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ANTIBODIES TARGETING, AND OTHER MODULATORS OF, THE CD276 ANTIGEN, AND USES THEREOF

20230007977 · 2023-01-12

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention pertains to antibodies or other antigen binding proteins targeting the CD276 antigen, also known as B7-H3. The invention provides an improved set of antibodies which bind at new positions within the CD276 antigen and are of particular use as therapeutics in the treatment of CD276 positive cancer. Further the invention provides antibody conjugate and bispecific antibodies which were developed on basis of the novel anti-CD276 antibodies of the invention. Furthermore, the invention discloses the therapeutic use of the antibodies and other modulators in the treatment of CD276 positive cancer. Finally, the nucleic acid constructs encoding the molecules of the invention, recombinant cells expressing them, as well as particular uses and methods are provided.

    Claims

    1. An isolated antigen binding protein (ABP) which specifically binds to a CD276 protein, or a variant thereof, and wherein the isolated ABP is able to induce an antibody dependent cell-mediated cytotoxicity (ADCC) against a cell expressing the CD276 protein; wherein the ABP comprises at least one, preferably two, Complementary Determining Region (CDR) 3 having an amino acid sequence with at least 80% sequence identity to, or having no more than three or two, preferably one amino acid substitution(s), deletion(s) or insertion(s) relative to, a sequence selected from SEQ ID NOs. 3, 7, 11, 15, 19, 23, 27, 31, 35, and 39.

    2. The isolated ABP of claim 1, wherein said ABP further comprises at least one, preferably two, CDR1, and at least one, preferably two, CDR2.

    3. The isolated ABP of claim 1, comprising an antibody heavy chain sequence and/or an antibody light chain sequence, or an antigen binding fragment thereof; wherein the antibody heavy chain sequence, or the fragment thereof, comprises a CDR3 having at least 80% sequence identity to, or having no more than three or two, preferably one amino acid substitution(s), deletion(s) or insertion(s) relative to, a sequence selected from SEQ ID NOs. 3, 11, 19, 27, and 35, and/or wherein antibody light chain sequence, or the fragment thereof, comprises a CDR3 having at least 80% sequence identity to, or having no more than three or two, preferably one amino acid substitution(s), deletion(s) or insertion(s) relative to, a sequence selected from SEQ ID NOs. 7, 15, 23, 31, and 39.

    4. The isolated ABP of claim 1, comprising an antigen binding fragment of an antibody, wherein said antigen binding fragment comprises CDR1, CDR2 and CDR3, optionally selected from the CDR1, CDR2 and CDR3 sequences having the respective amino acid sequences of SEQ ID Nos. 1, 2, 3, or 5, 6, 7, or 9, 10, 11, or 13, 14, 15, or. 17, 18, 19, or 21, 22, 23, or 25, 26, 27, or. 29, 30, 31, or 33, 34, 35, or 37, 38, 39; in each case independently, optionally with not more than three or two, preferably one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

    5. The isolated ABP of claim 1, wherein the ABP is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences; and the ABP has at least one antigen binding domain which: (A) comprises an antibody heavy chain CDR1 sequence shown in SEQ ID NO: 1, an antibody heavy chain CDR2 sequence shown in SEQ ID NO: 2, and an antibody heavy chain CDR3 sequence shown in SEQ ID NO: 3; and an antibody light chain CDR1 sequence shown in SEQ ID NO: 5, an antibody light chain CDR2 sequence shown in SEQ ID NO: 6, and an antibody light chain CDR3 sequence shown in SEQ ID NO: 7; in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences; or (B) comprises an antibody heavy chain CDR1 sequence shown in SEQ ID NO: 9, an antibody heavy chain CDR2 sequence shown in SEQ ID NO: 10, and an antibody heavy chain CDR3 sequence shown in SEQ ID NO: 11; and an antibody light chain CDR1 sequence shown in SEQ ID NO: 13, an antibody light chain CDR2 sequence shown in SEQ ID NO: 14, and an antibody light chain CDR3 sequence shown in SEQ ID NO: 15; in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences; or (C) comprises an antibody heavy chain CDR1 sequence shown in SEQ ID NO: 17, an antibody heavy chain CDR2 sequence shown in SEQ ID NO: 18, and an antibody heavy chain CDR3 sequence shown in SEQ ID NO: 19; and an antibody light chain CDR1 sequence shown in SEQ ID NO: 21, an antibody light chain CDR2 sequence shown in SEQ ID NO: 22, and an antibody light chain CDR3 sequence shown in SEQ ID NO: 23; in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences; or (D) comprises an antibody heavy chain CDR1 sequence shown in SEQ ID NO: 25, an antibody heavy chain CDR2 sequence shown in SEQ ID NO: 26, and an antibody heavy chain CDR3 sequence shown in SEQ ID NO: 27; and an antibody light chain CDR1 sequence shown in SEQ ID NO: 29, an antibody light chain CDR2 sequence shown in SEQ ID NO: 30, and an antibody light chain CDR3 sequence shown in SEQ ID NO: 31; in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences; or (E) comprises an antibody heavy chain CDR1 sequence shown in SEQ ID NO: 33, an antibody heavy chain CDR2 sequence shown in SEQ ID NO: 34, and an antibody heavy chain CDR3 sequence shown in SEQ ID NO: 35; and an antibody light chain CDR1 sequence shown in SEQ ID NO: 36, an antibody light chain CDR2 sequence shown in SEQ ID NO: 37, and an antibody light chain CDR3 sequence shown in SEQ ID NO: 38; in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

    6. The isolated ABP according to claim 1, wherein said ABP is modified or engineered to increase antibody-dependent cellular cytotoxicity (ADCC), preferably wherein said ABP comprises the SDIE mutations and/or is afucosylated.

    7. The isolated ABP according to claim 1, which comprises one or more additional antigen binding domain(s) that bind(s) to antigen(s) other than said CD276, or the variant thereof; such as antigen(s) present on a mammalian T-cell, and most preferably human CD3.

    8. The isolated ABP of claim 1, which further comprises a moiety which enhanced antibody dependent cell cytotoxicity (ADCC), preferably wherein the moiety is an immunocytokine (MIC) such as Interleukin-15 (IL-15) or modified IL-15.

    9. A bispecific antigen binding protein (ABP) which comprises a first antigen binding domain capable of binding to the CD276 antigen, or the variant thereof, and a second antigen binding domain capable of binding to an antigen expressed on an immune cell, preferably CD3; wherein the first antigen binding domain is an antigen binding domain of the ABP according to claim 1.

    10. The bispecific ABP of claim 9, having an activity to bind to a T-cell expressing CD3 and to a CD276 expressing tumor cell, or a tumor cell adjacent cell expressing CD276, such as a tumor vascular cell, preferably wherein the bispecific ABP increases the recruitment of cytotoxic cells to a CD276 expressing cell by binding to CD276 and CD3.

    11. An isolated nucleic acid comprising a sequence encoding for an ABP, or for an antigen binding fragment or a monomer, such as a heavy or light chain, of an ABP, of claim 1.

    12. A nucleic acid construct (NAC) comprising a nucleic acid of claim 11 and one or more additional sequence features permitting the expression of the encoded ABP or bispecific ABP, or a component of said ABP or bispecific ABP (such as an antibody heavy chain or light chain) in a cell.

    13. A recombinant host cell, comprising a nucleic acid of claim 11.

    14. A pharmaceutical composition comprising an ABP or bispecific ABP of claim 1, and a pharmaceutically acceptable carrier, stabilizer and/or excipient.

    15. (canceled)

    16. A method for enhancing T cell-mediated killing of and/or inhibiting the proliferation of CD276 positive tumor or tumor associated cells, or tumor cells or tumor associated cells positive for the variant of CD276 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an ABP or a bispecific ABP of claim 1.

    17. A method for treating or preventing a proliferative disorder characterized by the expression of CD276 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an ABP or a bispecific ABP of claim 1.

    Description

    BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

    [0219] The figures show:

    [0220] FIG. 1: shows the antibodies and antibody constructs of the invention; (A) a summary of epitope mapping results with the newly generated B7-H3 antibodies. The scheme shows the crystal structure of B7-H3 antigen (Vigdorovich V et al., Structure 2013), arrows identify the binding sites of the various proprietary B7-H3 mAb. Note that the 7C4 antibody contained in CC-3 binds an epitope close to the cell membrane. The indicated antibodies no longer bound to CD276 mutants carrying the indicated amino acid exchanges. These changes were selected on the bases of differences between the CD276 sequence of non human primates and humans. (B) Cartoons of monospecific antibodies (left), immunocytokines (MIC proteins, middle) and bispecific IgGsc antibodies (right). Note that monospecific antibodies were used either with a wildtype human IgG1 Fc part or a SDIE modified version thereof (as depicted).

    [0221] FIG. 2: shows Biacore® sensorgrams determining the binding of B7-H3 antigen to the indicated B7-H3xCD3 bsAbs. Note that constructs containing 7C4 or 8H8 exhibit particularly long off-rates.

    [0222] FIG. 3: shows binding of different B7-H3xCD3 bsAbs to B7-H3 and CD3 expressed on NALM-16 (A) and Jurkat cells (B), respectively, as determined by flow cytometry. Note that in all constructs the anti CD3 antibody affinity was attenuated to reduce off target T cell activation, while binding to the target antigen B7-H3 is profoundly higher for all constructs.

    [0223] FIG. 4: shows NK cell activation and tumor cell depletion by various monospecific CD276 antibodies (A,B) or MIC proteins containing SDIE-modified Fc-parts (C,D) Methods: Tumor cells (LNCap) were incubated with human PBMC and the indicated antibodies. After 3 days NK cell activation and depletion of tumor cells was determined by flow cytometry.

    [0224] FIG. 5: shows NK cell activation, -proliferation and tumor cell depletion by various CD276 antibodies (A-C) or MIC proteins (D-F) containing wildtype human IgG1 Fc-parts Methods: Tumor cells (LNCap) were incubated with human PBMC and the indicated antibodies. After 3 days NK cell activation and depletion of tumor cells was determined by flow cytometry.

    [0225] FIG. 6: shows T cell activation and tumor cell killing by various CD276 antibodies incorporated into bispecific antibodies in the IgGsc format (see FIG. 1B); Methods: B7-H3 expressing NALM-16 tumor cells were incubated with PBMC of healthy donors in the presence of increasing concentrations of the indicated B7-H3xCD3 constructs or a control bsAb with an unrelated specificity and activation of CD4 and CD8 T cells as well as tumor cells killing was determined by flow cytometry after 3 days (B). Induction of T cell proliferation (C) was assessed using a 3H-thymidine incorporation assay. Note that CC-3 (7C4xCD3) in all assays mediates markedly superior T cell stimulation compared to the other B7-H3xCD3 bsAbs. Long time xCELLigence tumor lysis assay (D) was performed with B7-H3 expressing LNCaP tumor cells and PBMC of healthy donors in the presence of the indicated B7-H3xCD3 bsAb. Note that CC-3 (7C4xCD3) mediates superior T cell activation and tumor cell killing compared to the other B7-H3xCD3 bsAbs

    [0226] FIG. 7: shows in vivo antitumor activity of different CD276 antibodies incorporated into bispecific IgGsc antibodies (FIG. 1B) Methods: LNCaP cells were injected s.c. into the right flank of female NSG mice to establish tumors with a diameter of 5 mm. Then (day 1) PBMC with or without B7-H3xCD3 antibodies (2 μg per dose) or an irrelevant controls bsAb were applied i.v. at days 1, 8 and 15 (indicated with arrows). Tumor growth was measured twice weekly, and mice were euthanized when tumors had reached a diameter of 15 mm. Note that in this in vivo setting, CC-3 (7C4xCD3) is again markedly more effective than 8D9xCD3 confirming the in vitro data presented.

    [0227] The sequences show:

    TABLE-US-00005 Table 1 Antibody Sequences of the Invention: SEQ ID Antibody Sequence- NO: Name: Domain: Amino Acid Sequence:  1 7C4 VH-CDR1 EYTMH  2 7C4 VH-CDR2 GINPNNGGTTYNQIFKN  3 7C4 VH-CDR3 RGYHVSSWYFDV  4 7C4 VH-Variable EVQLEQSGPELVKPGTSVKISCKTSGYTFTEY Full Length TMHWVKQSHGKSLEWIGGINPNNGGTTYN QIFKNKATLTVDKSSSTAYMELRSLTSEDSA VYYCARRGYHVSSWYFDVWGAGTTVTVSS  5 7C4 VL-CDR1 SASSSVSYMH  6 7C4 VL-CDR2 DTSKLAS  7 7C4 VL-CDR3 LQWHSNPLT  8 7C4 VL-Variable DIVLTQSTAIMSASPGEKVTMTCSASSSVSY Full Length MHWYQQKSGTSPKRWIYDTSKLASGVPARF SGSGSGTSYSLTISSMETEDSATYYCLQWHS NPLTFGAGTKLELKR  9 11A7 VH-CDR1 NYWMN 10 11A7 VH-CDR2 EIRLKSNNYATHYAESVKG 11 11A7 VH-CDR3 HAD 12 11A7 VH-Variable EVQLEESGGGLVQPGGSMKLSCVVSGFTFR Full Length NYWMNWVRQSPEKGLEWVAEIRLKSNNYA THYAESVKGRFTISRDDSKSSVYLQMNNLR AEDTGIYYCNSHADWGQGTLVSVSA 13 11A7 VL-CDR1 RASENIYYTLA 14 11A7 VL-CDR2 NANSLED 15 11A7 VL-CDR3 KQGYDVPYT 16 11A7 VL-Variable DIVLTQTTASLAASVGETVTITCRASENIYYT Full Length LAWYQQKQGKSPQLLIYNANSLEDGVPSRF SGSGSGTQYSMKINSMQPEDTATYFCKQGY DVPYTFGGGTKLEIKR 17 8D9 VH-CDR1 SYAMS 18 8D9 VH-CDR2 TISTGGSYTYYADSVKG 19 8D9 VH-CDR3 HLYLYFDV 20 8D9 VH-Variable EVKLEQSGGGLVKPGGSLKLSCAASGFTFSS Full Length YAMSWVRQKPEKRLEWVATISTGGSYTYYA DSVKGRFTISRDDARNTLNLQMSSLRSEDT AMYYCARHLYLYFDVWGAGTTVTVSS 21 8D9 VL-CDR1 RASENIYSYLA 22 8D9 VL-CDR2 NAKTLAE 23 8D9 VL-CDR3 QHHYGTPPYT 24 8D9 VL-Variable DIVMTQSTASLSASVGETVTITCRASENIYSY Full Length LAWYQQKQGKSPQLLVYNAKTLAEGVPSRF SGSGSGTQFSLKINSLQPEDFGSYYCQHHYG TPPYTFGGGTKLEIKR 25 10A7 VH-CDR1 SYYIH 26 10A7 VH-CDR2 WIYPGNVNTNYNERFKG 27 10A7 VH-CDR3 GTYFFAY 28 10A7 VH-Variable EVQLQQSGPELVKPGASVRISCKASGYTFTS Full Length YYIHWVKQRPGQGLEWIGWIYPGNVNTNY NERFKGKATLTADKSSSTAYMQLSSLTSEDS AVYFCAGGTYFFAYWGQGTLVTVSA 29 10A7 VL-CDR1 RASKSISKYLA 30 10A7 VL-CDR2 SGSTLQS 31 10A7 VL-CDR3 QQHNEYPLT 32 10A7 VL-Variable DIVLTQSPSHLAASPGETITINCRASKSISKYL Full Length AWYQEKPGKTNKLLIYSGSTLQSGVPSRFSG SGSNTDFTLTISSLEPEDFAMYYCQQHNEYP LTFGAGTELELKR 33 8H8 VH-CDR1 DFDIN 34 8H8 VH-CDR2 WIFPGDGSTKYDETFKD 35 8H8 VH-CDR3 PRYGGSWFAY 36 8H8 VH-Variable EVQLEQSGPDLVKPGASVKLSCKASGYTFTD Full Length FDINWVRQRPEQGLEWIGWIFPGDGSTKYD ETFKDKATLTTDKSSSTAYMQLSRLTSEDSA VYFCARPRYGGSWFAYWGQGTLVSVSA 37 8H8 VL-CDR1 KSSQSLLYNTIQRSYLA 38 8H8 VL-CDR2 WASTRES 39 8H8 VL-CDR3 QQNYQYPWT 40 8H8 VL-Variable DIVLTQSPSSLTVSVGEKVTMSCKSSQSLLYN Full Length TIQRSYLAWYQQKPGQSPKLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVKAEDLAVYFC QQNYQYPWTFGGGTKLEIKR Abbreviations: VH: “variable heavy chain”, VL: “variable light chain”; CDR: “Complementary Determining Region”

    TABLE-US-00006 SEQ ID NO: 41 shows the amino acid sequence of human CD276 isoform 1:         10         20         30         40         50 MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC         60         70         80         90        100 SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL        110        120        130        140        150 AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE        160        170        180        190        200 PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG        210        220        230        240        250 LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ        260        270        280        290        300 VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG        310        320        330        340        350 RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA        360        370        380        390        400 AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ        410        420        430        440        450 GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH        460        470        480        490        500 GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN        510        520        530 AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA SEQ ID NO: 42 shows the amino acid sequence of human CD276 isoform 2:         10         20         30         40         50 MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC         60         70         80         90        100 SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL        110        120        130        140        150 AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE        160        170        180        190        200 PNKDLRPGDT VTITCSSYRG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG        210        220        230        240        250 LFDVHSVLRV VLGANGTYSC LVRNPVLQQD AHGSVTITGQ PMTFPPEALW        260        270        280        290        300 VTVGLSVCLI ALLVALAFVC WRKIKQSCEE ENAGAEDQDG EGEGSKTALQ        310 PLKHSDSKED DGQEIA SEQ ID NO: 43 shows the amino acid sequence of human CD276 isoform 3:         10         20         30         40         50 MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC         60         70         80         90        100 SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL        110        120        130        140        150 AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE        160        170        180        190        200 PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG        210        220        230        240        250 LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ        260        270        280        290        300 VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG        310        320        330        340        350 RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA        360        370        380        390        400 AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ        410        420        430        440        450 GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH        460        470        480        490 GSVTITGQPM TFPPGPASSA VPLSPAHPPH GSMCWSHWFS RGL SEQ ID NO: 44 shows the amino acid sequence of human CD276 isoform 4:         10         20         30         40         50 MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC         60         70         80         90        100 SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL        110        120        130        140        150 AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE        160        170        180        190        200 PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG        210        220        230        240        250 LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ        260        270        280        290        300 VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG        310        320        330        340        350 RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA        360        370        380        390        400 AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ        410        420        430        440        450 GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH        460        470        480        490        500 GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN        510        520        530 AGAEDQDGEG EGSKTALQPL KHSDSKEGKD TWA

    EXAMPLES

    [0228] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

    [0229] In course of the present invention a series of seven antibodies was generated after immunization of mice with recombinant CD276 protein using a hybridization and screening procedure. The binding epitope of the antibodies was then characterized (see FIG. 1A) and representative antibodies for each epitope were selected for construction of the following derivatives using recombinant antibody technology (see FIG. 1B): [0230] monospecific antibodies containing wildtype or SDIE-modified human IgG1 Fc-parts) [0231] immunocytokines containing a modified IL-15 part and wild type or SDIE-modified human IgG1 Fc-parts (MIC proteins) [0232] bispecific antibodies with CD276xCD3 specificity in the previously developed IgGsc-format

    [0233] The antitumor activity of the different constructs was then tested in vitro and in vivo, and CD276 binders with optimal activity in the various formats were identified according to the following examples.

    [0234] The examples show:

    Example 1: Generation of Antibodies

    [0235] Balb/c mice were immunized with recombinant CD276 protein and spleen cells were fused according to standard protocols. Female, 6 month old BALB/C mice were immunized using soluble CD276-Fc fusion protein. The protein used, consists out of the extracellular domain of human CD276 Isoform 2 (NM_001024736.1) with Met1-Thr461, fused to the N-terminus of the Fc region of human IgG1. 50 μg of the Fc fusion protein was repeatedly applied intraperitoneally in a volume of 100 μl PBS for each immunization. The mice were immunized every 10 days for a total of 3 to 4 immunizations with a final intravenous application 4 days prior of the fusion. Supernatants of fused cells were screened for production of specific antibodies by evaluating binding to CD276-transfected mouse cells by flow cytometry. A total of 7 hybridomas were then recloned and grown in advanced DMEM medium supplemented with 1.25% FCS. Antibodies were purified from culture supernatant by protein affinity chromatography and size exclusion chromatography (SEC) on Superdex S200. Purity was evaluated by SDS page and analytical SEC. Sequences of the generated antibodies are provided in table 1.

    Example 2: Epitope Mapping of the New CD276-Antibodies

    [0236] The human CD276 molecule exists as two isoforms, namely 2IgB7-H3 and 4IgB7-H3, with the latter one being the result of an exon duplication and that constitutes the predominant form in humans. The extracellular structure of the protein is characterized by IgV-IgC-like (2IgB7-H3) or IgV1-IgC1-IgV2-IgC2-like (4IgB7-H3) domains. Mice express only the 2IgB7- H3 isoform. For the epitope mapping, we first generated truncated versions of the 4IgB7-H3 molecule, IgV1-IgC1 and IgV2-IgC2, as Fc fusion proteins. We noted that all antibodies recognized both truncated versions. In addition, none of the antibodies were cross-reactive with the murine protein. Therefore, a sequence alignment between the highly conserved human and murine protein was performed. Finally, we replaced the variable amino acids in the human IgV1-IgC1-Fc protein with the respective amino acids of the murine molecule. This resulted in the epitope map and grouping of the antibodies as shown in FIG. 1A. In addition, FACS based competition assays were performed, confirming that antibodies within the same group crossblock each other whereas antibodies of different groups don not.

    Example 3: Generation and Characterization of Recombinant Antibody Derivatives

    [0237] The variable regions of representative antibodies of each group were sequenced, incorporated into different antibody formats and the antitumor activity of the antibodies was tested in suitable functional assays:

    (1) SDIE optimized monospecific antibodies: ADCC Graph with 3 SDIE antibodies. Results are provided for the Fe enhanced version of antibodies 7C4 and 8D9 in FIG. 4. The antibody 7C4 outperforms the other, with significantly higher NK cell activation, and tumor cell depletion.
    (2) Immunocytokine containing a modified IL-15 moiety: shown is NK cell activation and tumor cell killing of antibodies MIC 7C4, 8H8 and 8D9 in FIGS. 4 and 5. As can be seen all antibodies perform excellent with the 7C4 being the most active antibody.
    (3) bispecific antibodies with CD265xCD3-specificity Biacore, FACS binding and tumor cell killing: results are shown in FIGS. 2, 3, and 6 to 7.

    REFERENCES

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