain (clone PRO 7201_evi-5 UMG.1.CH-hk.LC) [SEQ ID NO: 35]

[0645] ●QIALTQSPAIMSASPGEKVTMTCSASSSVSSMYWYQLKPGSSPRLLIYDTSKMASGVPIRFSGSGSGTSFSLTVSRVEAEDAATYYCQQWSSYPPITFGAGSKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0646] Human heavy chain (VH3) (clone PRO 7683_evi-5 UMG.HUM3-h1.HC) [SEQ ID NO: 36]

[0647] ●EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0648] Human light chain (VL4) (clone PRO 7700_evi-5 UMG.VL4-hk.LC) [SEQ ID NO: 37]

[0649] ●QVVMTQSPAFLSVTPGEKVTITCSASSSVSSMYWYQLKPDQAPKLLIYDTSKMASGVPIRFSGSGSGTDFTFTVSSVEAEDAATYYCQQWSSYPPITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0650] Mouse heavy chain (clone PRO 29709_evi-5 UMG.VH-m1.HC) [SEQ ID NO: 38]

[0651] ●DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0652] Mouse light chain (clone PRO 29710_evi-5 UMG.VL-mk.LC) [SEQ ID NO: 39]

[0653] ●`QIALTQSPAIMSASPGEKVTMTCSASSSVSSMYWYQLKPGSSPRLLIYDTSKMASGVPIRFSGSGSGTSFSLTVSRVEAEDAATYYCQQWSSYPPITFGAGSKLEL KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0654] Bispecific human heavy chain-CD3 (clone PRO 32827_evi-5 UMG.VH3-h1.HC-CD3.scFv) [SEQ ID NO: 40]

[0655] ●EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFK DKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0656] CAR-T plasmid sequence, nucleic acid [SEQ ID NO:41]:

[0657]

[0658] ●

[0659] Recombinant human protein CD43 (aa 20 to 253) [SEQ ID NO: 42]:

[0660] ●STTAVQTPTSGEPLVSTSEPLSSKMYTTSITSDPKADSTGDQTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPHATSHPAVPITANSLGSHTVTGGTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSGPPVTMATDSLETSTGTTGPPVTMTTGSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSR

[0661] UMG1 heavy chain CDR2 - long [SEQ ID NO:43]

[0662] ●YISSGSGNFYYVDTVKG

[0663] UMG1 VH (murine) – alternative sequence [SEQ ID NO:44]

[0664] ●DVQVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSGNFYYVDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSTYYHGSRGAMDYWGQGTSVTVSS

[0665] Humanized VH1 - alternative sequence [SEQ ID NO:45]

[0666] ●EVQVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVSYISSGSGNFYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWGQGTLVTVSS

[0667] Neutral peptide linker [SEQ ID NO: 46]

[0668] ●GSGSGSG

[0669] Synthetic peptide from influenza hemagglutinin (HA) epitope [SEQ ID NO:47]

[0670] ●YPYDVPDYAG

[0671] Linear epitope of human CD43 – aa 71-78 [SEQ ID NO:48]

[0672] ●INEGSPLW

[0673] Linear epitope of human CD43 – highly basic sequence 1 [SEQ ID NO:49]

[0674] ●RRRQKR

[0675] Linear epitope of human CD43 – highly basic sequence 2 [SEQ ID NO:50]

[0676] ●RRPTLTTFFGRRK

[0677] Linear epitope of human CD43 for substitution screening [SEQ ID NO:51]

[0678] ●PPSTSINEGSPLWTS

[0679] Linear epitope of non-human CD43 [SEQ ID NO:52]

[0680] ●PPSTSVNEGSPLGTS

[0681] Linear epitope of human CD43 – aa 73-78 [SEQ ID NO:53]

[0682] ●EGSPLW

[0683] Linear peptide sequence of human CD43 - aa 66-72 [SEQ ID NO: 54]

[0684] ●PPSTSIN

[0685] Linear peptide sequence of human CD43 – aa 79-80 [SEQ ID NO:55]

[0686] ●TS

[0687] Table 8: UMG1 CDR sequences

[0688]

[0689]

[0690] 7. Incorporation by Reference

[0691] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0692] 8. Equivalent forms

[0693] Although various specific embodiments have been shown and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. Many changes will be clearly thought of by those skilled in the art after reading this specification.

Claims

1. A bispecific antibody comprising: a first antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the first antibody is an anti-CD43 antibody, According to the Chothia definition, the VH domain contains: The VH CDR1 sequence of SEQ ID NO: 62; the VH CDR2 sequence of SEQ ID NO: 63; and The VH CDR3 sequence of SEQ ID NO: 64; and wherein according to the Chothia definition, the VL domain comprises: The VL CDR1 sequence of SEQ ID NO: 65; the VL CDR2 sequence of SEQ ID NO: 66; and the VL CDR3 sequence of SEQ ID NO: 67; and A second antibody, which binds CD3, wherein the bispecific antibody comprises a heavy chain having the sequence of SEQ ID NO:

40.

2. The bispecific antibody of claim 1, wherein the first antibody comprises a human variable domain framework region.

3. The bispecific antibody of any one of claims 1-2, wherein the first antibody is IgG1 or IgG2.

4. Use of the bispecific antibody according to any one of claims 1 to 3 in the preparation of a medicament for treating a CD43-positive cancer, wherein the CD43-positive cancer is selected from the group consisting of T-ALL, diffuse large B-cell lymphoma, MALT lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma and testicular cancer.

5. The use according to claim 4, wherein the testicular cancer is seminoma.

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