WO2020254670A1 - Monoclonal antibody targeting a unique cancer-associated epitope of cd43 - Google Patents
Monoclonal antibody targeting a unique cancer-associated epitope of cd43 Download PDFInfo
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- WO2020254670A1 WO2020254670A1 PCT/EP2020/067258 EP2020067258W WO2020254670A1 WO 2020254670 A1 WO2020254670 A1 WO 2020254670A1 EP 2020067258 W EP2020067258 W EP 2020067258W WO 2020254670 A1 WO2020254670 A1 WO 2020254670A1
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Definitions
- a hybridoma secreting mAh UMG1 was deposited under the terms of the Budapest Treaty on August 4, 2016 at the Centro di Biotecnologie Avanzate (CBA) Interlab Cell Line Collection (ICLC) under ICLC accession number ICLC PD n° 16001.
- CBA Centro di Biotecnologie Avanzate
- ICLC Interlab Cell Line Collection
- CD43 is a leukocyte marker normally restricted to cells of the hematopoietic lineage. CD43 is widely expressed on most peripheral and bone marrow-derived cell components.
- CD43 migrates with an apparent molecular weight of 54 kD. In its mature form, CD43 is heavily glycosylated, having a molecular weight between 115 and 200 kD. CD4 + thymocytes and monocytes express the 115 kD form, while activated CD4 + and CD8 + T cells, B cells, neutrophils and platelets express a 130 kD form. CD43 is involved in multiple functions, such as cell adhesion, apoptosis and migration (Ostberg et al .,
- a murine anti-human CD43 monoclonal antibody, UNI was first described 25 years ago. Originally selected for high reactivity against human immature thymocytes (Tassone et al. , Tissue Antigens 44:73-82, 1994), the UNI mAh 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.
- UMG1 monoclonal antibody
- the UMG1 antibody binds to a small subset of lymphocytes in peripheral blood mononuclear cells (PBMCs) from healthy human donors (Example 1).
- PBMCs peripheral blood mononuclear cells
- the UMG1 positive lymphocytes are mostly CD45 + CD3 + CD4 + CD8 CD127 + CCR7 + T lymphocytes (Example 2).
- the UMG1 antibody binds to T-ALL cell lines mostly belonging to EGIL T3 classification (Example 3). Unlike the UNI antibody, however, the UMG1 antibody does not bind to breast cancer cells (Example 3). In the first test, UMG1 antibody did not show any binding to cancer cells in lung cancer, colorectal cancer, and breast cancer tumors (Example 5), in contrast to prior observations with UNI ( see de Laurentiis et al., Molecular & Cellular Proteomics 10: 1-12, 2011, FIG. 9). UMG1 does, however, bind to cellular immune infiltrates in a variety of tumors, including lung cancer, colorectal cancer, and breast cancer tumors (Example 5).
- UMG1 does not bind to myeloid-derived cells in PBMCs from healthy donors (Example 1), the UMG1 epitope is expressed in tumor- associated macrophages, and UMG1 epitope expression is elevated when macrophages are co-cultured and interact with cancer cells (Example 6).
- UMG1 also binds to some B-cell derived malignancies, including Waldenstrom’s macroglobulinemia cell lines (Example 3).
- UMG1 binds to a small group of neutrophils from healthy donors (Example 9).
- UMG1 does not bind to activated T lymphocytes from healthy donors (Example 10).
- Tissue microarray of human healthy tissue demonstrate a peculiar distribution of UMG1 mAh binding, limited mostly to thymus (mostly on cortical thymocytes) and rare scattered immune infiltrate in organs such as lymph nodes, gut, and lung (Example 11).
- Tissue microarray data show that in additional to lymphomas, the UMG1 epitope is also expressed in melanoma and testicular cancer of various origin (Example 11). Expanded screening on multiple tissue microarrays also binding of UMG1 antibody to neoplastic cells and immune infiltrates are pediatric tumor of different origins (Example 11).
- Chimeric antibodies constructed by fusing the variable regions of the UMG1 murine antibody to human IgG Fc regions were capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against the T-ALL cell line HPB-ALL and T lymphoma cell line H9 in the presence of effector cells from human PBMCs (Example 17).
- ch-UMGl antibodies were also capable of inducing ADCC against Waldenstrom’s Macroglobulinemia cells (Example 18).
- Humanized antibodies constructed by grafting the CDRs from the UMG1 heavy and light chains into human frameworks (h-UMGl) were able to reduce growth of HPB-ALL xenografts in an NSG mouse model (Example 21).
- third generation chimeric antigen receptor (CAR) T cells in which the CAR targeting moiety is a scFv having all 6 CDRs of the UMG1 antibody were activated in the presence of H9 T lymphoma cells (Example 22), predicting that UMG1 -directed CAR-T therapy will be effective in treating T cell lymphoma.
- the UMG1-CD3 bispecific antibody was capable of binding to UMG1 + or CD3 + positive cells and redirecting T-cell cytotoxicity to target cancer cells (Examples 23 to 30).
- lymphomas such as non-Hodgkin lymphomas derived from peripheral B cell or peripheral T cell, 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 aberrantly expressing the epitope such as pediatric cancers or in which depletion of tumor-associated macrophages would prove therapeutically beneficial.
- 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 the 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 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, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma,
- hepatoblastoma medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.
- the anti-CD43 antibody or antigen-binding fragment binds amino acids 71-78 of wild-type CD43. In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds amino acids 73-78 of wild-type CD43.
- the anti-CD43 antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain
- VH domain comprises: the VH CDR1 sequence of SEQ ID NO: 1; the VH CDR2 sequence of SEQ ID NO: 43; and the VH CDR3 sequence of SEQ ID NO: 3
- the VL domain comprises: the VL CDR1 sequence of SEQ ID NO: 4; the VL CDR2 sequence of SEQ ID NO: 5; and the VL CDR3 sequence of SEQ ID NO: 6.
- the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 12.
- the anti-CD43 antibody is a murine antibody produced by the hybridoma cell line deposited under ICLC accession number ICLC PD number 16001 (UMG1).
- the anti-CD43 antibody is a chimeric antibody further comprising human constant region domains.
- the human constant region domains are IgG domains.
- the antibody heavy chain sequence is SEQ ID NO: 34 and the antibody light chain sequence is SEQ ID NO: 35.
- the anti-CD43 antibody or antigen-binding fragment comprises human variable domain framework regions.
- the VH domain has a sequence selected from: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:
- VL domain has a sequence selected from: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
- the anti-CD43 antibody is a monoclonal antibody.
- the anti-CD43 antibody or antigen-binding fragment is an F(ab), an F(ab)’2, an scFv, a diabody, a single domain antibody, a tandab, or a flexibody.
- the anti-CD43 antibody or antigen-binding fragment is capable of inducing antibody dependent cellular cytotoxicity (ADCC) in the presence of an effector cell. In some embodiments, the anti-CD43 antibody or antigen-binding fragment is capable of depleting tumor-associated macrophages (TAMs).
- ADCC antibody dependent cellular cytotoxicity
- TAMs tumor-associated macrophages
- the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug.
- the patient has diffuse large B cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, or mantle cell lymphoma.
- the patient has multiple myeloma.
- the patient has melanoma.
- the patient has testicular cancer.
- the testicular cancer is selected from the group consisting of: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma.
- the patient has nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, or leiomyosarcoma.
- a bispecific antibody for use in a method of treating a CD43 positive cancer, comprising: administering a therapeutically effective amount of the 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’s lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma,
- the bispecific antibody has a second binding specificity for CD3.
- a CAR-T cell for use in a method of treating a CD43 positive cancer, comprising: administering a therapeutically effective amount of the CAR-T cell to a patient having a CD43 positive cancer, wherein the CAR-T cell binds 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, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, epen
- an anti-CD43 antibody or antigen-binding fragment thereof for use in a method of identifying a CD43 positive cancer, comprising: detectably contacting a sample comprising a CD43 positive cancer cell with the anti-CD43 antibody or antigen-binding fragment, wherein the anti-CD43 antibody or antigen-binding fragment binds 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, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plex
- an anti-CD43 antibody or antigen-binding fragment thereof for use in a method of diagnosing and treating a CD43 positive cancer, comprising: detectably contacting a sample from a patient with the anti-CD43 antibody or antigen-binding fragment, diagnosing the patient with a CD43 positive cancer if a 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 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, test
- provided herein is a method of treating a CD43 positive cancer.
- the method comprises: administering a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment thereof to a patient having a CD43 positive cancer, wherein the anti-CD43 antibody or antigen-binding fragment binds 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, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, al
- the anti-CD43 antibody or antigen-binding fragment binds amino acids 71-78 of wild-type CD43. In some embodiments, the anti-CD43 antibody or antigen-binding fragment binds amino acids 73-78 of wild-type CD43.
- the anti-CD43 antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain
- VH domain comprises: the VH CDR1 sequence of SEQ ID NO: 1; the VH CDR2 sequence of SEQ ID NO: 43; and the VH CDR3 sequence of SEQ ID NO: 3
- the VL domain comprises: the VL CDR1 sequence of SEQ ID NO: 4; the VL CDR2 sequence of SEQ ID NO: 5; and the VL CDR3 sequence of SEQ ID NO: 6.
- the VH sequence is SEQ ID NO: 7 and the VL sequence is SEQ ID NO: 12.
- the anti-CD43 antibody is a murine antibody produced by the hybridoma cell line deposited under ICLC accession number ICLC PD number 16001 (UMG1).
- the anti-CD43 antibody is a chimeric antibody further comprising human constant region domains.
- the human constant region domains are IgG domains.
- the antibody heavy chain sequence is SEQ ID NO: 34 and the antibody light chain sequence is SEQ ID NO: 35.
- the anti-CD43 antibody or antigen-binding fragment comprises human variable domain framework regions.
- the VH domain has a sequence selected from: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:
- VL domain has a sequence selected from: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
- the anti-CD43 antibody is a monoclonal antibody.
- the anti-CD43 antibody or antigen-binding fragment is an F(ab), an F(ab)’2, an scFv, a diabody, a single domain antibody, a tandab, or a flexibody.
- the anti-CD43 antibody or antigen-binding fragment is capable of inducing antibody dependent cellular cytotoxicity (ADCC) in the presence of an effector cell. In some embodiments, the anti-CD43 antibody or antigen-binding fragment is capable of depleting tumor-associated macrophages (TAMs).
- ADCC antibody dependent cellular cytotoxicity
- TAMs tumor-associated macrophages
- the anti-CD43 antibody or antigen-binding fragment is conjugated to a toxic drug.
- the patient has diffuse large B cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, or mantle cell lymphoma. [0043] In some embodiments, the patient has multiple myeloma.
- the patient has melanoma.
- the patient has testicular cancer.
- the testicular cancer is selected from the group consisting of: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma.
- the patient has nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, or leiomyosarcoma.
- 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’s lymphoma, anaplastic large cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, melanoma, testicular cancer, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma,
- the bispecific antibody has a second binding specificity for CD3.
- a method of treating a CD43 positive cancer comprising: administering a therapeutically effective amount of a CAR-T cell to a patient having a CD43 positive cancer, wherein the CAR-T cell binds 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, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neurode
- a method of identifying a CD43 positive cancer comprising: detectably contacting a sample comprising a CD43 positive cancer cell with an anti-CD43 antibody or antigen-binding fragment thereof, wherein the anti-CD43 antibody or antigen-binding fragment binds 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, nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma,
- hepatoblastoma medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.
- a method of diagnosing and treating a CD43 positive cancer comprising: detectably contacting a sample from a patient with the anti-CD43 antibody or antigen-binding fragment, diagnosing the patient with a CD43 positive cancer if a binding to the anti-CD43 antibody or antigen-binding fragment is detected, and administering a therapeutically effective amount of an anti-CD43 antibody or antigen-binding fragment to the patient thereof, wherein the anti-CD43 antibody or antigen binding fragment binds 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, nephroblastoma, neuroblasto
- hepatoblastoma medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, and leiomyosarcoma.
- FIG. 1A and IB demonstrate expression of the epitope recognized by the UMG1 antibody on peripheral blood mononuclear cells of a panel of healthy donors and comparison to a commercial CD43 antibody.
- the scatterplot of FIG. 1A presents data obtained by flow cytometry.
- the x-axis presents the forward scatter detected (FSC)
- the y-axis depicts the side scatter (SSC). Each dot corresponds to one cell.
- the histogram in FIG. IB depicts on the x- axis the phycoerythrin signal intensity.
- the y-axis relates the signal intensities to the maximum signal intensity (i.e. 100%) of the unstained sample.
- the red curve represents the unstained control
- the blue curve represents the scramble IgGl stained cells (i.e. the negative control)
- the orange curve represents the mAb UMG1 stained cells
- the green curve represents the commercial anti-CD43 antibody stained cells.
- FIGS. 2A-2D show four representative scatterplots of cell populations recognized by the UMG1 antibody produced by the hybridoma cell deposited according to the invention.
- FIGS. 2A and 2C show two scatterplots belonging to lymphocytes.
- FIG. 2B and 2D are from lymphocytes detected by the UMG1 antibody produced by the hybridoma cell deposited according to the invention.
- the x-axis represents CD4 signal intensity
- the y-axis depicts CD8 signal intensity.
- the x-axis represents CD45ro signal intensity
- the y-axis represents CCR7 signal intensity.
- FIG. 3A presents UMG1 expression detected by UMG1 antibody on BCWM.1 cell line.
- FIG. 3B presents UMG1 expression on MWCL.1 cell line.
- the unfilled curve represents the unstained control
- the curve filled with horizontal stripes represents the secondary mAb stained cells
- the curve filled with vertical stripes represents the scramble IgG plus secondary mAb stained cells
- the curve filled with diagonal stripes represents cells stained by mAb UMG1.
- FIG. 4 shows tumor associated macrophages (TAM) recognized by the UMG1 antibody.
- TAM tumor associated macrophages
- FIG. 5A show THP1 -derived macrophages.
- FIG. 5A show THP1 -derived macrophages stained with: control IgGl in absence of tumor cells (first row), ch- UMG1 (chimeric antibody according to aspect 2 of this invention, where the original murine Fc region was replaced with a fully human IgGl Fc region) in absence of tumor cells (second row) and ch-UMGl in presence of PANC1 pancreatic cancer cell line (third row and shown in greater detail in FIG. 5B).
- the first column represents the DAPI staining
- the second column the antibody plus Alexa-Fluor 488 labeled secondary antibody
- the third column represents the superimposed image.
- Figures 6A-6B are bar graphs showing the results of the degranulation assay to evaluate Antibody-Dependent Cell mediated Cytotoxicity (ADCC) in HPB-ALL (FIG. 6A) and H9 cell lines (FIG. 6B).
- ADCC Antibody-Dependent Cell mediated Cytotoxicity
- the numbers on the x-axis represent the different samples tested: no target is indicated by (1), effector plus target cells (E+T) (2), Negative control (NC) 200 pg/ml (3), ch-UMGl 10 pg/ml (4), ch-UMGl 50 pg/ml (5), ch-UMGl 100 pg/ml (6), ch-UMGl 200 pg/ml (7), Positive control (PC) 200 pg/ml (8).
- the y-axis represents the percentage of CD107a + NK cells affected by ADCC related to the whole number of CD107a + NK cells tested per sample.
- Figure 7 is a bar graph showing the results of the degranulation assay to evaluate Antibody-Dependent Cell mediated Cytotoxicity (ADCC) in BCWM.1 cell line.
- the numbers on x-axis represent the different samples.
- the numbers on the x-axis represent the different samples tested: no target is indicated by (1), effector plus target cells (E+T) (2), Negative control (NC) 200 pg/ml (3), ch-UMGl 10 pg/ml (4), ch-UMGl 50 pg/ml (5), ch-UMGl 100 pg/ml (6), ch-UMGl 200 pg/ml (7), Positive control (PC) 200 pg/ml (8).
- the y-axis represents the percentage of CD107a + NK cells affected by ADCC related to the whole number of CD107a + NK cells tested per sample.
- Figure 8 is a bar graph illustrating that the CD3 + expressing lymphocytes (CAR-T) were able to release significantly higher amount of Interferon gamma (IFNy) in the presence of H9 cells.
- the y-axis shows the concentration of IFNy expressed in ng/ml.
- the numbers indicated represent the different cells tested: (1) indicates non-transduced T cells (negative control); (2) indicates, T cells transduced with a control CAR (vehicle control); and (3) indicated T cells transduced with CAR-UMGl .
- Figure 9 is a bar graph illustrating that the CAR-T were able to release significantly higher amount of Interleukin 2 (IL-2) in the presence of H9 cells.
- the y-axis represents the concentration of IL2 expressed in ng/ml.
- the numbers indicated represent the different cells tested: (1) indicates non-transduced T cells (negative control); (2) indicates, T cells transduced with a control CAR (vehicle control); and (3) indicated T cells transduced with CAR-UMG1.
- Figure 10 is a bar graph showing that the CAR-T were able to induce selective killing of H9 cells.
- the y-axis reports the dead/live cells 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 CAR-UMGl, also referred to as UMGl-CAR (4).
- Figure 11 is a line graph representing the tumor volume curves of an in vivo experiment comparing a control IgGl (Rituximab) versus the humanized version of UMG1- mAb (h-UMGl) and the afucosylated version of UMGl-mAb (a-h-UMGl).
- h- UMG1 is indicated with a line with squares
- a-h-UMGl is indicated with (a line with triangles)
- the control IgGl is indicated with a line with circles.
- Figures 12A and 12B show representative flow cytometry results of direct staining of h-UMGl -PE and three commercially available CD43 antibodies.
- FIG. 12A shows staining in the ALL-SIL human cell line.
- FIG. 12B shows staining in the KE-37 cell line.
- FIG. 13A shows competitive binding assays.
- FIG. 13A shows
- FIG. 13B shows representative results from a competitive binding assay between h-UMGl, h-UMGl-PE, and three commerically available CD43 antibodies on the HPB-ALL cell line.
- Figures 14A-14C show representative images of m-UMGl staining in the
- FIG.14A shows m-UMGl staining in colorectal adenocarcinoma.
- FIG. 14B shows m-UMGl staining in lung cancer adenocarcinoma.
- FIG. 14C shows m-UMGl staining in breast cancer.
- FIG. 15A-15F show representative results from Example 12.
- FIG. 15A shows the amino acid sequence of full-length CD43 (SEQ ID NO: 17).
- FIG. 15B is an illustration depicting CD43 protein variants used to transfect the HEK293T cells.
- FIGs. 15C and 15E show the western blot results on the protein lysates of transfected HEK293T cells.
- FIGs. 15D and 15F are bar graphs showing FACS results on transfected HEK293T cells.
- Figure 16 shows screening of the h-UMGl antibodies for their affinity to the antigent on HPB-ALL and H9 cell lines, which are known to be positive for the UMG1 epitope.
- FIGS 17A-17B show comparative flow cytometric profiles of h-UMGl and UNI in four different cell lines of the hematopoietic lineage.
- FIG. 17A shows the reported UNI flow cytometric profiles in cell lines of the hematopoietic lineage as provided by (Tassone et al. , Tissue Antigens 44:73-82, 1994).
- FIG. 17B shows UMG1 flow cytometric profiles in cell lines of the hematopoietic lineage as provided by Example 8.
- Figures 18A-18B show representative FACS images of treatment with the UMG1- CD3 bispecific antibody to conduct T-cell cytotoxicity assays on cell lines ALL-SIL (FIG. 18B) and KE-37 (FIG. 18A), as provided by Example 23.
- Figure 19 shows evaluation of the binding kinetics of h-UMGl mAh to recombinant human CD43 analyte (aa 20-253, SEQ ID NO: 42) expressed in E. coli vector, an
- Figure 20 depicts the plasmid map for the construct used to make various
- Figures 21A-21B show the efficacy of a-h-UMGl mAh (afucosylated h-UMGl) and UMG1-CD3 bispecific antibody on T-ALL cells, with FIG. 21A showing the efficacy of a-h- UMGl mAh and UMG1-CD3 bispecific antibody on the CEM cell line and FIG. 21B showing the efficacy of a-h-UMGl mAh and UMG1-CD3 bispecific antibody on T-ALL primary blasts.
- FIGS 22A-22C show the efficacy of UMG1-CD3 bispecific antibody on T-ALL cell lines at different concentrations, with FIG. 22A showing the efficacy of UMG1-CD3 bispecific antibody on the CEM cell line, FIG. 22B showing the efficacy of UMG1-CD3 bispecific antibody on the KE37 cell line, and FIG. 22C showing the efficacy of UMG1-CD3 bispecific antibody on the ALL-SIL cell line.
- Figure 23 shows the apoptosis induction by different doses of UMG1-CD3 bispecific compared to a non-treated control (NC).
- Figure 24 shows the role of the CD8 + and CD4 + T-cells in inducing a response to UMG1-CD3 bispecific treatment.
- Figure 25 shows the proliferation of PBMCs in the absence or in the presence of increasing concentrations of UMG1-CD3 bispecific antibody.
- Figures 26A-26B show the proliferation of PBMCs in the absence or in the presence of UMG1-CD3 bispecific antibody, with FIG. 26A showing the proliferation of PBMCs in the absence of UMG1-CD3 bispecific antibody and FIG. 26B showing the proliferation of PBMCs in the presence of UMG1-CD3 bispecific antibody.
- FIG. 27A-27B show the expression of T cell activation markers in the absence or in the presence of increasing concentrations of UMGl-CD3-bispecific antibody, with FIG.
- FIG. 27 A showing the percentage of CD69 positive cells and FIG. 27B showing the percentage of CD25 positive cells.
- FIGS 28A-28D show the induction of IFNy and TNFa in CD4 + and CD8 + T cells, with FIG. 28A showing the induction of IFNY in CD4 + T cells, FIG. 28B showing the induction of IFNY in CD8 + T cells, FIG. 28C showing the induction of TNFa in CD4 + T cells, and FIG. 28D showing the induction of TNFa in CD8 + T cells.
- Figure 29 shows the effect of UMG1-CD3 bispecific treatment on NFKB protein expression in PBMCs and CCRF-CEM cell line respectively.
- FIGS 30A-30F show the in vitro efficacy of UMG1-CD3 bispecific on multiple myeloma cell lines, with FIG. 30A showing the epression of UMG1 epitope on Delta 47 cell line, FIG. 30B showing the in vitro efficacy of UMG1-CD3 bispecific on Delta 47 cell line, FIG. 30C showing the epression of UMG1 epitope on H929 cell line, FIG. 30D showing the in vitro efficacy of UMG1-CD3 bispecific on H929 cell line, FIG. 30E showing the epression of UMG1 epitope on KMS26 cell line, and FIG. 30C showing the in vitro efficacy of UMG1-CD3 bispecific on KMS26 cell line.
- FIG. 31A-31C show the in vitro efficacy of UMG1-CD3 bispecific on a testicular cancer (seminoma) cell line, TCAM2, with FIG. 31A showing the expression of the UMG1 epitope on the TCAM2 cell line, FIG. 31B the in vitro efficacy of UMG1-CD3 bispecific on the TCAM2 cell line compared to the negative control (NC), and FIG. 31C showing the in vitro efficacy of UMG1-CD3 bispecific on the TCAM2 cell line compared to the negative control (NC) and the a-h-UMGl monoclonal antibody.
- Figure 32 shows the binding activity of UMG1-CD3 bispecific at different concentrations on CEM, Jurkat, and KE37 cell lines.
- Figure 33 shows the binding of h-UMGl mAh on inactivated and activated neutrophils.
- FIGS 34A-34D show the FACS results of IgG isotype control and h-UMGl mAh binding on activated T cells, with FIG. 34A showing the binding of IgG isotype control on CD25 positive cells, FIG. 34B showing the binding of IgG isotype control on CD69 positive cells, FIG. 34C showing the binding of h-UMGl mAh on CD25 positive cells, and FIG. 34D showing the binding of h-UMGl mAh on CD69 positive cells.
- Figure 35 shows the results of the epitope mapping of the binding of h-UMGl mAh (humanized anti-CD43 mAh) on human CD43 peptide microarray.
- Figure 36 shows the results of the epitope mapping of the binding of h-UMGl mAh (humanized anti-CD43 mAh) on PPSTSINEGSPLWTS (SEQ ID NO: 51) peptide microarray.
- Figures 37A-C show the heat map, substitution matrix and amino acid plot representing the conserved amino acids for the h-UMGl mAh binding, with FIG. 37A showing the heat map, FIG. 37B showing the substitution matrix, and FIG. 37C showing the amino acid plot.
- FIG. 38A-C show UMG1 mAh binding on human tissues, with FIG. 38A showing the binding of UMG1 on thymocytes with a characteristic increased presence of positive cells in the cortical thymus (inlet), FIG. 38B showing the UMG1 mAh membrane and cytoplasmic binding on a human tonsil, and FIG. 38C showing the cytoplasmic binding observed on intra-tissue macrophages in the lung.
- Figures 39A-B show UMG1 mAh binding on diffuse large B-cell lymphomas and T- lymphomas, with FIG. 39A showing UMG1 binding on diffuse large B-cell lymphomas and FIG. 39B showing UMG1 binding on T-cell lymphomas. Representative images from Ly2084 tissue microarray.
- Figures 40A-B show UMG1 mAb binding on melanoma and seminoma tissues, with FIG. 40A showing UMG1 binding on the melanoma tissue (ME2081) and FIG. 40B
- Monoclonal antibody“UMG1” is a murine anti-human CD43 antibody produced by the hybridoma cell line deposited under ICLC accession number ICLC PD n° 16001.
- the term“antibody” has its broadest art- recognized meaning and includes all known formats, including, without limitation: 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 flexibodies.
- the terms "treat” or “treatment” are used in their broadest accepted clinical sense. The terms include, without limitation, lessening a sign or symptom of disease; improving a sign or symptom of disease; alleviation of symptoms; diminishment of extent of disease; stabilization (i.e., not worsening) of the state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; remission (whether partial or total), whether detectable or undetectable; cure; prolonging survival as compared to expected survival if not receiving treatment. Unless explicitly stated otherwise,“treat” or“treatment” do not intend prophylaxis or prevention of disease.
- subject or “individual” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
- Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
- patient intends a human“subject.”
- the term“sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.
- the term“therapeutically effective amount” is an amount that is effective to treat a disease.
- A“prophylactically effective amount” is an amount that is effective to slow onset of or prevent a disease.
- Ranges provided herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints.
- a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2,
- the current disclosure provides novel humanized and murine CD43 antibodies and binding molecules derived therefrom that have different expression and binding properties in comparison to the properties of other previously disclosed and commercially available CD43 antibodies.
- the invention relates to a monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.
- the hybridoma cell was deposited under the terms of the Budapest Treaty at Centro Biotecnologie Avanzate (CBA), Interlab Cell Line Collection (ICLC), Largo Rosanna, 10, 16132 Genova, Italy under accession number ICLC PD n° 16001 on August 4, 2016.
- the antibody was tested in the examples given below. As shown in the examples, the antibody binds to a specific epitope on CD43 in a portion of the protein that could be
- the invention further relates to an antibody, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences GFTFSSFGMH (SEQ ID NO: 1),
- the antibody comprises all three heavy chain Complementary Determining Regions (CDRs) and all three light chain CDRs from: the antibody
- the CDR sequences mentioned above are the CDR sequences from the monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001, as determined by sequencing.
- CDR complementary metal-oxide-semiconductor determining region
- CDR complementarity determining region
- the term "CDR" or "complementarity determining region” means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Rabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Rabat 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 the definitions include overlapping or subsets of amino acid residues when compared against each other.
- CDR is a CDR as defined by Rabat, based on sequence comparisons.
- CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRLl, CDRL2 and CDRL3 denote the light chain CDRs.
- This monoclonal antibody may have framework sequences from any species.
- it may have a mouse or human framework.
- framework (FR) amino acid residues refers to those amino acids in the framework region of an immunoglobulin chain.
- framework region or "FR region” as used herein, includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Rabat definition of CDRs).
- Methods for producing a monoclonal antibody with the CDR sequences as mentioned above are known in the art and include the introduction of the nucleic acid sequences encoding the CDRs into suitable expression vectors encoding the desired framework sequences. Further methods are described below.
- the invention relates to an antibody which recognizes the same epitope as the antibody according to the first aspect.
- an antibody is a protein belonging to the protein family of immunoglobulins and is composed in its variable regions of framework regions and complementarity determining regions as defined above. Naturally, antibodies are produced by plasma cells in response to a certain antigen.
- each antibody has two identical heavy chain immunoglobulins and two identical light chain immunoglobulins. Each heavy and each light chain may have a variable and a constant region.
- the constant region of a heavy chain may be one of five types of mammalian Ig heavy chains: a, d, e, g and m.
- the type of the heavy chain present usually defines the class (isotype) of the antibody: IgA, IgD, IgE, IgG and IgM antibodies, respectively.
- the constant region of a light chain may be one of two types of mammalian Ig light chains: k and l.
- the variable regions of heavy and light chains are usually made of a unique combination of numerous protein sequences allowing the binding to a particular antigen.
- the term“antibody” also covers an isolated antibody.
- each heavy chain is connected to one of the light chains, whereby the variable regions of a heavy and a light chain combine to form one of the two identical antigen-binding sites and their constant regions combine to form the constant region of the antibody.
- both constructs of one heavy and one light chain may be connected via the constant regions of their heavy chains, forming a“Y”-shaped molecule, whereby the two arms depict the antigen-binding variable region and the stem depicts the constant region.
- the antibody according to the second aspect may be a complete antibody, meaning that it usually comprises a heavy chain of three or four constant domains and a light chain of one constant domain as well as the respective variable domains, whereby each domain may comprise further modifications, such as mutations, deletions or insertions, which do not change the overall domain structure.
- the antibody according to the second aspect of the present invention may form a homo- or heterodimer or a homo- or heteromultimer, whereby“dimer” and “multimer” means that two and at least three antibodies, respectively, may combine to form a complex.
- the prefix“homo” means that a complex may be formed of identical antibody molecules, whereby the prefix“hetero” means that a complex may be formed of different antibody molecules.
- the term“antibody” is intended to comprise all above-mentioned immunoglobulin isotypes, i.e. the antibody may be an IgA, IgD, IgE, IgG or IgM antibody, including any subclass of these isotypes.
- the antibody is an IgG antibody.
- the antibody may also comprise two different constant regions of heavy chains, e.g. one IgGl and one IgG2 heavy chain, or heavy chains from different species. However, the heavy chains preferably are from the same species.
- the antibody may comprise either a lambda or a kappa light chain.
- the antibody which recognizes the same epitope as one of the antibodies of the first aspect of the invention my further be an antibody, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 have the amino acid sequences GFTFSSFGMH (SEQ ID NO: 1), YISSGSGNFYYVDTVKG (SEQ ID NO: 43), STYYHGSRGAMDY (SEQ ID NO: 3), SASSSVSSMYWY (SEQ ID NO: 4),
- the antibody which recognizes the same epitope as one of the antibodies of the first aspect of the invention may be an antibody wherein the CDRs, in comparison to the sequences mentioned above has at least one conservative amino acid exchange, e.g., a similar amino acid with similar chemical structure and properties and/or function as the original amino acid.
- the antibody which recognizes the same epitope as one of the antibodies of the first aspect of the invention may also be an antibody which has an increased or lowered affinity or specificity in comparison to one of the antibodies of the first aspect of the invention.
- Such antibodies are readily obtained by methods known in the art and further described herein below.
- the antibody according to the second aspect of the invention may have a sequence, especially in its variable regions, that 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 that of monoclonal mouse antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.
- the mouse antibody comprises a variable heavy chain to an amino acid sequence having 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 DVQLVESGGGLVQPGGSRKLSCVASGFTFSSFGMHWVRQAPEKGLEW
- the mouse antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO:
- the mouse antibody comprises an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:
- the mouse antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:
- the mouse antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the mouse antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:
- the mouse antibody comprises an amino acid sequence having 99% or greater 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%,
- the mouse antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 7.
- the mouse antibody comprises a variable light chain to an amino acid sequence having 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 QIALTQ SP AIMS ASPGEK VTMTC S AS S S VS SMYW YQLKPGS SPRLLIYDT SKMASGVP IRF SGSGSGT SF SLT V SRVEAED AAT Y Y CQQ W S S YPPITF GAGSKLELK (SEQ ID NO: 12).
- the mouse antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 12.
- the mouse antibody comprises an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 12. In some embodiments, the mouse antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 12.
- 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.
- the mouse antibody comprises a variable heavy chain to an amino acid sequence having 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
- the antibody according to the invention may be a monoclonal, a bispecific, or a multispecific antibody. Such antibodies are known in the art.
- the term“monoclonal” may be understood in the broadest sense describing antibodies produced by a single clone of B lymphocytes or antibodies having the same or a similar amino acid sequence.
- the term“bispecific”, as used herein, may be understood in the broadest sense describing antibodies interacting with two different epitopes.
- the bispecific antibody may be derived from two monoclonal antibodies.
- these two different epitopes may be localized on the same antigen, but they may also be localized on two different antigens.
- the term“multispecific”, as used herein, may be understood in the broadest sense describing antibodies interacting with three or more different types of epitopes.
- these epitopes may be localized on the same antigen or on two or more antigens.
- the antibody according to aspect two of the present invention is a monoclonal antibody.
- the antibody according to aspect two of the present invention preferably is a bispecific or a multispecific antibody.
- antibodies are produced by making hybridoma cells.
- Methods for the production of hybridoma cells as well as methods for the production of antibodies with the help of hybridoma cells are well-known to the person skilled in the art.
- mice are injected with the desired antigen and killed after a few days in order to isolate the spleen cells secreting the antibody against the desired antigen.
- fusion of these antibody- secreting spleen cells with immortal non-secreting myeloma cells results to hybridoma cells.
- These hybridoma cells are then usually screened and the hybridoma producing the desired antibody is selected.
- the selected hybridoma may then be cultured in vivo or in vitro and the desired antibody can be isolated.
- bifunctional, or bispecific, antibodies may have antigen binding sites of different specificities.
- Various forms of bispecific antibodies and their production are known to the person skilled in the art.
- these include BSIgG, which are IgG molecules comprising two distinct heavy chains and two distinct light chains that are secreted by so- called “hybrid hybridomas”, and heteroantibody conjugates produced by the chemical conjugation 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 by reference in its entirety).
- Bispecific antibodies may be generated to deliver cells, cytotoxins, or drugs to specific sites.
- An important use may be to deliver host cytotoxic cells, such as NK or cytotoxic T cells, to specific cellular targets.
- Another important use may be the delivery of cytotoxic proteins to specific cellular targets (V. Raso, T. Griffin, Cancer Res. 1981, 41 :2073; S. Honda et al., Cytotechnology, 1990, 4:59 each of which is incorporated by reference in its entirety).
- a further important use may be to deliver anti-cancer non-protein drugs to specific cellular targets (J. Corvalan et al., Inti. J. Cancer Suppl. 1988, 2:22; M.
- bispecific antibodies may be prepared by chemical cross-linking (M.
- Quadromas may be constructed by fusing hybridomas that secrete two different types of antibodies against two different antigens (Milstein and Cuello, Nature, 1983, 305: 537-539; which is incorporated by reference in its entirety).
- epitope may be understood in the broadest sense as a portion of a CD43 molecule capable of being recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 at one or more of the antibody’s antigen binding regions.
- the part of an antibody that binds to the epitope is called a paratope.
- epitopes have conformational properties that specifically generate binding sites for the paratope.
- Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three dimensional structural characteristics as well as specific charge characteristics.
- the interaction between the epitope and the antibody may generally be based on the primary structure of the antigen, i.e. a continuous sequence of amino acids. Usually, the interaction may also be based on the secondary structure, the tertiary structure or the quaternary structure of the epitope as well as post-translational modifications, such as glycosylation.
- the interaction between the epitope and the antibody may further be based on the three-dimensional structure and resulting surface features of the antigen, which may involve a discontinuous section of the amino acid sequence comprising amino acids at distant locations into the interaction with the antibody.
- an antibody recognizes "the same epitope" as the antibody according the first aspect, when the two antibodies recognize identical or sterically overlapping epitopes.
- competition assays which usually may be configured in all number of different formats, using either labeled antigen or labeled antibody.
- the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.
- An antibody that recognizes“the same epitope” as the antibody according to the first aspect usually refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody usually blocks binding of the antibody to its antigen in a competition assay by 50% or more.
- the epitope recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 may be identified by any suitable epitope mapping method known in the art in combination with the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.
- Examples of such a method include screening peptides of varying lengths derived from CD43 for binding to the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001, whereby the smallest fragment that can specifically bind to the antibody usually contains the sequence of the epitope recognized by the antibody.
- CD43 peptides may be produced synthetically or by proteolytic digestion of CD43.
- Methods for the identification of peptides binding to the antibody are well-known to the person skilled in the art, such as mass spectrometric analysis.
- NMR spectroscopy can be used to identify residues which interact with an antibody of the present invention.
- a CD43 peptide that has been uniformly 15N and 2H labelled can be mixed with an unlabelled antibody and those amino acids in the labelled peptide that interact with the unlabelled antibody can be detected as their position within the NMR spectra change.
- the difference between the two spectra enables the identification of the amino acids in CD43 that are involved in the interaction with the antibody.
- mass spectrometric analysis is used for the identification of peptides binding to the antibody.
- the epitope recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 may also be identified by a method comprising amplification of various DNA fragments of CD43 DNA by polymerase chain reaction (PCR), integration of these fragments into an expression vector comprising their connection to a histidine fusion protein and, following protein expression, detection of the epitope, for example by western blot.
- PCR polymerase chain reaction
- an expression vector cloned with CD43 may be introduced with deletion mutation by PCR method to prepare mutant series, such as Escherichia coli (E. coli) mutant series, that express proteins having various deleted sites in CD43.
- E. coli mutants may be cultured and induced for expression.
- Western blot analysis may be carried out using the cell lysate as an antigen.
- Further methods for the identification of the epitope recognized by and bound by the antibody produced by the hybridoma cell deposited under ICLC PD n° 16001 may comprise detection via immunoassays, such as enzyme-linked immunosorbent assay (ELISA).
- immunoassays such as enzyme-linked immunosorbent assay (ELISA).
- affinity may be understood in the broadest sense as the strength of the interaction between an epitope and an epitope-binding site of an antibody.
- Methods for determining an absolute value for antibody affinity i.e. the affinity constant, are well known to the person skilled in the art.
- relative values of antibody affinities may generally be determined, i.e. the affinity of two antibodies is compared without determining their absolute values.
- Methods for comparing the affinities of antibodies are well-known to the person skilled in the art. For example, flow cytometry may be used, whereby cells having the desired epitope may independently be brought into contact with different antibodies, which are subsequently marked with an immunofluorescent secondary antibody. Usually, after detection with flow cytometry, the intensity of the signals of the antibodies can be compared.
- the antibody of the invention recognizing the same epitope may also be a human antibody.
- the antibody according to the second aspect is a chimeric antibody. In a more preferred embodiment, the antibody according to the second aspect is a chimeric antibody according to the first aspect.
- a chimeric antibody is an antibody, in which at least one region of an
- immunoglobulin of a species is fused to another region of an immunoglobulin of another species by genetic engineering in order to reduce its immunogenicity (see, e.g., U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397).
- the antibody according to the second aspect is a humanized antibody.
- the antibody according to the second aspect is a chimeric or humanized antibody according to the antibody of the first aspect.
- humanized antibodies are a particular type of chimeric antibodies.
- humanized antibodies may be produced by grafting DNA of a human antibody into the mouse antibody framework coding DNA or by grafting DNA of a mouse antibody into human antibody framework coding DNA.
- DNA of a human antibody is grafted into the mouse antibody framework coding DNA.
- grafting of DNA comprises grafting of one or more DNA sequences into the target antibody framework coding DNA.
- the variable and constant regions as well as heavy and light chains may be partially or fully humanized.
- the heavy chain variable region and the light chain variable region of a mouse antibody are humanized.
- the heavy chain variable region and the light chain variable region of a mouse antibody are humanized by changing a DNA sequence encoding 1 to 50, preferably, 1 to 30, more preferably 1 to 20 amino acids.
- the DNA grafted may generally comprise DNA regions of the six
- hypervariable loops determining antigen specificity also called complementarity-determining regions (CDR)
- CDR complementarity-determining regions
- the humanization comprises grafting of DNA not comprising CDRs.
- the resulting DNA construct may then be used to express and produce antibodies that are usually less or not immunogenic in comparison to the non-human parental antibody.
- modified antibodies such as aglycosylated antibodies or afucosylated antibodies.
- the antibody of the invention recognizing the same epitope may also be an aglycosylated antibody or a afucosylated antibody.
- the disclosure also provides engineered humanized antibodies that recognize CD43.
- the h-UMGl antibody can comprise one or more of the variable heavy or light regions provided in SEQ ID NOs: 8-11, and SEQ ID NOs:13-16, respectively.
- a person skilled in the art can generated various embodiments by making one or more conservative substitutions of amino acid residue provided by the present disclosure.
- A“conservative substitution” or a “conservative amino acid substitution,” refers to the substitution an amino acid with a chemically or functionally similar amino acid.
- the antibody is IgGl, IgG2, IgG4, or IgM.
- the antigen binding protein is an Fv fragment, a Fab fragment, a F(ab’) 2 fragment, a Fab’ fragment, an scFv fragment, an scFv-Fc fragment, and/or a single-domain antibody.
- the h-UMGl 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
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:
- the h-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the h-UMGl 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-UMGl antibody the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8.
- the h-UMGl 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
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO:
- the h-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the h-UMGl 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-UMGl antibody the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO:
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9.
- the h-UMGl 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
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO:
- the h-UMGl antibody comprises an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 10.
- the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 10.
- the h-UMGl antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 1 1, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 1 1.
- the h-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h- UMG1 antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h-UMGl antibody the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 1 1. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
- the h-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 11
- the h-UMGl antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 13, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to 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: 13. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:
- the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 13. In some embodiments, the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 13.
- the h-UMGl antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 14, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to 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: 14. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO:
- the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 14. In some embodiments, the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 14.
- the h-UMGl antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 15, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to
- the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to 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: 15. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 15.
- the h-UMGl antibody comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 97% or greater sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 15. In some embodiments, the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 15.
- the h-UMGl antibody comprises an amino acid sequence having 60-100% sequence identity to SEQ ID NO: 16, such as 70-100%, 80-100%, 85-100%, 90-100%, 95-100%, 97-100%, or 99-100% sequence identity to
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 8 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 16. [00176] In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 13.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 14.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 15.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 15. [00183] In some embodiments, the h-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 60% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl 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-UMGl antibody comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the h-UMGl 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-UMGl antibody comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 16.
- the monoclonal antibody according to the antibody of aspect two is capable of inducing antibody dependent cellular cytotoxicity (ADCC) against the EGIL T3 subgroup of T cell acute lymphoblastic leukemia (T-ALL), against T cell lymphoblastic lymphoma cells and against Waldenstrom’s macroglobulinemia (WM) cells.
- ADCC antibody dependent cellular cytotoxicity
- 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.
- B- and T-cells can abnormally develop to B- and T- cell tumors. Due to the numerous developmental stages of developing B- and T-cells, there are various kinds of tumors. Both, B-cells and T-cells, originate from lymphoid progenitor cells.
- this lymphoid progenitor cell develops via many B cell developmental stages each comprising a certain definable cell type until a plasma cell is formed.
- One of these stages includes the so-called“IgM-secreting B cell”, which finally develops into an antibody-producing plasma cell.
- a tumor originating from an“IgM- secreting B-cell” is called“Waldenstrom’s macroglobulinemia” (WM).
- WM is a rare, indolent and incurable disease. It is characterized by bone marrow accumulation of clonal IgM secreting lymphoplasmacytic cells.
- T-cells develop from lymphoid progenitor cells to mature T-cells in only a few developmental stages. Tumors may especially evolve from mature T-cells or lymphoid progenitor cells, the latter leading to B- or T-cell acute lymphoblastic leukemia, (B-ALL) and (T-ALL), respectively.
- B-ALL B- or T-cell acute lymphoblastic leukemia
- T-ALL 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 differential diagnosis between the two diseases is based on prevalent localization in specific sites, such as bone marrow in T-ALL or secondary lymphoid organ in T-LBL.
- T-LBL T-cell lymphoblastic lymphoma
- EGIL T1 pro-
- cCD3 cytoplasmic positivity for CD3
- CD7 CD7
- EGIL T4 mature leukemia
- ADCC Antibody-dependent cellular cytotoxicity
- NK cells CD3 /CD56 + /CD107a + cells inducing target cells lysis (CD107a + ) are measured.
- An increase of CD3 /CD56 + /CD107a + cells according to increasing antibody concentrations therefore confirms the potential of an antibody to induce ADCC.
- the resulting data allow to design immune targeting approaches, which e.g. are an urgent and unmet clinical need in T cell acute lymphoblastic leukemias/lymphoblastic lymphomas. Further methods to examine, whether an antibody is capable of inducing ADCC, can also be used and are well-known to the person skilled in the art.
- the invention provides a binding molecule derived from an UMG1 antibody according to aspect one or aspect two.
- a binding molecule is a molecule derived from the monoclonal mouse UMG1 antibody produced by the hybridoma cell deposited under ICLC PD n° 16001.
- the binding molecule is an immunoglobulin comprising molecule, i.e. it comprises at least one Immunoglobulin (Ig) domain.
- the binding molecule of the invention is being selected from the group consisting of single chain antibodies.
- the binding molecule is being selected from the group consisting of a single chain variable fragment (scFv), a multimer of a scFv, such as a diabody, a triabody or a tetrabody, antibody fragments, preferably a Fab, a tandab, and a flexibody.
- scFv single chain variable fragment
- a multimer of a scFv such as a diabody, a triabody or a tetrabody
- antibody fragments preferably a Fab, a tandab, and a flexibody.
- VH Fv and VL Fv the two antigen binding variable regions of the light and heavy chain (VH Fv and VL Fv) of an antibody are in general artificially connected by a linker peptide, designated as 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 are incorporated by reference in their entirety).
- the antigen binding site can be made up of the variable domains of light and heavy chains of a monoclonal antibody.
- diabodies are scFv with two binding specificities and can either be monospecific and bivalent or bispecific and bivalent.
- Tandabs and flexibodies are further antibody formats which are e.g. defined in US2007031436 and EP1293514B1, respectively, which are incorporated by reference in their entirety.
- Antibody fragments that contain the idiotypes of the protein can be generated by techniques known in the art.
- such fragments include, but are not limited to, the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragment that can be generated by reducing the disulfide bridges of the F(ab')2 fragment; the Fab fragment that can be generated by treating the antibody molecular with papain and a reducing agent; and Fv fragments.
- the antibody or binding molecule of the invention can further be linked to an active substance, preferably a toxin, a nanoparticle, a cytokine, or a radionucleotide.
- an active substance preferably a toxin, a nanoparticle, a cytokine, or a radionucleotide.
- ADCs antibody drug conjugates
- Wu AM Senter PD. Nature Biotechnol.
- the binding molecule is conjugated to a therapeutic agent (i.e. drug) to form a binding molecule-drug conjugate.
- therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g. radioisotopes), immune modulators (e.g. cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g. cytotoxic agents).
- the therapeutic agents are attached to the binding molecule through a linker peptide, as discussed in more detail below in Section 6.7.3.
- ADCs antibody-drug conjugates
- the disclosure also provides a chimeric antigen receptor (CAR) comprising a binding molecule of aspect three linked to an intracellular domain preferably comprising one or more signaling domains.
- the invention relates to a chimeric antigen receptor (CAR) comprising the scFv of the preferred embodiment of the binding molecule of aspect three linked to an intracellular region comprising the CD3z chain, the signaling region of the T cell receptor, and to the two co-stimulatory domains CD28 and 4-1BB.
- CAR chimeric antigen receptor
- the CAR according to the invention is a relevant tool for targeting malignant cells bearing the epitope recognized and bound by the monoclonal antibody of aspect one or aspect two, when expressed in T-cells or NK cells.
- the term“Chimeric antigen receptors” (CAR), as used herein, refers to synthetic receptors comprising a targeting moiety that is associated with one or more signaling domains in a single fusion molecule.
- the binding moiety of a CAR comprises scFv, but it may also comprise other binding entities. Binding moieties based on receptor or ligand domains have also been used successfully.
- the signaling domains for CARs can be derived from the cytoplasmic region of the CD3z or the Fc receptor gamma chains, but may also be derived from other cytoplasmic regions.
- First generation CARs have been shown to successfully redirect T-cell cytotoxicity.
- Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (Porter DL et ak, N Eng J Med, 2011).
- CAR-T provided by the disclosure can be designed for particular applications provided by the disclosure (D. Xu et al. Qncotarget. 2018 Mar 2; 9(17)), which is hereby incorporated by reference in its entirety.
- the CAR is a 1 st generation CAR (Eshhar et al. Proc Natl Acad Sci USA (1993) 90(2)); in various embodiments, the CAR is a co-stimulatory CAR (Krause et al. J ExpMed.(1998) 188(4)); in various embodiments, the CAR is a 2nd 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.
- the CAR is a 3rd generation CAR (Pule et al. (2005) MolTher. 12(5); Geiger et al. Blood (2001) 98; Wilkie et al. (2008) J Immunol. 180(7)); in various embodiments, the CAR is a 4th generation TRUCKS CAR (Chmielewski et al. Cancer Res (2011) 71.); in various embodiments, the CAR is an Armored CAR generation CAR (Pegram et al. (2012) Blood 119; Curran et al. (2015) MolTher. 2015 Apr;23(4)); in various embodiments, the CAR is a engineered co-stimulation generation CAR (Zhao et al.
- the CAR is a SynNotch/sequential AND gate generation CAR (Roybal et al. (2016) Cell 164); in various embodiments, the CAR is a co-stimulation in cis and in trans generation CAR (Stephan et al. (2007) Nat Med 13(12)); in various embodiments, the CAR is a dual-targeted generation CAR (Wilkie et al. (2012) J Clin Immunol. 32(5)); in various embodiments, the CAR is a Combinatorial CARs/ AND gate generation CAR (Kloss et al.
- the CAR is a TanCAR generation CAR (Ahmed et al. (2013) MolTher Nucleic Acids. 2:el05); in various embodiments, the CAR is a Go-CART generation CAR (Foster et al, (2014)); the disclosures of which are incorporated herein by reference in their entireties.
- the CAR is a pCAR, as described in US pre-grant publication US 2019/0002521, incorporated by reference herein in its entirety.
- CAR constructs (CAR-UMG1) with a Primary intracellular signaling domain
- the CAR construct comprises a primary intracellular signaling domain.
- a primary intracellular signaling domain produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain, to which it is fused binds cognate antigen.
- the primary intracellular signaling domain is derived from a primary stimulatory molecule, e.g., it comprises intracellular sequence of a primary stimulatory molecule.
- the primary intracellular signaling domain comprises sufficient primary stimulatory molecule sequence to produce an intracellular signal, e.g., when an antigen binding domain to which it is fused binds cognate antigen.
- a primary stimulatory molecule is a molecule, that upon binding cognate ligand, mediates an immune effector response, e.g., in the cell in which it is expressed. Typically, it generates an intracellular signal that is dependent on binding to a cognate ligand that comprises antigen.
- the TCR/CD3 complex is an exemplary primary stimulatory molecule; it generates an intracellular signal upon binding to cognate ligand, e.g., an MHC molecule loaded with a peptide.
- cognate ligand e.g., an MHC molecule loaded with a peptide.
- the generation of an intracellular signal by a primary intracellular signaling domain is dependent on binding of the primary stimulatory molecule to antigen.
- Primary stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-b, and/or reorganization of cytoskeletal structures, and the like.
- Stimulation can, e.g., in the presence of co-stimulation, result in an optimization, e.g., an increase, in an immune effector function of the CART cell.
- Stimulation e.g., in the context of a CART cell, can mediate a T cell response, e.g., proliferation, activation, differentiation, and the like.
- the primary intracellular signaling domain comprises a signaling motif, e.g., an immunoreceptor tyrosine-based activation motif or IT AMs.
- a primary intracellular signaling domain can comprise IT AM containing cytoplasmic signaling sequences from (for example) TCR zeta (CD3 zeta, CDz), common FcR gamma, (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS”), FcsRI, DAPIO, DAP 12, and CD66d.
- IT AM containing cytoplasmic signaling sequences from (for example) TCR zeta (CD3 zeta, CDz), common FcR gamma, (FCER1G), Fc gamma Rlla, FcR beta (Fc
- a primary intracellular signaling domain comprises a functional fragment, or analog, of a primary stimulatory molecule (e.g., CD3 zeta, O ⁇ 3z).
- the primary intracellular signaling domain can comprise the entire intracellular region or a fragment of the primary stimulatory molecule
- the primary intracellular signaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99 % sequence identity with the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring primary stimulatory molecule, e.g., a human, or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular primary stimulatory molecule.
- a naturally occurring primary stimulatory molecule e.g., a human, or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular primary stimulatory molecule.
- the primary intracellular signaling domain has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, 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, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring human primary stimulatory molecule, e.g., a naturally occurring human primary stimulatory molecule disclosed herein. 6.3.7.3. CAR constructs (CAR-UMG1) with a naturally occurring human primary stimulatory molecule.
- the CAR construct comprises a costimulatory signaling domain which produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain, to which it is fused binds cognate ligand.
- the costimulatory signaling domain is derived from a costimulatory molecule.
- the costimulatory signaling domain comprises sufficient primary costimulatory molecule sequence to produce an intracellular signal, e.g., when an extracellular domain, e.g., an antigen binding domain, to which it is fused binds cognate ligand.
- the costimulatory domain can be one which optimizes the performance, e.g., the persistence, or immune effector function, of a T cell that comprises a CAR which comprises the costimulatory domain.
- Costimulatory molecules are cell surface molecules, other than antigen receptors or their counter ligands that promote an immune effector response. In some cases they are required for an efficient or enhanced immune response.
- a costimulatory molecule generates an intracellular signal that is dependent on binding to a cognate ligand that is, in certain embodiments, other than an antigen, e.g., the antigen recognized by an antigen binding domain of a CART cell.
- signaling from a primary stimulatory molecule and a costimulatory molecule contribute to an immune effector response, and in some cases both are required for efficient or enhanced generation of an immune effector response.
- a costimulatory domain comprises a functional fragment, or analog, of a costimulatory molecule (e.g., ICOS, CD28, or 4-1BB). It can comprise the entire intracellular region or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, e.g., when an antigen binding domain to which it is fused binds cognate antigen.
- a costimulatory molecule e.g., ICOS, CD28, or 4-1BB.
- the costimulatory domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99 % sequence identity with the entire intracellular region, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of a naturally occurring costimulatory molecule, e.g., a human, or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular costimulatory molecule.
- a naturally occurring costimulatory molecule e.g., a human, or other mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murine intracellular costimulatory molecule.
- Exemplary co-stimulatory domains include, but are no limited to, those selected from CD27, CD27, CD28, 4-1BB (CD137), QX40, CD30, CD40, ICQS (CD278), ICAM-1, LFA-1 (CDlla/CD18), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD8, CDS, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY55), CD 19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, IT GAD, CDlld, ITGAE,
- CD 103 IT GAL, IT GAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IP0-3), BLAME (SLAMF8), SELPLG
- the costimulatory signaling domain has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, 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, or a fragment of the intracellular region which is sufficient for generation of an intracellular signal, of, a naturally occurring human costimulatory molecule, e.g., a naturally occurring human costimulatory molecule disclosed herein.
- Immune effector cell comprising the chimeric antigen receptor (CAR)
- the invention provides a CD3 + lymphocyte, an NK lymphocyte, a Cytokine induced killer (CIK) cell, a gamma-delta lymphocyte, an NKT cell or another immune effector cell comprising the chimeric antigen -ch-UMGl according to aspect four or the expression vector according to aspect five.
- CIK Cytokine induced killer
- CD3 is a complex of four signaling chains associated to the a:b heterodimer of the T-cell receptor in a functional T-cell receptor complex.
- the CD3 complex is usually required for T-cell receptor signaling.
- the group of CD3 + lymphocytes exclusively contain thymocytes and T-cells. Detection of CD3 + cells can be achieved by e.g., flow-cytometry. 6.3.8. Bispecific T-cell engagers (BiTEs)
- CAR chimeric antigen receptors
- BiTE bispecific T-cell engagers
- BiTE-UMGl constructs Humanehls AM et al.,“ Bispecific T-cell engagers for cancer immunotherapy” Immunol Cell Biol. 2015 Mar;93(3):290-6; Zhukovsky EA et al.,“ Bispecific antibodies and CARs:
- BiTEs are constructed of two single-chain variable fragments (scFv) connected in tandem by a flexible linker.
- scFv single-chain variable fragments
- One scFv binds to a T-cell-specific molecule, usually CD3, whereas the second scFv binds to a tumor-associated antigen.
- This structure and specificity allows a BiTE to physically link a T cell to a tumor cell, ultimately stimulating T- cell activation, tumor killing and cytokine production.
- the BiTE-UMGl constructs target a hematological cancer.
- the BiTE-UMGl constructs target a solid tumor cancer type. In some embodiments, the BiTE-UMGl constructs target tumor-associated macrophages in a solid tumor.
- the invention provides a pharmaceutical composition comprising the monoclonal UMG1 antibody according to aspects 1 or 2 or the UMG1 binding molecule according to aspect three or the CD3+ lymphocyte, the NK lymphocyte, the Cytokine induced killer (CIK) cell, the gamma-delta lymphocyte, the NKT cell or the other immune effector cell according to aspect six.
- CIK Cytokine induced killer
- the pharmaceutical composition is an antibody or antigen binding fragment thereof.
- the antibody is monoclonal.
- the monoclonal antibody is a chimeric antibody.
- the monoclonal antibody is a humanized antibody.
- the monoclonal antibody is a human antibody.
- the pharmaceutical composition is an antibody-drug conjugate.
- the pharmaceutical compositions are described in more 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, US application 10/813,483, WO 2014/066468, WO 2011/104381, and WO 2016/180941, each of which is incorporated herein in its entirety.
- UMG1 binding molecules (antibodies, protein, antigens, etc.) provided by the present disclosure can be manufactured using standard methods known in the art.
- UMG1 binding molecules can be made by expression using standard cell free translation, transient transfection, and stable transfection approaches currently used for antibody manufacture.
- Expi293 cells ThermoFisher
- Expi293 cells can be used for production of the binding molecules using protocols and reagents from
- ThermoFisher such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethylenimine as described in detail in Fang et al. ( Biological Procedures Online , 2017, 19: 11), which is incorporated herein in its entirety.
- Expressed proteins can be readily purified using standard methods known in the art such as, for example, a CHI affinity resin, such as the CaptureSelect CHI resin and provided protocol from ThermoFisher. Further purification can be accomplished using ion exchange chromatography as is routinely used in the art.
- the UMG1 pharmaceutical composition provided by the present disclosure may be administered by any suitable route of administration.
- Suitable routes of administration include, but are not limited to, parenteral administration, including subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, nasal, and pulmonary routes.
- the present disclosure also provides combination therapeutics. In some embodiments
- the pharmaceutical composition provided herein is given in combination with another therapeutic treatment.
- the therapeutic treatment may be, surgical, radiation, holistic, cellular therapy, tissue regeneration, or another pharmaceutical composition known for the treatment of a cell proliferation disease or cancer.
- Therapeutically-effective dosages vary in some embodiments when the
- compositions provide by the present disclosure are used in treatment combinations.
- Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens include the use of metronomic dosing, i.e., by providing more frequent, lower doses in order to minimize toxic side effects.
- Combination treatment regimens encompass treatment regimens in which administration of a compound described herein is initiated prior to, during, or after treatment with a second agent described above, and continues until 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 the second agent being used in combination are administered simultaneously or at different times and/or at decreasing or increasing intervals during the treatment period.
- Combination treatments further include periodic treatments that start and stop at various times to assist with the clinical management of the patient.
- a compound described herein in the combination treatment is administered weekly at the onset of treatment, decreasing to biweekly, and decreasing further as appropriate.
- the disclosure also provides various UMG1 pharmaceutical formulations comprising an effective amount of an UMG1 antigen, antibody, or binding molecule or protein.
- compositions are formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are optionally used as suitable.
- Pharmaceutical compositions comprising UMG1 antibody or UMG1 binding molecule are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee making, levigating, emulsifying, encapsulating, entrapping or compression processes.
- UMG1 pharmaceutical compositions can optionally include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, buffers, and/or other therapeutically valuable substances.
- Methods for the preparation of compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid.
- Solid formulation of compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
- Liquid formulation compositions include solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein.
- Semi-solid compositions include, but are not limited to, gels, suspensions and creams.
- the form of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.
- the content of the antibody, the binding molecule or the CD3+ lymphocyte in the pharmaceutical composition is not limited as far as it is useful for treatment or prevention, but preferably contains 0.0000001-10% by weight per total composition.
- the antibody, the binding molecule or the CD3+ lymphocyte described herein are preferably employed in a carrier.
- the choice of carrier may depend upon route of administration and concentration of the active agent(s) and the carrier may be in the form of a lyophilized composition or an aqueous solution. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic.
- the carrier include but are not limited to saline, Ringer's solution and dextrose solution.
- acceptable excipients, carriers, or stabilizers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g.
- polyvinylpyrrolidone amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides;
- disaccharides e.g. sucrose, mannitol, trehalose or sorbitol
- chelating agents e.g. EDTA
- non-ionic surfactants e.g. Tween, Pluronics or polyethylene glycol
- antioxidants including methionine, ascorbic acid and tocopherol
- preservatives e.g.
- composition may also contain at least one further active compound, such as a chemotherapeutic agent.
- the antibody, the binding molecule, the CD3 + lymphocyte and/or the active compound are included in an effective amount.
- effective amount refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is to be administered.
- the invention provides a nucleic acid or polynucleotide, encoding a UMG1 antibody according to aspects one or two or the UMG1 binding molecule according to aspect three.
- polynucleotides encoding an antibody that are optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements.
- Methods to generate optimized nucleic acids encoding an antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and
- potential splice sites and instability elements within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression.
- the alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid.
- it can be desirable to alter one or more codons to encode a conservative mutation e.g., a similar amino acid with similar chemical structure and properties and/or function as the original amino acid.
- Such methods can increase expression of an antibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more relative to the expression of an antibody encoded by polynucleotides that have not been optimized.
- an optimized polynucleotide sequence encoding an antibody described herein or a fragment thereof can hybridize to an antisense (e.g., complementary) polynucleotide of an unoptimized
- an optimized nucleotide sequence encoding an antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding an antibody described herein or a fragment thereof.
- an optimized nucleotide sequence encoding an antibody described herein or a fragment thereof hybridizes under high stringency, intermediate or lower stringency hybridization conditions to an antisense polynucleotide of an unoptimized nucleotide sequence encoding an antibody described herein or a fragment thereof.
- Information regarding hybridization conditions has been described, see, e.g., U.S. Patent Application Publication No. US 2005/0048549 (e.g., paragraphs 72-73).
- the polynucleotides of the invention can be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
- Nucleotide sequences encoding antibodies described herein, and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody.
- Such a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et ah, (1994), BioTechniques 17: 242-6; which is incorporated by reference in its entirety), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
- chemically synthesized oligonucleotides e.g., as described in Kutmeier G et ah, (1994), BioTechniques 17: 242-6; which is incorporated by reference in its entirety
- a polynucleotide encoding an antibody described herein can be generated from nucleic acid 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, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and/or heavy chain of an antibody.
- a suitable source e.g., a hybridoma
- methods well known in the art e.g., PCR and other molecular cloning methods.
- PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest.
- Such PCR amplification methods can
- Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and/or the variable heavy chain region of an antibody.
- the amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies.
- immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of the sequence or by cloning using an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected
- oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
- Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
- 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 binding specifically to genes encoding the heavy and light chains of the antibodies).
- Hybridoma cells can serve as a source of such DNA.
- 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., CHO cells from the CHO GS SystemTM (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the antibodies in the recombinant host cells.
- host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS SystemTM (Lonza)), or myeloma cells that do not otherwise produce immunoglob
- PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones or other clones.
- the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions.
- the vectors for expressing the VH or VL domains comprise a promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selection marker such as neomycin.
- the VH and VL domains can also be cloned into one vector expressing the necessary constant regions.
- the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
- the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the murine sequences, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
- mutagenesis methods can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
- affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies.
- the invention provides a hybridoma cell that produces the monoclonal antibody according to the antibody of aspects one or two.
- the invention also provides a hybridoma composition, deposited under ICLC PD n° 16001. 6.12. Method for producing the UMG1 monoclonal antibody
- the invention provides a method for producing the
- monoclonal antibody according to aspects one or two, said method comprising isolating said antibody from the hybridoma cell deposited under ICLC PD n° 16001.
- the invention provides a method for the identification or isolation of T-cell acute lymbhoblastic leukemia cells, T lymphoma cells, Waldenstrom’s Macroglobulinemia cells or tumor-associated macrophages, comprising contacting a cell sample comprising said cells with the monoclonal antibody according to aspects one or two or with the binding molecule according to aspect three.
- TAM Tumor associated macrophages
- T-cell acute lymbhoblastic leukemia cells T lymphoma cells
- Waldenstrom Macroglobulinemia cells or tumor-associated macrophages, based on antibodies or binding molecules in general are well- known to the person skilled in the art, such as methods based on fluorescent cell sorting by flow cytometry, magnetic cell isolation or single cell sorting, e.g. by cell sorters.
- the invention provides a method for producing CD3+ lymphocytes, NK lymphocyte, the Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or the other immune effector cells expressing a chimeric antigen receptor according to the chimeric antigen receptor of aspect four comprising the introduction of the expression vector according to the expression vector of aspect five into said CD3+ lymphocytes, NK lymphocyte, the Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or the other immune effector cells. 6.15. Expression Vector Compositions
- the invention provides an expression vector comprising a nucleic acid sequence which encodes the chimeric antigen receptor according to aspect four, the antibody according to the aspects one and two or the binding molecule according to the binding molecule according to aspect three.
- expression vectors are plasmids which are used to introduce a desired nucleic acid sequence, such as a gene, into a target cell, resulting in the transcription and translation of the protein encoded by the nucleic acid sequence, i.e. the chimeric antigen receptor, the antibody or the binding molecule.
- the expression vector in general comprises regulatory sequences, such as promoter and enhancer regions, as well as a polyadenylation site in order to direct efficient transcription of the nucleic acid sequence on the expression vector.
- the expression vector may further comprise additional necessary or useful regions, such as a selectable marker for selection in eukaryotic or prokaryotic cells, a purification tag for the purification of the resulting protein, a multiple cloning site or an origin of replication.
- the expression vector may be a viral or a non-viral vector.
- various kinds of viral vectors such as retroviral vectors, e.g. lentiviral or adenoviral vectors, or plasmids may be used.
- the expression vector according to aspect five is a viral vector.
- the expression vector is a lentiviral vector.
- methods of treatment comprising administering a binding molecule or antibody as described herein to a patient in an amount effective to treat the patient.
- the method comprises administering a binding molecule or antibody as described herein to a patient in an amount effective to treat the patient using a CAR or CAR-T.
- the method comprises administering a binding molecule or antibody as described herein to a patient in an amount effective to treat the patient using a BiTE.
- the method comprises administering a binding molecule or antibody as described herein to a patient in an amount effective to treat the patient using an antibody-drug conjugate.
- an antibody or binding molecule of the present disclosure may be used to treat a proliferation disease or cancer.
- the cancer is a solid tumor.
- the cancer is a blood cancer including, but not limited to, T-cell malignancies, T-cell leukemia, T-cell lymphoma, T-cell acute lymphoblastic leukemia, multiple myeloma, B cell malignancies, myeloid malignancies, acute myeloid leukemia and chronic myeloid leukemia.
- the cancer or proliferation disease may be a cancer from the bladder, blood, blood immune cells (e.g., T-cell or B-cells, monocytes, and the like), bone, bone marrow, brain, breast, colon, colorectal, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, pancreas, skin, stomach, testis, tongue, or uterus.
- blood immune cells e.g., T-cell or B-cells, monocytes, and the like
- the cancer or tumor treated with the antibody or binding molecule of the present disclosure may be a neoplasm, malignant; non-malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma;
- adenocarcinoma gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar
- adenocarcinoma papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary
- cystadenocarcinoma papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia;
- thymoma malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma;
- rhabdomyosarcoma embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma;
- hemangiopericytoma malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal
- chondrosarcoma giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma;
- pinealoma malignant; chordoma; glioma, malignant; ependymoma; astrocytoma;
- astrocytoma protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma;
- oligodendroglioma oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor;
- meningioma malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas;
- the invention provides a method for producing CD3+ lymphocytes, NK lymphocyte, the Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or the other immune effector cells expressing a chimeric antigen receptor according to the chimeric antigen receptor of aspect four comprising the introduction of the expression vector according to the expression vector of aspect five into said CD3+ lymphocytes, NK lymphocyte, the Cytokine induced killer (CIK) cells, gamma-delta lymphocytes, NKT cells or the other immune effector cells.
- CIK Cytokine induced killer
- the cancer to be treated by the antibody disclosed herein is a non-Hodgkin lymphoma derived from peripheral B cell or peripheral T cell, including but not limited to Diffuse large B cell lymphoma, MALT lymphoma, Burkitt lymphoma, anaplastic large cell lymphoma, follicular lymphoma, and mantle cell lymphoma.
- the cancer to be treated by the antibody disclosed herein is multiple myeloma.
- the cancer to be treated by the antibody disclosed herein is melanoma.
- the cancer to be treated by the antibody disclosed herein is testicular cancer, including but not limited to seminoma, embryonal carcinoma, yolk sac tumor, and teratoma.
- the cancer to be treated by the antibody disclosed herein is a pediatric malignant tumors such as nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, or leiomyosarcoma.
- a pediatric malignant tumors such as nephroblastoma, neuroblastoma, endodermal sinus carcinoma, retinoblastoma, hepatoblastoma, medulloblastoma, choroid plexus papilloma, glioblastoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosar
- PBMCs Peripheral blood mononuclear cells
- PBMCs Peripheral blood mononuclear cells
- FITC fluorescein isothiocyanate
- the UMG1 antibody was able to recognize a lymphocyte subpopulation having variable prevalence (range: 0-15%) in different human donors.
- the UMG1 antibody did not show any reactivity with any other cell populations within the PBMCs, including myeloid-derived cells, demonstrating that myeloid-derived cells in PBMCs from healthy subjects are negative for expression of the UMG1 epitope (see FIG. 1A and FIG. IB).
- the epitope on CD43 recognized by the UMG1 antibody exhibits a specific, restricted, expression pattern in PBMC cells that is different from the pattern of expression of the epitope recognized by a commercial anti-CD43 antibody (S7).
- This example further characterizes the lymphocyte sub-population detected by the UMG1 antibody using an immune-magnetic sorting of the respective lymphocytes.
- Methods Briefly, 15 pg of the UMG1 antibody were mixed with components provided by the manufacturer (EasySepTM“Do-it-yourself’ Selection Kit, STEMCELL Technologies) to obtain a solution ready for immunomagnetic separation. This solution was added to PBMCs from 3 different donors having at least 10% of lymphocytes detected by the antibody, after FcR blocking, and cells were incubated at room temperature (r.t.) for 15 minutes. Subsequently, EasySep® Magnetic Nanoparticles were added to the solution and cells were incubated for further 10 minutes at r.t. The solution was then placed in a magnet and unbound cells were removed.
- CD45 + CD3 + CD4 + CD8 CD 127 + CCR7 + T lymphocytes (see FIGs. 2A-2D and Table 1).
- T-ALL cell lines belonging to EGIL T3 classification and Waldenstrom’s macroglobulinemia were all positive for expression of the UMG1 epitope, while the other cell lines assayed were negative for the UMG1 epitope (see Table 2).
- the UMG1 antibody recognizes T-ALL and Waldenstrom’s Macroglobulinemia cell lines, but not other hematopoietic cancers and non-hematopoietic tumors.
- Example 4 UMG1 binding specificity - UMG1 binds to T- ALL human cell lines with binding pattern different from commercially available CD43 antibodies [00294] 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.
- CD43 antibodies Commercially available CD43 antibodies: clones, CD43 1G10 (Becton Dickinson), CD43 MEM-59 (Invitrogen), and CD43 L-10 (Invitrogen) were compared to the UMGl antibody.
- the secondary fluorochrome-labeled antibody was diluted following the manufacturer’s instructions in a final volume of 100 pL of cells and incubated for at least 15 minutes at 2-8 °C, protect from light. Cells were washed twice as indicated above and then resuspend in 500 pL of PBS IX and analyzed by flow cytometry. [00297] Results: We observed different expression density and intensity by FACS analysis for the UMG1 antibody, and CD43 1G10 (Becton Dickinson), CD43 MEM-59 (Invitrogen), and CD43 L-10 (Invitrogen) in both ALL-SIL and KE-37 cell lines. See Figures 12A and
- CD43 than three different CD43 commercial antibodies.
- 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.
- Antigen unmasking was carried out using Novocastra Epitope Retrieval Solutions, pH 9 (Leica Biosystems) in Thermostatic bath at 98° C for 30 minutes. Neutralize endogenous peroxidase using Peroxidase Block for 10 minutes. Peroxidase Block. 3/4%,
- the sections were stained using the primary antibody UMG1 (“m-UMGl”) overnight at 4 C°, at a dilution of 1 :300. Next, the stained sections were washed in PBS for 2 times for 5 minutes each wash. After washing, the samples were incubated with rabbit anti mouse IgG from 30 mins and then washed in PBS for 2 times for 5 minutes each wash. After washing, the samples were incubated with Novolink Polymer for 30 minutes, anti -rabbit Poly-HRP-IgG, and then washed in PBS for 2 times for 5 minutes for each wash.
- Microsystems and microphotographs were collected using a digital camera (Leica).
- TAM macrophages
- UMG1 epitope expression changes were assessed in a model of macrophage differentiation in the presence or absence of co-cultured cancer cells.
- THP-1 monocytic leukemia cells were used; as shown in Example 3, these cells do not express the UMG1 epitope.
- THP-1M differentiated human unpolarized M0 macrophages
- PMA phorbol 12-myristate 13-acetate
- PANC1 pancreatic cancer cell line cells were added at a 1 : 1 ratio selected wells and incubated for 48hrs.
- THP-1M cells were then prepared for immunofluorescence analysis. Briefly, after fixation, THP-1M cells were stained with a chimeric antibody derived from UMG1, ch-UMGl, which is further described in Example 16, or human IgGl control, and incubated at 4°C overnight. A FITC anti-human secondary mAh was then added to the cells for 2 hours. After washing, anti-fade mounting medium with DAPI (Vectashield®, Vectorlabs) was added to cells and coverslips and then analyzed. [00313] Results: As shown in FIG. 5A, THP-l-derived macrophages stained with control
- THP1 -derived macrophages showed strong (bright) UMG1 expression.
- FIG. 5A One particular interaction between THEM -derived macrophages (white arrow) and PANC1 cells (red arrow) is shown (FIG. 5A, on the left).
- UMG1 epitope is a suitable target for therapeutic approaches focused on purging tumor- associated macrophages. Beyond this relevant potential as therapeutic tool, UMG1 might also prove useful for detection, analysis of prognostic role and predictive studies.
- h-UMGl a humanized version of the UMG1 antibody, further described in Example 19, below
- h-UMGl -PE phycoerythrin-conjugated h-UMGl
- h-UMGl and three commercially available CD43 antibodies were conducted on two different cell lines, CEM and HPB-ALL.
- CEM and HPB-ALL cells were incubated for 20 minutes, on ice, in the dark with h-UMGl unconjugated at increasing concentrations (0.016 pg/ml, 0.08 pg/ml, 0.4 pg/ml, 1 pg/ml, 2 pg/ml) in the presence of 1 pg/ml of one of the CD43 clones or h-UMGl -PE (positive control).
- unconjugated h-UMGl does not compete with the binding of other commercially available CD43 antibodies (MEM-59 (Invitrogen), L-10 (Invitrogen), and 1G10 (Becton Dickinson). Indeed, the number of stained cells marked with anti-CD43 was not reduced by increasing the concentration of unconjugated h-UMGl antibody. See, Figures 13A and 13B (line with up-facing triangles, line with down-facing triangles, line with squares).
- Methods Cells from 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 1,200 rpm for 5 minutes at room temperature to pellet and supernatant was discarded. [00324] The h-UMGl primary antibody was added at the concentration of 1 pg/ml in the final staining volume of 100 pL of cells. Next, cells were mixed by pulse vortex and incubated for 15 minutes at 2-8°C, protected from light.
- the excess primary antibody was then washed off twice, by adding 2 mL of staining buffer and centrifuge cells at 1,200 rpm for 5 minutes at room temperature to pellet, and the supernatant was discarded.
- the secondary fluorochrome-labeled antibody was added at the recommended dilution in a final volume of 100 pL of cells, and incubated for at least 15 minutes at 2-8 °C, protected from light.
- FIG. 17A shows the historical flow cytometric profiles of UNI performed in 1994 by the Tassone lab (Tassone et al, Tissue Antigens 44:73-82, 1994), in JURKAT, MOLT-4, CEM, and HPB-ALL cell lines.
- FIG. 17B shows the results of flow cytometric profiles of h-UMGl antibody in JURKAT, MOLT-4, CEM, and HPB-ALL cell lines.
- UMGl epitope expression was evaluated on inactivated and activated neutrophils isolated from human healthy donor peripheral blood.
- lymphocytes from healthy donor peripheral blood.
- PBMCs Peripheral blood mononuclear cells
- Lymphocytes from 3 HD were activated with PMA (25 ng/ml) and ionomycin (1 pg/ml) for 24 hours, 37°C, 5% CO2. After activation, cells were collected, washed with lx PBS and stained with early and late T cell activation marker, specifically CD25-BV515, CD69-PE and h-UMGl-APC or IgG isotype control-APC. Flow cytometry analysis was performed to evaluate h-UMGl expression on CD25 + T cells and CD69 + T cells.
- h-UMGl mAb does not target activated T lymphocytes. 6.17.11.
- Example 11 UMG1 binding specificity - immunohistochemical analysis of mUMGl binding in healthy and neoplastic tissues
- Tissue microarrays used in the immunohistochemical analysis include: Dog Female Normal Organ (DGF281); Rat Normal Organ (RAT901a); Mouse
- M0541c Rhesus Monkey Normal Organ (RhFDAla); Cynomolgus Monkey Normal Organ (CyFDAlc); Multiple Organ Cancer and Normal Tissue (MC5003c);
- PC701 Malignant Tumor (PC701) produced by US Biomax, Inc, and FDA Standard paraffin Tissue Array Human Normal Organ (Catalog No: T8234701) produced by BioChain. The description of selected TMAs is provided below.
- MC5003c Multiple Organ Cancer and Normal Tissue: High-density multiple organs tumor with normal tissue microarray, containing 20 types of organs, each organ was taken from 25 individuals (20 cases of tumor and 5 normal tissue), single core per case.
- Malignant Melanoma ME2081: Malignant melanoma and skin tissue microarray, containing 88 cases of malignant melanoma, 16 skin tissue, duplicate cores per case.
- Lymphoma Survey Tissue (Ly2084): Lymphoma tumor survey tissue microarray (slide 4 of 520 cases of lymphoma survey slide set), containing 104 cases of malignant tumor (64 B-cell lymphoma, 24 mucosa associated lymphoma tissue, 6 T-cell lymphoma, 4
- Hodgkin's lymphoma 4 anaplastic large cell lymphoma, 1 each of mantle cell lymphoma and Burkitt lymphoma), duplicate cores per case.
- Testis Disease Testis tumor tissue microarray, containing 46 cases of seminoma, 8 yolk sac tumor, 16 embryonal carcinomas, 5 teratomas, 3 tuberculosis, 6 atrophy, 15 adjacent normal tissue and 5 normal tissue, duplicate cores per case.
- PC701 Human Pediatric Malignant Tumor: Pediatric malignant tumor tissue microarray with normal tissue, containing 21 cases of nephroblastoma, 13 neuroblastoma, 7 endodermal sinus carcinoma, 4 retinoblastoma, 3 hepatoblastoma, 2 medulloblastoma, 4 lymphoma, 1 each of choroid plexus papilloma, glioblastoma, adrenocortical carcinoma, embryonal rhabdomyosarcoma, ependymoma, primitive neuroectodermal tumor, alveolus rhabdomyosarcoma, immaturity teratoma, leiomyosarcoma, plus 7 normal tissue, single cores per case.
- PC701 Human Pediatric Malignant Tumor
- tissue sections were dewaxed and rehydrated.
- the antigen unmasking technique was performed using Novocastra Epitope Retrieval Solutions, pH 9 EDTA-based buffer in thermostatic bath (FALC Instruments S.r.L, Treviglio (BG) Italy, Model WB-MD 5) at 98°C for 30 minutes.
- TMAs were incubated overnight with the primary UMG1 mAh (m-UMGl mAh was used for this application, dilution 1 :300) at 4°C.
- the immunostaining was revealed by either a polymer detection method (Novolink Polymer Detection Systems Novocastra Leica Biosystems Newcastle Ltd Product No: RE7280-K) or AEC (3-amino-9-ethylcarbazole,
- UMG1 mAb target epitope was clear also on malignant cells in multiple observed spots, although with different degree of intensity and distribution.
- UMG1 mAb target was expressed on the malignant cell membrane and on TAMs (FIG. 40A, Table 6). Also, intratumor leukocytes infiltrate stained positive in some samples as expected by preliminary data on the epitope expression on tumor associated macrophages.
- CD43 protein variants The sequences of CD43 protein clones tested are provide in Figure 15A, Table 4 and in the sequence listing as SEQ ID NOs: 17-24. Wildtype CD43, indicated as“CD43 #1” was generated using the full 400 amino acid region. For engineering the CD43 protein variants, the N-terminal domain was sequentially truncated. The first CD43 truncated variant,“CD43 #2,” was generated using aa from 31 to 400 of full-length CD43. The second CD43 variant indicated as,“CD43 #3” was generated using aa from 41 to 400 of the full length CD43. The third CD43 variant, indicated as“CD43 #4” was generated using aa from 61 to 400 from full-length CD43. The fourth CD43 variant, indicated as“CD43 #5”, consists of aa 91-400 from full length CD43. The fifth CD43 variant, indicated as“CD43 #6”, has a deletion from aa 64 to 78.
- the sixth CD43 variant indicated as“CD43 #7” has a deletion of a single amino acid at aa 69, which is thought to be the GalNac site.
- CD43 protein constructs were expressed using pLenti-CMV-(insert)- Histag-GFP-2A-Puro expression vectors from Applied Biological Materials (ABM) Inc. service (Vancouver, Canada). His-Tag and/or GFP detections served as a positive control for successful transfection and/or protein expression.
- HEK293T cells were transiently expressed in HEK293T cells by 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 (ThermoFisher Scientific, MA, USA). 72h after transfection, cells were subjected to western blot or flow cytometry (FACS) analysis.
- FACS flow cytometry
- Proteins were transferred bv electro-transferred for 30 minutes with the Trans-Blot® TurboTM 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 (#G020) from abm (Vancouver, Canada) and h-UMGl primary antibody (both at 1 :500 dilution). Goat anti-mouse and rabbit anti-human HRP-conjugated antibodies (Invitrogen) were used as secondary antibodies (1 :3,000 dilution). Immunoreactive bands were revealed by enhanced chemiluminescence detection method using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, MA, USA).
- Flow cytometry (FACS) analysis FACS was conducted to determine if the antibody could detect the CD43 wildtype and CD43 variants, as expressed in HEK293T cell line cells.
- FACS assay was conducted following standard procedures, by using 1 pg/ml of h- UMG1-PE conjugated antibody to detect the percentage of h-UMGl positive cells among the GFP positive cells. Samples were acquired by flow cytometry (LSRFortessaTM X-20, BD) and analyzed by DIVA software (BD FACSDivaTM software). A minimum of 20,000 events were gated for each measurement.
- Results The results from the western analysis suggest that the UMG1 epitope binding site is located between aa 61 to 91 (as numbered in the wildtype CD43). See, boxed sequence in Figure 15A, which shows the hypothesized binding site for the h-UMGl antibody. Furthermore, these studies show that the UMG1 antibody binds specifically to UMG1 rather than to CD43 His-tagged proteins that lack of specific extracellular regions that UMG1 recognizes. See, Figures 15C and 15E.
- Treonine 69 is deleted (CD43 #7) or substituted with an amino acid that is not O-glycosylated (CD43 #8). These results in the binding of an epitope that should not have any sugar group.
- Microarray Content The sequence of human CD43 (SEQ ID NO: 17) was elongated with neutral GSGSGSG (SEQ ID NO: 46) linkers at the C- and N-terminus to avoid truncated peptides. The elongated antigen sequence was translated into linear 15 amino acid peptides with a peptide-peptide overlap of 14 amino acids. The resulting CD43 peptide microarrays contained 400 different peptides printed in duplicate (800 peptide spots) and were framed by additional HA (YPYDVPDYAG (SEQ ID NO: 47), 82 spots) control peptides. The microarrays synthesis and analysis was performed by PEPperPRINT GmbH, Heidelberg.
- a CD43 peptide microarray was initially incubated with the secondary and control antibodies in the incubation buffer (washing buffer with 10% blocking buffer) at room temperature (RT) for 45 min to investigate background interactions with the linear CD43 peptides that could interfere with the main assays.
- Microarray Content The epitope substitution scan of wild type peptide PPSTSINEGSPLWTS (SEQ ID NO: 51) was based on an exchange of all amino acid positions by the 20 main amino acids.
- the resulting peptide microarray contained 300 different peptide variants of the wild type peptide printed in triplicate (900 peptide spots), 9 spots of custom control peptide PPSTSVNEGSPLGTS (SEQ ID NO: 52) and a frame of additional HA control peptides (YPYDVPDYAG, 82 spots (SEQ ID NO: 47)).
- the microarrays synthesis and analysis was performed by PEPperPRINT GmbH, Heidelberg.
- Pre-staining of the peptide microarray copy was done with secondary and control antibodies. After 15 min pre-swelling in washing buffer and 30 min incubation in blocking buffer, a peptide microarray copy was initially incubated with the secondary and control antibodies for 45 min at room temperature to analyze background interactions with the variants of wild type peptide that could interfere with the main assays.
- substitution matrix highlighted the preference for a given amino acid by a color code (red for preferred amino acids, blue for less preferred amino acids) and were calculated by dividing the spot intensity of a given peptide by the averaged spot intensities of all 20 peptides that were substituted at the same position.
- the amino acid plot was calculated by dividing the spot intensity of a given peptide by the spot intensity of the wild type peptide. The position of an amino acid thus reflected the intensity ratio compared to the amino acid of the native wild type peptide.
- Amino acid positions 76 (P) and 77 (L) of human CD43 were essential for antibody binding and did not tolerate any amino acid exchange at all.
- Amino acid positions 73 (E) and 78 (W) were highly conserved and tolerated only a conserved exchange by D and F respectively. Exchange of W by F at position 78 even resulted in an apparent increase in antibody binding.
- Amino acid position 74 (G) showed a lower degree of sequence conservation and was susceptible for replacement by M, acidic amino acids D and E as well as by aromatic amino acids W, F and Y.
- variable amino acid positions including amino acid position 75 (S) exhibited a variable character.
- the variable amino acid positions showed an objection for substitution by basic amino acids K and H, but surprisingly not R.
- Example 15a UMG1 binding specificity - binding to the aglycosylated extracellular portion of CD43 in
- This example shows the binding affinity measurement between a humanized-UMGl (h-UMGl) (H3-L4) and of the extracellular portion of CD43 (aa 20-253). The result is reported as dissociation constant KD.
- the chip was then blocked with 5% (w/v) non-fat milk in water overnight, and washed with 10* PBST for 10 min, 1 x PBST for 10 min, and deionized water twice for 10 min before being dried under a stream of nitrogen prior to use.
- SPRi measurements were performed with PlexAray HT (Plexera Bioscience, Seattle, WA, US). Collimated light (660 nm) passes through the coupling prism, reflects off the SPR-active gold surface, and is received by the CCD camera.
- Various concentrations of analyte human recombinant CD43 extracellular portion (from aa 20-253) produced in E.
- coli vector of CD43 SEQ ID NO: 42
- Buffers and samples were injected by a non-pulsatile piston pump into the 30 pL flowcell that was mounted on the coupling prim.
- Each measurement cycle contained four steps: washing with PBST running buffer at a constant rate of 2 uL/s to obtain a stable baseline, sample injection at 5 uL/s for binding, surface washing with PBST at 2 uL/s for 300 s, and regeneration with 0.5% (v/v) H3P04 at 2 uL/s for 300 s. All the measurements were performed at 25°C. The signal changes after binding and washing (in AU) are recorded as the assay value.
- Results The SPR binding result showed a KD value of 99.4 nM between the extracellular portion of CD43 and h-UMGl ( Figure 19). The result indicates strong binding affinity to the target.
- the binding to the aglycosylated extracellular portion of CD43 differentiate UMG1 from other anti-CD43 antibodies that bind only to glycosylated or neuraminidase-sensitive epitope, such as UNI and MEM-59 respectively (de Laurentiis A, et al ., Mol Cell Proteomics. 2011 May; 10(5)).
- Table 5 compares properties of UMG1 to historically reported data on UNI.
- a chimeric antibody with the binding specificity of UMG1 was constructed by fusing the murine UMG1 VH (SEQ ID NO: 34) to human VH constant region and the murine UMG1 VL (SEQ ID NO: 35) to a human light chain constant region using standard techniques. 6.17.18.
- ADCC antibody-dependent cell-mediated cytotoxicity
- PBMCs from healthy donors were co-cultured with T-ALL cell line HPB-ALL or T lymphoma cell line H9 (target cells) in the presence of different concentration of the ch-UMGl as follows.
- Chimeric mAb ch-UMGl is an active immunotherapeutic tool for T cell acute lymphoblastic leukemias/lymphoblastic lymphomas.
- Example 18 ch-UMGl - ch-UMGl induces antibody- dependent cell mediate cytotoxicity (ADCC) of
- Methods We performed a degranulation assay by co-culturing purified NK cells from healthy donors (effector cells) and the BCWM.1 cell line (target cells) in the presence of different concentrations of ch-UMGl antibody or negative/positive controls. We selected the mAh cetuximab as negative control and the mAh rituximab as positive control.
- Humanized UMGl antibodies were constructed using combination of human heavy chain SEQ ID NO: 8 to SEQ ID NO: 11 and human light chain SEQ ID NO: 13 to SEQ ID NO: 16, provided herein.
- DNA concentration was determined by measuring the absorption at a wavelength of 260 nm. Correctness of the sequences was verified with Sanger sequencing (with up to two sequencing reactions per plasmid depending on the size of the cDNA.)
- Suspension-adapted CHO K1 cells (originally received from ATCC and adapted to serum-free growth in suspension culture) were used for production.
- afucosylated antibody (a-h-UMGl) the GlymaxX technology was used (ProBioGen) and it was transiently expressed in CHO cells (Evitria).
- the seed was grown in a chemically defined, animal- component free, serum-free medium.
- Cells were transfected with custom-made, proprietary transfection reagent, and cells were grown after transfection in an animal-component free, serum -free medium.
- Endotoxin content was measured with the Charles River Endosafe PTS system. Titers were measured with ForteBio Protein A biosensors (kinetic assay) and calculated based on a human IgGl standard.
- H 1-4 and L 1-4 humanized heavy chain (H 1-4) and four humanized light chain (L 1-4) variants were generated by identifying murine complementary determinant regions (CDRs) and grafting the CDRs into a human antibody framework by replacing selected residues in the closest human germ line sequence of the framework regions, with the aim to preserve potentially structurally important residues of the murine counterpart.
- 16 humanized antibodies were construction by combining each of the four humanized heavy chains (SEQ ID NOs: 8-11) with each of the four humanized light chains (SEQ ID NOs: 13-16).
- the IgGl isotype was used for all heavy chain variants.
- 8 hybrid CHL(l-4) and H(1-4)CL variants were generated the 8 hybrids variants include 4 with the mouse heavy chain and a human light chain selected between Ll-4 (SEQ NOs: 13-16) and 4 with the mouse light chain and a human heavy chain selected from HI -4 (SEQ NO: 8-11).
- Example 20 h-UMGl - Screening of h-UMGl antibodies for binding to HPB-ALL and H9 cell lines
- This example reports tumor volume curves of an in vivo experiment comparing a control IgGl versus the humanized version of UMGl-mAb (h-UMGl) and an afucosylated version of UMGl-mAb (a-h-UMGl).
- Methods In this experiment 15 NOD-SCID-g-chain-null (NSG) mice were engrafted subcutaneously with 5xl0 6 HPB-ALL cells. Mice were then randomized to receive weekly intra-peritoneal administration of 15 mg/kg of control IgGl, h-UMGl or a-h-UMGl starting from day 1 until either death, tumor volume >2000mm A 3, or unacceptable toxicity. Tumor volume was assessed every other day and the average volume of the tumor for each treatment group at each time point is reported and summarized in Figure 11.
- Antigen Receptor-T cells (CAR-UMGl) induce T-cell activation in the presence of H9 cells
- a third generation CAR was designed by coupling an extracellular domain consisting of a scFv derived from the sequence of the UMG1 antibody (SEQ ID NO: 7 for the heavy chain and SEQ ID NO: 12 for the light chain) with an intracellular region consisting of the CD3z chain (the signaling region of the TCR), and two co-stimulatory domains, CD28 and 4-1BB, thus mimicking physiological T-cell activation.
- a map of the CAR construct is provided in Figure 20 (circularized map) and the complete sequence of the CAR construct is provided in SEQ ID NO: 41.
- the construct was cloned as a CAR cassette in a lentivirus vector (Qin DY et al., Anticancer Drugs. 2016 Sep;27(8):711-22). Subsequently, viral particles were used to transduce CD3 + lymphocytes from healthy donors at a multiplicity of infection (MOI) of 5 and transduction efficiency was evaluated by flow cytometry (about 38%). These CAR-T cells were assayed for their ability to release IFNy and IL-2 in the presence of target cells and for their selective cytotoxicity capability.
- MOI multiplicity of infection
- Chimeric antigen receptor CAR-UMGl induces significant cytotoxicity against cells expressing the UMG1 epitope.
- UMG1-CD3 bispecific antibody To test the specificity of a UMG1-CD3 bispecific antibody, and its ability to redirect T-cell cytotoxicity to UMG1 positive cells, assays were conducted on KE37 cell line which expresses the UMG1 CD43 epitope but is negative for CD3 (UMG1 + , CD3 ), and ALL-SIL cell line which is negative for both the UMG1 antigen and CD3 (UMG1-, CD3 ).
- a UMG1-CD3 construct comprising SEQ ID NO: 40 was used to generate a UMG1-CD3 bispecific antibody. Redirected T-cell cytotoxicity was assayed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and the KE37 cell line (UMG1 + , CD3 ) and ALL-SIL cell line (UMGU, CD3 ).
- FIGs. 18A-18B Representative FACS images from experiments using 1 pg/ml UMG1-CD3 bispecific antibody and E:T cell ratios of 20: 1 are showed in FIGs. 18A-18B.
- Results Increased killing was observed in both cell lines, KE37 (see FIG. 18A) and ALL-SIL (see FIG. 18B) treated with UMG1-CD3 bispecific antibody compared to untreated cells (indicated as NT).
- the KE37 cell line expressing the UMG1 antigen showed higher cell death, ⁇ 86 % of the cell population assayed, while ALL-SIL cell line which does not express the UMG1 antigen, has a lower cell death %, of -22% of the cell population assayed.
- This example tests the binding of the UMG1-CD3 bispecific antibody on KE37 cell line (UMG1 + , CD3 ), CCRF-CEM cells (UMGE, CD3 + ) and Jurkat cell line (UMGE,
- Binding activity was assayed by flow cytometry using KE37 cell line (UMG1 + , CD3 ), CCRF-CEM cells (UMGE, CD3 + ) and Jurkat cell line (UMGE, CD3 + ).
- T-ALL cell lines were incubated with increasing concentrations of UMG1-CD3 bispecific antibody for 20 minutes. After two lx PBS pH 7.4 (Gibco, 10010-015) washes (5 min, RT, 1300 rpm), AF647 anti-human IgG (PE) was used to stain T-ALL cells for 20 minutes. After two lx PBS pH 7.4 (Gibco, 10010-015) washes (5 min, RT, 1300 rpm), binding activity was evaluated by flow cytometry as percentage of PE positive cells in treated cells as compared to negative control.
- This example tests the efficacy of the UMG1-CD3 bispecific antibody on T-ALL cell lines and patient-derived primary T-ALL cells.
- a UMG1-CD3 construct comprising SEQ ID NO: 40 was used to generate a UMG1-CD3 bispecific antibody. Redirected T-cell cytotoxicity was assayed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and a panel of T- ALL cell lines and T-ALL primary blasts.
- Results Increased killing was observed mainly in UMG1 positive cells after treatment with UMG1-CD3 bispecific antibody compared to untreated cells (FIGs. 21A-21B; FIGs. 22A-22C). Further, the CCRF-CEM (see FIG. 21A and FIG. 22A), KE37 cell line (see FIG. 22B) and EGILIII T-ALL blasts (see FIG. 21B) expressing UMG1 antigen showed higher cell death, (80 to 95%) of the cell population assayed, while ALL-SIL cell line which does not express the UMG1 antigen, has a lower cell death (10%) of the cell population assayed (see FIG. 22C).
- T-ALL cell lines were co-cultured with total human PBMCs or PBMCs depleted of CD8 + T cells or CD4 + T cells by immunomagnetic beads.
- This example shows the ability of the UMG1-CD3 bispecific to induce apoptosis in a T-ALL cell line.
- Apoptosis was evaluated by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and CCRF-CEM cells.
- PBMC activation was evaluated by flow cytometry analysis of CD69 and CD25 positivity on CD4 and CD8 cells after 24 hours of treatment with UMG1-CD3 bispecific antibody or negative control. Protein expression of phospho-NFKB-p65 (A-8, Santacruz) was also evaluated by Western-blot analysis, 96 hours after UMG1-CD3 bispecific antibody treatment of PBMC alone or CCRF-CEM alone.
- Cytokines release was evaluated after 4 hours of PBMC incubation with Brefeldin A (Santacruz) as Intracellular IFNy and TNFa (BD Pharmingen) positivity on CD4 and CD8 cells 24h from UMG1-CD3 bispecific antibody treatment.
- UMG1-CD3 bispecific on a testicular cancer (seminoma) cell line
- Methods Redirected T-cell cytotoxicity was assayed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) and seminoma cell lines. In particular, TCAM2 seminoma UMG1 epitope positive cell line was tested.
- ⁇ V is A in germ line
- CD43 Clone #4 truncated CD43 (aa 61 to 400) [SEQ ID NO: 20]: • QTSALPPSTSINEGSPLWTSIGASTGSPLPEPTTYQEVSIKMSSVPQETPH AT SHP AVPIT AN SLGSHT VT GGTITTN SPETS SRTSGAP VTT AAS SLET S RGT S GPPLTM AT V SLET SKGT S GPP VTM ATD SLET S T GTTGPP VTMTT GSLEPSSGASGPQVSSVKLSTMMSPTTSTNASTVPFRNPDENSRGMLP
- AGT C C AGT GG A A AGT GG AC A AT GC TC T GC AG AGT GGG A AT T
- VH3 Human Heavy Chain (clone PRO 7683_evi-5 UMGHUM3-hl .HC)
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BR112021025908A BR112021025908A2 (en) | 2019-06-21 | 2020-06-19 | anti-cd43 antibody or antigen-binding fragment thereof, bispecific antibody, car-t cell, method of treating a cd43 positive cancer, method of identifying a cd43 positive cancer and method of diagnosing and treating a cd43 positive cancer |
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