EA042568B1 - NEW B7-H3 BINDING MOLECULES CONTAINING THEIR ANTIBODY-DRUG CONJUGATES AND METHODS FOR THEIR APPLICATION - Google Patents

Description

Cross-reference to related applications

This application claims priority under U.S. Patent Applications No. 62/432314 (filed December 9

2016; pending), 62/323249 (filed April 15, 2016; pending), 62/323228 (filed April 15, 2016; pending), each of which is incorporated herein by reference in its entirety.

Link to sequence listing

This application includes one or more sequence listings in accordance with 37 CFR 1.821 ff, which are disclosed on computer-readable media (file name: 1301_01430144PCT_ST25.txt, created March 28, 2017 and has a size of 104,762 bytes), which is incorporated herein by reference in full.

The field of technology to which the invention belongs

The present invention is directed to novel B7-H3 binding molecules capable of binding human and non-human B7-H3, and in particular to those molecules that are cross-reactive with B7-H3 of non-human primates (e.g. Javanese toque). The invention further relates to B7-H3 binding molecules that include light chain variable domains and/or heavy chain variable domains (VH) that have been humanized and/or deimmunized to exhibit reduced immunogenicity when administered to recipient subjects. The invention specifically relates to bispecific, trispecific, or multispecific B7-H3 binding molecules, including bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules, etc., which include: (i) such B7-H3 binding variable domains; and (ii) a domain capable of binding to an epitope of a molecule present on the surface of the effector cell. The invention also relates to pharmaceutical compositions that include any of such B7-H3 binding molecules, and to methods involving the use of any of such B7-H3 binding molecules in the treatment of cancer and other diseases and conditions. The invention also specifically relates to a molecule that includes a human B7-H3 binding domain of a humanized anti-human B7-H3 antibody conjugated to at least one drug moiety (B7-H3-ADC). The invention also relates to pharmaceutical compositions that include such B7-H3-ADCs and to methods associated with the use of any of these B7-H3-ADCs in the treatment of cancer and other diseases and conditions.

Prior Art

The growth and metastasis of tumors largely depend on their ability to evade host immune surveillance and overcome host defenses. Most tumors express antigens that can be recognized to a large extent by the host's immune system, but in many cases an inadequate immune response is due to inefficient activation of effector T cells (Khawli, L.A. et al. (2008) Cytokine, Chemokine, and Co- Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors Exper Pharmacol 181:291-328).

I. Superfamily B7 and B7-H3.

B7-H3 is a member of the B7-CD28 superfamily and is expressed on antigen-presenting cells. It binds to T cells, but the B7-H3 counter-receptor on the surface of such T cells has not yet been fully characterized.

B7-H3 is unique in that the basic human form contains two extracellular domains with IgV-IgC tandems (i.e. IgV-IgC-IgV-IgC) (Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands Genome Biol 6:223.1-223.7). Although originally thought to contain only 2 Ig domains (IgV-IgC), a variant with four extracellular immunoglobulin domains (4Ig-B7-H3) has been identified and recognized as the more common form of the protein in humans (Sharpe, A.H. et al., (2002) The B7-CD28 Superfamily, Nature Rev. Immunol. 2:116-126). However, the natural form in mice (2Ig) and the 4Ig form in humans show a similar function (Hofmeyer K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278). The 4Ig-B7-H3 molecule inhibits NK cell-mediated lysis of cancer cells (Castriconi, R. et al. Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl Acad Sci (U.S.A.) 101(34): 12640-12645). Human B7-H3 (2Ig form) is reported to promote T cell activation and IFN-γ production by binding to a putative receptor on activated T cells (Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2:269-274), however more recent studies point to an inhibitory role for mouse B7-H3 and human B7-H3 (Prasad, D.V., et al. (2004) Murine B7- H3 Is A Negative Regulator Of T Cells, J Immunol 173:2500-2506 Leitner, J., et al (2009) B7-H3 Is A Potent Inhibitor Of Human T-Cell Activation: No Evidence For B7-H3 And TREML2 Interaction, Eur J Immunol 39:1754-1764 Veenstra, R. G., et al (2015) B7-H3 expression in Dnor T Cells and Host Cells Negatively Regulates Acute Graft-Versus-Host Disease Lethality, Blood 125: 3335-3346.). B7-H3 mRNA expression has been found in cells of the heart, kidney, testis, lung, liver, pancreas, prostate, colon, and osteoblasts (Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome

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Biol. 6:223.1-223.7). At the protein level, B7-H3 is found in the liver, lungs, bladder, testis, prostate, breast, placenta, and lymphoid organs in humans (Hofmeyer K. et al.

(2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. sci. (U.S.A.) 105(30): 10277-10278).

Although B7-H3 is not expressed in resting B or T cells, monocytes or dendritic cells, it is induced on dendritic cells by IFN-γ and on monocytes by GM-CSF (Sharpe, A. H. et al. (2002 ) The B7-CD28 Superfamily, Nature Rev. Immunol. 2:116-126). The mechanism of action of B7-H3 is complex, and the protein has been reported to mediate both costimulation and co-inhibition of T cells (Hofmeyer K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. ( U.S.A.) 105(30): 10277-10278; Martin-Orozco, N. et al. (2007) Inhibitory Costimulation And Anti-Tumor Immunity, Semin. Cancer Biol. 17(4):288-298; Subudhi, S. K. et al. (2005) The Balance Of Immune Responses: Costimulation Verse Coinhibition, J. Mol. Med. 83:193-202). B7-H3 binds to as yet unidentified receptor(s), mediating T cell co-inhibition. In addition, B7-H3, through interactions with unknown receptor(s), is an inhibitor of NK cells and osteoblasts (Hofmeyer K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (U.S.A.) 105(30): 10277-10278). The inhibition may act through interactions with representatives of the major signaling pathways through which the T cell receptor (TCR) regulates gene transcription (eg, factors NFTA, NF-κB or AP-1). B7-H3 is believed to inhibit Th1, Th2 or Th17 in vivo (Prasad, DV et al. (2004) Murine B7-H3 Is A Negative Regulator Of T Cells, J. Immunol. 173:2500-2506; Fukushima, A. et al (2007) B7-H3 Regulates The Development Of Experimental Allergic Conjunctivitis In Mice Immunol Lett 113:52-57 Yi K.H et al (2009) Fine Tuning The Immune Response Through B7-H3 And B7-H4 , Immunol. Rev. 229:145151).

II. B7-H3-expressing tumors.

B7-H3 is also known to be expressed in various cancer cells (e.g., neuroblastoma, gastric, ovarian, non-small cell lung cancer, etc., see e.g. Modak, S., et al. (2001) Monoclonal antibody 8H9 targets a novel cell surface antigen expressed by a wide spectrum of human solid tumors Cancer Res 61:4048-54) and cultured cancer cells. Several independent studies have shown that human cancer cells exhibit a marked increase in B7-H3 protein expression and that this increased expression is associated with increased disease severity (Zang, X. et al. (2007) The B7 Family And Cancer Therapy: Costimulation And Coinhibition Clin. Cancer Res. 13:5271-5279; Sun, Y., et al. (2006) B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53:143-51; Tekle, C, et al. (2012) B7-H3 Contributes To The Metastatic Capacity Of Melanoma Cells By Modulation Of Known Metastasis-Associated Genes, Int. J. Cancer 130:2282-90 Wang, L., et al. Immunology: Friend Or Foe?, Int. J. Cancer 134(12):2764-2771), which confirms that B7-H3 is used by tumors as an escape route from immunosurveillance (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc Natl Acad Sci (U.S.A.) 105(30): 10277-10278).

B7-H3 protein expression has also been detected immunohistologically in tumor cell lines (Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production Nature Immunol. 2:269-274; Saatian , B. et al (2004) Expression Of Genes For B7-H3 And Other T Cell Ligands By Nasal Epithelial Cells During Differentiation And Activation, Amer J Physiol Lung Cell Mol Physiol 287:L217-L225 Mather (2004) Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl. Acad. Sci. (U.S.A.) 101(34): 12640-12645); Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol. 168:6294-6297).

The role of B7-H3 in inhibiting the immune system and the increase in B7-H3 expression on human tumors suggests that this molecule may serve as a therapeutic target for cancer treatment. Thus, the use of anti-B7-H3 antibodies and other molecules that modulate B7-H3 expression has been proposed for the treatment of tumors and/or positive modulation of the immune response (see Loo, D. et al. (2012) Development of an Fc -Enhanced Anti-B7-H3 Monoclonal Antibody with Potent Antitumor Activity Clin Cancer Res 18: 3834-3845 Ahmed, M. et al (2015) Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loop of B7-H3, J. Biol. Chem. 290: 3001830029 Nagase-Zembutsu, A. et al. (2016) Development of DS-5573a: A novel afucosylated monoclonal antibody directed at B7-H3 with potent antitumor activity Cancer Sci 2016, doi: 10.1111/cas.12915 Modak, S. et al.(March 1999) Disialoganglioside GD2 And Antigen 8H9: Potential Targets For Antibody-Based Immunotherapy Against Desmoplastic Small Round Cell Tumor (DSRCT) And Rhabdomyosarcoma (RMS) Proceedings Of The American Association For Cancer Research Annual Meeting, Vol 40:474 (9 ^0th Annual Mee ting Of The American Association For Cancer Research; Philadelphia, Pennsylvania, US; April 10-14, 1999; Modak, S. et al. (March 2000) Radioimmunotargeting To Human Rhabdomyosarcoma Using Monoclonal Antibody 8H9 Proc. Am. Assoc. Cancer Res.41:724; Modak, S. et al. (2001) Monoclonal Antibody 8H9 Targets A Novel Cell Surface Antigen Expressed By A Wide Spectrum Of Human Solid Tumors, Cancer Res. 61(10):4048-4054; Steinberger, P. et al. (2004) Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains, J. Immunol. 172(4):2352-2359; Xu, H. et al. (2009) MicroRNA miR-29 Modulates

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Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors, Cancer Res. 69(15):5275-6281; see also US Pat. US Nos. 7279567, 7358354, 7368554, 7527969, 7718774, 8216570, 8779098, 8802091, 9150656, publ. Pat. USA No. 2002/0168762; 2005/0202536, 2008/0081346, 2008/0116219, 2009/0018315, 2009/0022747, 2009/0087416, 2013/0078234, 2015/0274838, public. PCT No. WO 2008/066691; WO2006/016276; WO2008/116219; WO 04/001381, WO 2001/094413, WO 2002/10187, WO 2002/32375, WO 2004/093894, WO 2006/016276, WO 2008/116219/WO 2011/109400 and EP 1292619B.

Despite all such preliminary successes, there is a need for additional therapeutic agents that target and kill tumor cells expressing B7-H3. The present invention aims to solve this and other problems.

The essence of the invention

The present invention is directed to novel B7-H3 binding molecules capable of binding to human and non-human B7-H3, and in particular to those molecules that are cross-reactive with B7-H3 of non-human primates (e.g. cynomolgus monkey) . The invention further relates to B7-H3 binding molecules that include light chain variable domains and/or heavy chain variable domains (VH) that have been humanized and/or deimmunized to exhibit reduced immunogenicity when administered to recipient subjects. The invention particularly relates to bispecific, trispecific, or multispecific B7-H3 binding molecules, including bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules, and the like. which include: (i) such B7-H3 binding variable domains; and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell.

The invention also relates to pharmaceutical compositions that include any of such B7-H3 binding molecules, and to methods involving the use of any of such B7-H3 binding molecules in the treatment of cancer and other diseases and conditions. The invention also specifically relates to a molecule that includes a human B7-H3 binding domain from a humanized anti-human B7-H3 antibody conjugated to at least one drug moiety (B7-H3-ADC). The invention also relates to pharmaceutical compositions that include such B7-H3-ADCs and to methods associated with the use of any of these B7-H3-ADCs in the treatment of cancer and other diseases and conditions.

In particular, one aspect of the present invention provides a B7H3 binding molecule that includes a light chain variable domain (VL) and a heavy chain variable domain (VH), wherein said variable chain domain comprises a CDR _H 1 domain, a CDR _H 2 domain, and a CDR _H 3, and said light chain variable domain comprises a CDRL1 domain, a CDRL2 domain, and a CDRL3 domain in which at least three of said domains, at least four of said domains, at least five of said domains, or all of said domains are selected from the group consisting of:

(1) CDRH1 domain, includes the amino acid sequence of SEQ ID NO:27;

(2) CDRH2 domain, includes the amino acid sequence of SEQ ID NO:28;

(3) CDRH3 domain, includes the amino acid sequence of SEQ ID NO:29;

(4) CDRL1 domain, includes the amino acid sequence of SEQ ID NO:23;

(5) CDRL2 domain, includes the amino acid sequence of SEQ ID NO:24; and (6) a CDRL3 domain comprising the amino acid sequence of SEQ ID NO:25.

The invention further relates to an embodiment of such a B7-H3 binding molecule which comprises said light chain variable domain (VL) which includes a CDRL1 domain, a CDRL2 domain and a CDRL3 domain and said heavy chain variable domain (VH) which includes a CDRH1 domain, a CDRH2 and CDRH3 domain, where:

(1) the specified CDRH1 domain includes the amino acid sequence of SEQ ID NO:27;

(2) the specified CDRH2 domain includes the amino acid sequence of SEQ ID NO:28;

(3) said CDRH3 domain includes the amino acid sequence of SEQ ID NO:29.

The invention further relates to an embodiment of such a B7-H3 binding molecule which comprises said light chain variable domain (VL) which includes a CDRL1 domain, a CDRL2 domain and a CDRL3 domain and said heavy chain variable domain (VH) which includes a CDRH1 domain, a CDRH2 and CDRH3 domain, where:

(1) the specified CDRL1 domain includes the amino acid sequence of SEQ ID NO:23;

(2) the specified CDRL2 domain includes the amino acid sequence of SEQ ID NO:24; and (3) said CDRL3 domain comprises the amino acid sequence of SEQ ID NO:25.

The invention further relates to an embodiment of such B7-H3 binding molecules, wherein said heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:31.

The invention further relates to an embodiment of such B7-H3 binding molecules, wherein said light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:30.

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The invention further relates to B7-H3 binding molecules that include a VL domain and a VH domain, wherein said VL domain includes the amino acid sequence of SEQ ID

NO:20.

The invention further relates to B7-H3 binding molecules that include a VL domain and a VH domain, wherein said VH domain comprises the amino acid sequence of SEQ ID NO:21.

The invention further relates to B7-H3 binding molecules which include a VL domain and a VH domain, wherein said VL domain comprises the amino acid sequence of SEQ ID NO:20 and said VH domain comprises the amino acid sequence of SEQ ID NO:21.

The invention also relates to an embodiment of such B7-H3 binding molecules, wherein the molecule is an antibody or an epitope-binding fragment thereof. The invention also relates to embodiments of such a B7-H3 binding molecule, wherein the molecule is a bispecific antibody or diabody, especially a diabody or diabody complex, which contains two, three, four or five polypeptide chains, each of which has an N-terminus and a C- an end where such polypeptide chains are linked to each other through one or more covalent bonds, and especially one or more covalent disulfide bonds. The invention further relates to an embodiment of such B7-H3 binding molecules, wherein the molecule is a trivalent binding molecule, and especially wherein the trivalent binding molecule is a covalently linked complex that contains three, four, five or more polypeptide chains. The invention also relates to an embodiment of such a B7-H3 binding molecule, wherein the molecule includes an Fc domain. The invention further relates to an embodiment of such B7-H3 binding molecules, wherein the molecule is a diabody and includes an albumin binding domain, and in particular a deimmunized albumin binding domain.

The invention also relates to embodiments of all such B7-H3 binding molecules which further comprise an Fc domain, and especially wherein the Fc domain is a variant Fc domain that contains one or more amino acid modifications that reduce the affinity of the variant Fc domain for FcyR and/or increase the period the serum half-life of the B7-H3 binding molecule, and more specifically, wherein the modifications comprise at least one substitution selected from the group consisting of:

(a) L234A;

(b) L235A;

(c) L234A and L235A;

(d) M252Y; M252Y and S254T;

(e) M252Y and T256E;

(f) M252Y, S254T and T256E;

(g) K288D and H435K;

where the numbering is the EU index, as in Kabat.

The invention also relates to an embodiment of such B7-H3 binding molecules wherein the molecule is bispecific, and particularly relates to an embodiment in which the molecule contains two epitope-binding sites capable of immunospecific binding to a B7-H3 epitope and two epitope-binding sites capable of immunospecific binding with an epitope of a molecule present on the surface of the effector cell, or an embodiment in which the molecule contains one epitope-binding site capable of immunospecific binding to a B7-H3 epitope and one epitope-binding site capable of immunospecific binding to an epitope of a molecule present on the surface of the effector cell.

The invention further relates to an embodiment of such B7-H3 binding molecules, wherein the molecule is a trivalent binding molecule, and in particular relates to embodiments in which the molecule contains one epitope-binding site capable of immunospecific binding to a B7-H3 epitope, one epitope-binding site capable of immunospecific binding to the epitope of the first molecule present on the surface of the effector cell; and one epitope-binding site capable of immunospecific binding to an epitope of a second molecule present on the surface of the effector cell, such first and second molecules being other than B7-H3.

The invention further relates to an embodiment of such a B7-H3 binding molecule, where the molecule is capable of simultaneously binding to B7-H3 and to a second epitope, and in particular relates to an embodiment in which the second epitope is an epitope of a second molecule present on the surface of an effector cell (especially wherein the second epitope is a CD2, CD3, CD8, CD16, TCR or NKG2D epitope and most specifically wherein the second epitope is a CD3 epitope). The invention further relates to an embodiment of such B7-H3 binding molecules wherein the effector cells are a cytotoxic T cell or a natural killer (NK) cell. The invention further relates to an embodiment of such B7-H3 binding molecules wherein the molecule is also capable of binding a third epitope, and in particular relates to an embodiment wherein the third epitope is

- 4 042568 CD8 epitope. The invention also relates to embodiments of such molecules, wherein the molecule mediates coordinated binding of a B7-H3 expressing cell to a cytotoxic T cell.

The invention also relates to pharmaceutical compositions containing an effective amount of any of the above B7-H3 binding molecules and a pharmaceutically acceptable carrier, excipient or diluent.

The invention is further directed to the use of any of the above described B7-H3 binding molecules, in the treatment of a disease or condition associated with B7-H3 or characterized by the expression of B7-H3, or a method of treating a disease or condition characterized by the expression of B7-H3, in particular, where the disease or the condition associated with B7-H3 expression or characterized by B7-H3 expression is cancer, and more specifically, where the cancer is selected from the group consisting of adrenal tumor, AIDS-associated cancer, soft tissue alveolar sarcoma, astrocytic tumor, adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, B-cell neoplasia, breast cancer, carotid tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, ulcerative cell carcinoma, cancer colon, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic and small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, imperfect bone fibrogenesis, fibrous bone dysplasia, cancer of the gallbladder or bile duct, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor , Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer , papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior pole uveal melanoma, renal metastatic cancer, rhabdoid tumor and, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

A second aspect of the present invention relates to a molecule that includes a human B7-H3 binding domain from a humanized anti-human B7-H3 antibody conjugated to at least one drug moiety (B7-H3-ADC). The invention also relates to pharmaceutical compositions that include such B7-H3-ADCs and to methods associated with the use of any of these B7-H3-ADCs in the treatment of cancer and other diseases and conditions.

In particular, the invention relates to a drug-anti-B7-H3 antibody conjugate (B7-H3-ADC) having the formula

Ab-(LM) _m -(D) _n , where Ab is an antibody that binds to B7-H3, comprising a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL), or its B7-H3- linking fragment;

D is a cytotoxic drug moiety;

LM is a linker molecule that covalently binds to Ab and D;

m is an integer from 0 to n and denotes the number of linker molecules in B7-H3ADC;

n is an integer from 1 to 10 and denotes the number of cytotoxic dosage forms covalently linked to the ADC.

In addition, the invention relates to such B7-H3-ADC, in which the LM linker molecule is absent (i.e. m = 0), and B7-H3-ADC, which have more than one LM linker molecule (i.e. m is an integer from 2 to n), where each of the linker molecules LM covalently links the cytotoxic drug component D to the Ab of such B7-H3-ADC. In addition, the present invention relates to those B7H3-ADCs whose Abs are covalently linked to more than one LM linker molecule, where all such linker molecules are identical. Cytotoxic D drug moieties that are covalently linked to Abs of such B7-H3-ADCs may be all the same or may include 2, 3, 4, or more non-identical cytotoxic D drug moieties. ADCs in which Ab is covalently linked to more than one LM linker molecule, where all such linker molecules are not identical. The cytotoxic D drug moieties that are covalently linked to the Abs of such B7-H3-ADCs may be all the same or may include 2, 3, 4 or more non-identical cytotoxic D drug moieties.

The invention further provides B7-H3-ADCs in which:

(A) (i) the humanized VL domain includes the amino acid sequence of SEQ ID NO:99 and (ii) the humanized VH domain includes the amino acid sequence of SEQ ID NO:104;

- 5 042568 or (B) (i) the humanized VL domain includes the amino acid sequence of SEQ ID NO:20 and (ii) the humanized VH domain includes the amino acid sequence of SEQ ID NO:21; or (C) (i) the humanized VL domain includes the amino acid sequence of SEQ ID NO:30 and (ii) the humanized VH domain includes the amino acid sequence of SEQ ID NO:31.

Further, the present invention relates to those B7-H3-ADCs wherein the humanized VL domain comprises the amino acid sequence of SEQ ID NO:99 and the humanized VH domain comprises the amino acid sequence of SEQ ID NO:104.

In addition, the present invention relates to those B7-H3-ADC, in which the humanized VL domain includes the amino acid sequence of SEQ ID NO:20, and the humanized VH domain includes the amino acid sequence of SEQ ID NO:21.

In addition, the invention provides those B7-H3-ADCs wherein the humanized humanized VL domain comprises the amino acid sequence of SEQ ID NO:30 and the humanized VH domain includes the amino acid sequence of SEQ ID NO:31.

In addition, the present invention relates to those B7-H3-ADC, in which Ab is an antibody or antigennegative fragment of an antibody.

The invention further provides such B7-H3-ADCs, wherein the B7-H3-ADC comprises an Fc domain from human IgG (especially human IgG1, IgG2, IgG3 or IgG4).

The invention further provides such B7-H3-ADCs, where the B7-H3-ADC contains a variant Fc domain that includes:

(a) one or more amino acid modifications that reduce the affinity of the variant Fc domain for FcyR; and/or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc domain.

The invention further provides B7-H3-ADCs that include a variant Fc domain, where modifications that reduce the affinity of the variant Fc domain for FcyR include substitution L234A; L235A; or L234A and L235A, where the numbering corresponds to the numbering of the EU index, as in Kabat.

The invention further provides B7-H3-ADCs that include a variant Fc domain, wherein modifications that improve the serum half-life of the variant Fc domain include the substitution M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, where the numbering corresponds to the EU index, like Kabat.

In addition, the present invention relates to those B7-H3-ADC, in which at least one of the LM is a linker molecule, and in particular, where the linker molecule is a peptide linker and/or a cleavable linker.

In addition, the present invention relates to those B7-H3-ADC, in which the molecule has the formula

Ab-[V-(W) _k -(X)iA]-D where V is a cleavable linker molecule LM;

(W) _k -(X)lA - elongated self-eliminating spacer system, which is self-eliminated by l,(4+2n)-elimination;

W and X each represent an electron cascade spacer and l,(4+2n) which are the same or different;

A is either a spacer group of formula (Y)m, where Y is a l,(4+2n) electron cascade spacer, or a group of formula U, which is a spacer to avoid cyclization;

k, l and m are independently an integer from 0 (inclusive) to 5 (inclusive), n is an integer from 0 (inclusive) to 10 (inclusive), provided that:

when A is (Y) _m : then k + l + m > 1, and if k + l + m = l, then n >1;

when A is equal to U: then k + 1 > 1;

W, X and Y are independently selected from compounds having the formula

or the formula

- 6 042568 where Q represents -R ⁵ C=CR ⁶ -, S, O, NR ⁵ , -R ⁵ C=N- or -N=CR ⁵ -;

P is NR ⁷ , O or S;

a, b and c are independently an integer from 0 (inclusive) to 5 (inclusive);

I, F and G are independently selected from compounds having the formula

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ independently represent H, C1.6 alkyl, C3-20 heterocyclyl, C5-20 aryl, C1.6 alkoxy, hydroxy ( OH), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF ₃ , CN, CONH2, SO ₂ Me, CONHMe, cyclic C ₁ . ₅ alkylamino, imidazolyl, C ₁ . ₆ alkylpiperazinyl, morpholino, thiol (SH), thioether (SR _x ), tetrazole, carboxy (COOH), carboxylate (COOR _x ), sulfoxy (S(=O) ₂ OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(=O) ₂ R _x ), sulfoxide (S(=O)OH), sulfinate (S(=O)ORx), sulfinyl (S(=O)Rx), phosphonooxy (OP(=O )(OH)2) and phosphate (OP(=O)(OR _x ) ₂ ) _x , where R _x , Rx ¹ and Rx ² are independently selected from C1. ₆ alkyl group, C ₃ . ₂₀ heterocyclic group or C _5-20 aryl group, two or more substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ or R ⁹ are optionally connected to each other to form one or several aliphatic or aromatic cyclic structures;

U is selected from compounds having the formula

where a, b and c are independently chosen as the integer 0 or 1; provided that a + b + c = 2 or 3;

R ¹ and/or R ² are independently H, C1.6 alkyl, where alkyl is optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF ₃ , CN, CONH2, SO ₂ Me, CONHMe, cyclic C _1-5 alkylamino, imidazolyl, C ₁ . ₆ alkylpiperazinyl, morpholino, thiol (SH), thioether (CPx), tetrazole, carboxy (COOH), carboxylate (COORx), sulfoxy (S(=O)2OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(=O) ₂ R _x ), sulfoxy (S(=O)OH), sulfinate (S(=O)OR _x ), sulfinyl (S(=O)R _x ), phosphonoxy (OP(=O) (OH) ₂ ) and phosphate (OP(=O)(OR _x ) ₂ ), where R _x , Rx ¹ and Rx ² are selected from C 1-6 alkyl group, C _3-20 heterocyclic group or C _5-20 aryl group; And

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently H, C1-6alkyl, C3-20heterocyclyl, C5-20aryl, C1-6alkoxy, hydroxy (OH), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF ₃ , CN, CONH2, SO ₂ Me, CONHMe, cyclic C _1-5 alkylamino, imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol ( SH), thioether (SR _x ), tetrazole, carboxy (COOH), carboxylate (COOR _x ), sulfoxy (S(=O) ₂ OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(= O) ₂ R _x ), sulfoxy (S(=O)OH), sulfinate (S(=O)ORx), sulfinyl (S(=O)Rx), phosphonooxy (OP(=O)(OH)2) and phosphate (OP(=O)(OR _x ) ₂ ), where R _x , Rx ¹ and Rx ² are selected from C 1-6 alkyl group, C _3-20 heterocyclic group or C _5-20 aryl group and two or more substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ or R ⁸ are optionally bonded to one another to form one or more aliphatic or aromatic ring structures.

In addition, the present invention relates to those B7-H3-ADC, in which the linker molecule LM includes:

(1) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl;

(2) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl;

(3) p-ammocinnamyloxycarbonyl;

(4) p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl;

(5) p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl;

(6) p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl;

(7) p-aminophenylpentadienyloxycarbonyl;

(8) p-aminophenylpentadienyloxycarbonyl-p-arninocinnamyloxycarbonyl;

(9) p-aminophenylpentadienyloxycarbonyl-paminobenzyloxycarbonyl;

(10) p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyloxycarbonyl;

(11) p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl;

(12) p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl;

(13) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(14) p-aminoindynamicoxycarbonyl-p-aminobenzyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl;

- 7 042568 (15) p-aminobenzyloxycarbonyl-p-arninocinnamyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(16) p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl;

(17) p-aminobenzyloxycarbonyl-p-aminobenzyl;

(18) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyl;

(19) p-aminocinnamyl;

(20) p-aminocinnanyloxycarbonyl-p-aminobenzyl;

(21) p-aminobenzyloxycarbonyl-p-aminocinnamyl;

(22) p-amino-cinnamyloxycarbonyl-p-aminocinnamyl;

(23) p-aminophenylpentadienyl;

(24) p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyl;

(25) p-aminophenylpentadienyloxycarbonyl-p-aminobenzyl; or (26) p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyl.

In addition, the invention provides those B7-H3-ADCs wherein the LM linker molecule is conjugated to the amino acid side chain of the Ab polypeptide chain and links Ab to the cytotoxic drug component D molecule, and in particular, where the cytotoxic drug component D comprises a cytotoxin, a radioisotope, an immunomodulator , cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, DNA, RNA, miRNA, RNAi, microRNA, photoactive therapeutic agent, antiangiogenic agent, proapoptotic agent, peptide, lipid, carbohydrate, chelating agent or combinations thereof.

In addition, the invention provides such B7-H3-ADCs wherein the LM linker molecule is conjugated to the amino acid side chain of the Ab polypeptide chain and links Ab to the cytotoxic drug component D molecule, and in particular, where the cytotoxic drug component D comprises a cytotoxin selected from the group consisting of tubulisin (especially tubulisin cytotoxin selected from the group consisting of tubulisin A, tubulisin B, tubulisin C and tubulisin D), auristatin (especially auristatin cytotoxin selected from the group consisting of MMAE (N-methylvaline-valin-dolaisoleuin-dolaproin- norephedrine) and MMAF (N-methylvaline-valine-dolaisoleuindolaproin-phenylalanine), maytansinoid (especially maytansinoid cytotoxin selected from the group consisting of maytansine, DM1 and DM4), calicheamicin (especially calicheamicin cytotoxin selected from the group consisting of calicheamicin γ1, calicheamycin e1Br, calicheamycin γ 1Br, calicheamycin a2I, calicheamycin a3I, calicheamycin βΠ, calicheamycin cin γH and calicheamicin A1I), pyrrolobenzodiazepine (especially pyrrolobenzodiazepine cytotoxin selected from the group consisting of vadastuximabataririn, SJG-136, SG2000, SG2285 and SG2274) and duocarmycin (especially duocarmycin cytotoxin selected from the group consisting of duocarmycin A, duocarmycin B1, duocarmycin ducarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adoselesin, bizelesin, carcelesin (U-80244) and spiro-duocarmycin (DUBA)).

The invention also relates to pharmaceutical compositions containing an effective amount of any of the B7-H3-ADCs described above and a pharmaceutically acceptable carrier, excipient or diluent.

The invention is further directed to the use of any of the B7-H3-ADCs described above in the treatment of a disease or condition associated with B7-H3 expression or characterized by B7-H3 expression, or in a method of treating a disease or condition characterized by B7H3 expression, particularly wherein the disease or the condition associated with B7-H3 expression or characterized by B7-H3 expression is a cancer, and more specifically, where the cancer is selected from the group consisting of acute myeloid leukemia, adrenal tumor, AIDS-associated cancer, soft tissue alveolar sarcoma, astrocytic tumor, adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, B-cell neoplasia, breast cancer, carotid tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, ulcerative carcinoma cells, colon cancer, colorectal cancer, skin benign fibrous gis thiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, imperfect bone fibrogenesis, fibrous bone dysplasia, cancer of the gallbladder or bile duct, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, tumor islet cells, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, cancer pancreas, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal

- 8 042568 metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus carcinoma, thymoma, metastatic thyroid cancer and uterine cancer.

Brief description of the drawings

In FIG. 1 is a diagram of a typical covalently linked diabody having two epitope-binding sites consisting of two polypeptide chains each having an E-helix or a K-helix to promote heterodimer formation (alternative heterodimer-facilitating domains are given below). A cysteine residue may be present in the linker and/or in the heterodimer promoting domain, as shown in FIG. 3b. The VL and VH domains that recognize the same epitope are shown using a single shading or shading scheme.

In FIG. 2 is a diagram of a typical covalently linked diabody molecule having two epitope binding sites consisting of two polypeptide chains each having a CH2 and CH3 domain such that the linked chains form all or part of an Fc domain. The VL and VH domains that recognize the same epitope are shown using a single shading or shading scheme.

In FIG. 3A-3C are diagrams showing representative covalently linked tetravalent diabodies having four epitope-binding sites consisting of two pairs of polypeptide chains (ie, a total of 4 polypeptide chains). One polypeptide of each pair has a CH2 and CH3 domain so that the linked strands form all or part of the Fc domain. The VL and VH domains that recognize the same epitope are shown using a single shading or shading scheme. The two pairs of polypeptide chains may be the same. In such embodiments where the two pairs of polypeptide chains are the same and the VL and VH domains recognize different epitopes (as shown in Figures 3A, 3B), the resulting molecule has four epitope binding sites and is bispecific and bivalent for each associated epitope. In such embodiments where the VL and VH domains recognize the same epitope (e.g., the same VL domain CDRs and the same VH domain CDRs are used in both strands), the resulting molecule has four epitope-binding sites and is monospecific and tetravalent for one epitope. Otherwise, the two pairs of polypeptides may be different. In such embodiments, where the two pairs of polypeptide chains are different, and the VL and VH domains of each pair of polypeptides recognize different epitopes (as shown by the different shading and patterns in Figure 3C), the resulting molecule has four epitope-binding sites and is tetraspecific and monovalent for each associated epitope. In FIG. 3A shows a diabody containing an Fc domain that includes a heterodimerization promoting domain peptide containing a cysteine residue. In FIG. 3B shows a diabody containing an Fc domain that includes E-helix and K-helix heterodimerization promoting domains that contain a cysteine residue and a linker (with an optional cysteine residue). In FIG. 3C shows a diabody containing an Fc region that includes the CH1 and CL domains of an antibody.

In FIG. 4A and 4B are diagrams of an exemplary covalently linked diabody molecule comprising two epitope-binding sites consisting of three polypeptide chains. The two polypeptide chains have CH2 and CH3 domains so that the linked chains form all or part of the Fc domain. Polypeptide chains containing VL and VH domains further include a heterodimerization promoting domain. The VL and VH domains that recognize the same epitope are shown using a single shading or shading scheme.

In FIG. 5 is a diagram of a typical molecule of a covalently linked diabody comprising four epitope-binding sites consisting of five polypeptide chains. The two polypeptide chains have CH2 and CH3 domains such that the linked chains form an Fc domain that includes all or part of the Fc domain. Polypeptide chains comprising linked VL and VH domains further comprise a heterodimerization promoting domain. The VL and VH domains that recognize the same epitope are shown using a single shading or shading scheme.

In FIG. 6A-6F are schematics of exemplary Fc domain containing trivalent binding molecules having three epitope binding sites. In FIG. 6A and 6B, respectively, schematically show the domains of trivalent binding molecules containing two binding domains of two antibodies and a Fab-type binding domain having different domain orientations, in which the diabody-type binding domains are N-terminal or C-terminal with respect to the Fc domain. . The molecules in Fig. 6A and 6B contain four chains. In FIG. 6C and 6D, respectively, schematically show the domains of trivalent binding molecules, comprising diabody-type binding domains that are N-terminal to an Fc domain, and a Fab-type binding domain in which the light chain and heavy chain are linked via a polypeptide spacer or binding domain. by scFv type. The trivalent binding molecules in FIG. 6E and 6F, respectively, schematically illustrate the domains of trivalent binding molecules comprising two diabody-type binding domains that are C-terminal to an Fc domain and a Fab-type binding domain in which the light chain and heavy chain are linked through a polypeptide spacer. or an scFv-type binding domain. The trivalent binding molecules in FIG. 6C-6F contain three chains. The VL and VH domains that recognize the same epitope are shown using a single shading or shading scheme.

- 9 042568

In FIG. 7 shows the results of screening for anti-B7-H3 antibodies capable of internalizing into Hs700T pancreatic cancer cells.

In FIG. 8A-8J show the results of a study on the ability of B7-H3-ADC of the present invention to mediate cytotoxicity in vitro against B7-H3 expressing JIMT-1 breast cancer cells (FIG. 8A), MDA-MB-468 breast cancer cells (FIG. 8B), A375.52 melanoma cells (Fig. 8C), Calu-6 non-small cell lung cancer cells (Fig. 8D), NCI-H1703 non-small cell lung cancer cells (Fig. 8E), NCI-H1975 non-small cell lung cancer cells (Fig. 8F), PA-1 ovarian cancer cells (Fig. 8G), Hs700T pancreatic cancer cells (Fig. 8H), DU145 prostate cancer cells (Fig. 8I), and B7-H3 negative Raji B-cell lymphoma cells ( Fig. 8J).

In FIG. 9 shows the results of a study on the ability of the B7-H3-ADC of the present invention to mediate in vivo cytotoxicity against MDA-MB-468 breast cancer tumor cells implanted in the mammary fat pad in a CD1 nude mouse model. Tumor growth curves are shown for mice treated ip with 10 mg/kg chmAb-B-vcMMAE, chmAb-C-vc-MMAE and chmAb-D-vc-MMAE or vehicle alone on day 25 (arrow).

In FIG. 10A-10C show the results of a study of the ability of the B7-H3-ADC of the present invention to mediate cytotoxicity in vivo against subcutaneously implanted NCI-H1703 NC1 non-small cell lung cancer tumor cells in a CD1 nude mouse model. Tumor growth curves are shown for mice injected ip with 10 mg/kg (Fig. 10A), 3 mg/kg (Fig. 1), 1 mg/kg (Fig. 10C) chmAb-B-vc-MMAE, chmAb-B -vc-MMAE and chmAb-B-vc-MMAE at 10 mg/kg or vehicle alone on day 52 (arrowed).

In FIG. 11A-11C show the results of a study of the ability of the B7-H3-ADC of the present invention to mediate in vivo cytotoxicity against subcutaneously implanted PA-1 ovarian cancer tumor cells in a CD1 nude mouse model. Tumor growth curves are shown for mice treated intraperitoneally with 10 mg/kg (Fig. 11A), 3 mg/kg (Fig. 11B), 1 mg/kg (Fig. 5C) chmAb-B-vcMMAE, chmAb-C-vc- MMAE and chmAb-D-vc-MMAE at 10 mg/kg or vehicle alone on day 42 (arrow).

In FIG. 12A-12C show the results of a study of the ability of the B7-H3-ADC of the present invention to mediate in vivo cytotoxicity against subcutaneously implanted Calu-6 non-small cell lung cancer tumor cells in a CD1 nude mouse model. Tumor growth curves are shown for mice injected ip with 10 mg/kg (Fig. 12A), 3 mg/kg (Fig. 1), 1 mg/kg (Fig. 12C) chmAb-B-vc-MMAE, chmAb-B -vc-MMAE and chmAb-D-vc-MMAE at 10 mg/kg or vehicle alone on day 20 (arrow).

In FIG. 13A-13C show the results of testing the ability of the B7-H3-ADC of the present invention to mediate cytotoxicity in vivo against subcutaneously implanted A375.S2 melanoma cells in a CD1 nude mouse model. Tumor growth curves are shown for mice injected ip with 10 mg/kg (Fig. 13A), 3 mg/kg (Fig. 1), 1 mg/kg (Fig. 13C) chmAb-B-vc-MMAE, chmAb- C-vc-MMAE and chmAb-D-vc-MMAE at 10 mg/kg or vehicle alone on day 30 (arrowed).

In FIG. 14A-14C show the results of a pharmacokinetic stability study of B7-H3-ADC molecules. Serum antibody concentration curves are shown for all antibodies (circles) and intact B7-H3-ADC (squares) derived from chmAb-B (Fig. 14A), chmAb-C (Fig. 14B), and chmAb-D (Fig. 14C). ).

In FIG. 15A-15C show retention of biological activity by hmAb-C B7-H3ADC having a typical duocarmycin (DUBA) moiety linked to the amino acid residue of the Ab moiety via a cleavable linker (hmAb-C-DUBA). Fig. 15A Calu-6 cells; fig. 15B, NCIH1703 cells; fig. 15C, Hs700T cells. The control molecule binds CD20 and is conjugated to DUBA (Ctrl-DUBA).

In FIG. 16 shows the results of an in vivo study of the efficacy of hmAb-C-DUBA on non-small cell lung carcinoma Calu-6 cells. hmAb-C-DUBA was administered to groups of mice (n=5) subcutaneously inoculated with non-small cell lung carcinoma Calu-6 cells. Doses of hmAb-C-DUBA (1 mg/kg x 3, 3 mg/kg x 3, or 6 mg/kg x 3) were administered intraperitoneally to mice on days 24, 31, 38, and 45 (shown by arrows) post-inoculation and animals were evaluated by tumor volume up to 62 days.

In FIG. 17 shows the results of an in vivo study of the efficacy of hmAb-C-DUBA against Calu-6 non-small cell lung carcinoma cells. hmAb-C-DUBA was administered to groups of mice (n = 7) that were subcutaneously inoculated with Calu-6 non-small cell lung carcinoma cells. The dose of hmAb-CDUBA or Ctrl-DUBA (3 mg/kg or 10 mg/kg) was administered to mice on the 20th day (arrow) after inoculation, and the animals were assessed for tumor volume up to 55 days.

In FIG. 18 shows the results of an in vivo study of the efficacy of hmAb-C-DUBA against PA-1 ovarian carcinoma cells. hmAb-C-DUBA or Ctrl-DUBA were administered to groups of mice (n=6) subcutaneously inoculated with PA-1 ovarian carcinoma cells. A dose of hmAb-C-DUBA or Ctrl-DUBA (3 mg/kg, 6 mg/kg, or 10 mg/kg) was administered to mice on day 25 (arrowed) post-inoculation, and animals

- 10 042568 was evaluated by tumor volume up to 60 days.

In FIG. 19 shows the results of an in vivo study of the efficacy of hmAb-C-DUBA against A375.S2 melanoma cells. hmAb-C-DUBA or Ctrl-DUBA were administered to groups of mice (n=7) that were subcutaneously inoculated with A375.S2 melanoma cells. The dose of hmAb-C-DUBA or Ctrl-DUBA (1 or 3 mg/kg) was administered to mice on the 25th day (arrow) after inoculation, and the animals were assessed for tumor volume up to 60 days.

In FIG. 20A-20D show the results of an in vivo study of the efficacy of hmAb-C-DUBA against fat body xenografts of MDA-MB468 breast carcinoma cells. hmAb-C-DUBA or Ctrl-DUBA was administered intraperitoneally to groups of mice on days 70, 74 and 78 after inoculation of MDA-MB468 mammary carcinoma cells into the mammary fat pad. A dose of hmAb-C-DUBA or Ctrl-DUBA (either a single dose of 3 mg/kg or 6 mg/kg) on day 70 or three doses of 3 mg/kg on days 70, 74 and 78 (shown by arrows) was given after inoculation, and animals were assessed for tumor volume up to 110 days. In FIG. 20A shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 6 mg/kg (single dose). In FIG. 20B shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 3 mg/kg (single dose). In FIG. 20C shows results for vehicle, hmAb-C-DUBA or CtrlDUBA at 3 mg/kg (three doses). In FIG. 20D shows all results in one graph.

In FIG. 21A-21D show the results of an in vivo study of the efficacy of hmAb-C-DUBA against subcutaneously implanted PA-1 ovarian carcinoma cell xenografts. hmAb-C-DUBA or Ctrl-DUBA was administered intraperitoneally (single dose of 3, 6 or 10 mg/kg) on day 24 post-inoculation or two doses of 10 mg/kg hmAb-C-DUBA or Ctrl-DUBA (on days 24 and 28 post-inoculation) or four doses of 6 mg/kg hmAb-C-DUBA or Ctrl-DUBA (on days 24, 28, 31 and 35 post-inoculation). Animals were assessed for tumor volume for 70 days. In FIG. 21A shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 10 mg/kg (single or double dose). In FIG. 21B shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 6 mg/kg (single or quadruple dose). In FIG. 21C shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 3 mg/kg (single doses). In FIG. 21D shows all results in one graph.

In FIG. 22 shows the pharmacokinetics of chmAb-C-DUBA administration in mice. The drawing shows total human IgG and intact ADC chmAb-C-DUBA at 3 mg/kg (n=3).

In FIG. 23A-23B show the pharmacokinetics of hmAb-C-DUBA administration in the cynomolgus monkey. The drawings show total human IgG (Fig. 23A) and intact ADC (Fig. 23B) hmAb-C-DUBA at 1 mg/kg (1 male, 1 female), 3 mg/kg (1 male, 1 female), 10 mg/kg (1 male, 1 female) or 27 mg/kg (2 males, 2 females)).

Detailed description of the invention

The present invention is directed to novel B7-H3 binding molecules capable of binding to human and non-human B7-H3, and in particular to those molecules that are cross-reactive with B7-H3 of non-human primates (e.g. cynomolgus monkey) . The invention further relates to B7-H3 binding molecules that include light chain variable domains and/or heavy chain variable domains (VH) that have been humanized and/or deimmunized such that they exhibit reduced immunogenicity when administered to recipient subjects. The invention particularly relates to bispecific, trispecific, or multispecific B7-H3 binding molecules, including bispecific diabodies, BiTEs, bispecific antibodies, trivalent binding molecules, and the like. which include: (i) such B7-H3 binding variable domains; and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell. The invention also relates to pharmaceutical compositions that include any of such B7-H3 binding molecules, and to methods involving the use of any of such B7-H3 binding molecules in the treatment of cancer and other diseases and conditions. The invention also specifically relates to a molecule that includes a human B7-H3 binding domain from a humanized anti-human B7-H3 antibody conjugated to at least one drug moiety (B7-H3-ADC). The invention also relates to pharmaceutical compositions that include such B7-H3-ADCs and to methods associated with the use of any of these B7-H3-ADCs in the treatment of cancer and other diseases and conditions.

The present invention also relates to a molecule that includes a human B7-H3 binding domain from a humanized anti-human B7-H3 antibody conjugated to at least one drug moiety (B7-H3-ADC). The invention also relates to pharmaceutical compositions that include such B7-H3-ADCs and to methods associated with the use of any of these B7-H3-ADCs in the treatment of cancer and other diseases and conditions.

The B7-H3-ADC molecules of the present invention include the formula

Ab-(LM) _m -(D)n, where Ab is an antibody that binds to B7-H3, which includes a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL) or is B7-H3 -binding fragment; And

D is a cytotoxic drug moiety;

LM is a bond or linker molecule that covalently links Ab and D;

- 11 042568 m is an integer from 0 to n and denotes the number of linker molecules in B7-H3ADC;

n is an integer from 1 to 10 and denotes the amount of cytotoxic drug moieties covalently linked to the B7-H3-ADC molecule.

I. Antibodies and their binding domains.

The antibodies of the present invention are immunoglobulin molecules capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. through at least one antigen recognition site located in the variable domain of an immunoglobulin molecule. The B7-H3-ADC molecules of the present invention thus comprise an antibody that binds to B7-H3, or a B7-H3 binding fragment. As used herein, the terms antibody and antibodies refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F(ab ')-fragments, disulfide-linked bispecific Fv (sdFv), intrabodies and epitope-binding fragments of any of the above. In particular, the term antibody includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie. molecules that include an epitope binding site. Immunoglobulin molecules can be of any type (eg IgG, IgE, IgM, IgD, IgA and IgY), class (eg IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 and IgA2) or subclass. Antibodies are capable of immunospecifically binding to (or binding to such a molecule in an immunospecific manner) a polypeptide or protein or non-protein molecule due to the presence of a particular domain or fragment or conformation (epitope) on such molecule. The epitope-containing molecule may have immunogenic activity such that it elicits an antibody production response in an animal; such molecules are called antigens. The past few decades have seen a resurgence of interest in the therapeutic potential of antibodies, and antibodies have emerged as one of the leading classes of biotechnologically derived drugs (Chan, CE et al. (2009) The Use Of Antibodies In The Treatment Of Infectious Diseases, Singapore Med. J. 50(7):663-666). More than 200 antibody-based drugs have been approved or are under development.

As used herein, an antibody, diabody, or other epitope-binding molecule is reported to immunospecifically bind a region of another molecule (i.e., an epitope) if it responds or associates more frequently, more rapidly, with greater duration, and/or with greater affinity to it. epitope relative to alternative epitopes. For example, an antibody that immunospecifically binds to a viral epitope is an antibody that binds that viral epitope with greater affinity, avidity, more readily, and/or longer duration than it immunospecifically binds to other viral epitopes or non-viral epitopes. It is also understood from reading this definition that, for example, an antibody (or fragment or epitope) that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target. Thus, immunospecific binding does not necessarily require (though it may include) exclusive binding. Typically, but not necessarily, reference to binding means immunospecific binding. Two molecules are said to be able to bind to each other in a physiospecific manner if such binding exhibits the specificity with which the receptors bind to their respective ligands.

The term monoclonal antibody refers to a homogeneous population of antibodies, where the monoclonal antibody consists of amino acids (naturally occurring or not occurring in nature) that participate in the selective binding of an antigen. Monoclonal antibodies are highly specific, directed against a single epitope (or antigenic site). The term monoclonal antibody covers not only intact monoclonal antibodies and full length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab') ₂ , Fv, etc.), single chain (scFv) binding molecules, their mutants , fusion proteins containing part of an antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the desired specificity and has the ability to bind to an antigen. The term is not intended to be limiting in relation to the source of the antibody or the method of its production (eg, using hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments, etc., as described above under the definition of antibody. Methods for producing monoclonal antibodies are known in the art. One method that may be used is that of Kohler, G. et al. (1975) Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity, Nature 256:495-497 or a modification thereof. Typically, monoclonal antibodies are produced in mice, rats, or rabbits. Antibodies are obtained by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations that include the epitope of interest. An immunogen can be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissue.

- 12 042568

Cells used for immunization may be cultured for a period of time (eg, at least 24 hours) prior to their use as an immunogen. Cells can be used as immunogens alone or in combination with a non-denaturing adjuvant such as Ribi (see, for example, Jennings, V.M. (1995) Review of Selected Adjuvants Used in Antibody Production, ILAR J. 37 (3): 119 -125). As a rule, the cells must be kept intact and preferably must be viable when used as immunogens. Intact cells may allow antigens to be better detected by immunized animals than in the case of damaged cell antigens. The use of denaturing or harsh adjuvants, such as Freud's adjuvant, may lead to cell rupture and is therefore not recommended. The immunogen may be administered several times at periodic intervals, such as twice a week or weekly, or may be administered in such a manner as to maintain viability in the animal (eg, in a tissue recombinant). Otherwise, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for the pathogenic epitope of interest can be sequenced and recombinantly produced by any means known in the art. In one embodiment, such an antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The antibody coding sequence can be maintained in the vector in the host cell and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of such antibodies can be used for genetic manipulation to produce monospecific or multispecific (e.g., bispecific, trispecific, and tetraspecific) molecules of the invention, as well as optimized affinity, chimeric antibody, humanized antibody, and/or caninized antibody, to improve affinity or other characteristics of the antibody. . The general principle of humanizing an antibody involves retaining the core sequence of the antigen-binding portion of the antibody while replacing the non-human residue of the antibody with human antibody sequences.

Natural antibodies (such as IgG antibodies) consist of two light chains complexed with two heavy chains. Each light chain contains a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH), three constant domains (CH1, CH2 and CH3) and a hinge region (H) located between the CH1 and CH2 domains. Thus, the basic structural unit of natural immunoglobulins (eg, IgG) is a tetramer having two light chains and two heavy chains, usually expressed as a glycoprotein of about 150,000 Da. The amino-terminal (N-terminal) portion of each chain includes a variable domain containing 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines a constant region, with light chains having one constant domain and heavy chains typically having three constant domains and a hinge domain. Thus, the structure of the light chains of the IgG molecule is n-VL-CL-c, and the structure of the heavy chains of IgG is n-VH-CH1-H-CH2-CH3-c (where n and c represent, respectively, the N-terminus and the C-terminus of the polypeptide).

A. Characteristics of antibody variable domains.

The variable domains of the IgG molecule are composed of complementarity determining regions (CDRs), which include epitope-contacting residues and non-CDR segments called framework fragments (FRs), which generally maintain the structure and determine the positioning of the CDR loops to allow said contact ( although some framework residues may also come into contact with the antigen). Thus, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR2FR3-CDR3-FR4-C. Polypeptides that are (or may be) the first, second, and third CDRs of an antibody light chain are designated, respectively: CDRL1 domain, CDRL2 domain, and CDRL3 domain. Similarly, polypeptides that (or may serve) as the first, second, and third CDRs of an antibody heavy chain are referred to as CDRH1 domain, CDRH2 domain, and CDRH3 domain, respectively. Thus, the terms CDRL1 domain, CDRL2 domain, CDRL3 domain, CDRH1 domain, CDRH2 domain, and CDRH3 domain refer to polypeptides that, when incorporated into a protein, result in that protein being able to bind to a particular epitope, whether or not that protein is an antibody having light and heavy chains, or is a diabody or single chain binding molecule (eg, scFv, BiTe, etc.) or is another type of protein. Accordingly, as used herein, the term epitope-binding fragment refers to a fragment of a molecule capable of immunospecific binding to an epitope. An epitope-binding fragment may contain any of 1, 2, 3, 4, or 5 CDRs of an antibody, or may contain all 6 CDRs of an antibody, and while capable of immunospecific binding to such an epitope, may exhibit immunospecificity, affinity, or selectivity for the epitope, which differs from the epitope of such an antibody. However, it is preferred that the epitope-binding fragment contains all 6 CDRs of such an antibody. The epitope-binding fragment of an antibody may be a single polypeptide chain (eg, scFv) or may contain two or more polypeptide chains, each having an amino-terminus and a carboxyl-terminus (eg, diabody, Fab fragment, Fab ₂ fragment, etc.). Unless otherwise indicated, the order of the domains of the protein molecules described herein is in the N direction.

- 13 042568 end-C-end.

The invention particularly relates to single chain variable fragments (scFv) containing the humanized anti-B7-H3-VL and/or VH domain of the present invention, as well as to multispecific binding molecules containing them. Single chain variable fragments contain VL and VH domains that are linked to each other by a short linker peptide. Such linkers can be modified to provide additional functionality, such as drug attachment or attachment to a solid support. Single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes an scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect, or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding scFvs of interest can be obtained using conventional manipulations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The invention also specifically covers CDR _H 1 , CDR _H 2 , CDR _H 3 , CDRL1, CDR _L 2 , CDR _L 3 or the VL domain and/or VH domain of the humanized B7-H3 antibody variants of the invention, as well as multispecific binding molecules containing the same. The term humanized antibody refers to a hybrid molecule, usually produced using recombinant techniques, having an immunoglobulin epitope-binding site from a non-human species and the rest of the immunoglobulin molecule structure that is based on the structure and/or sequence of human immunoglobulin. The anti-B7-H3 antibodies of the present invention specifically include humanized, hybridized, or caninized variants of mAb-A, mAb-B, mAb-C, or mAb-D antibodies. The polynucleotide sequence of the variable domains of such antibodies can be used for genetic manipulation in order to obtain such derivatives and improve the affinity or other characteristics of such antibodies. The general principle of humanizing an antibody involves maintaining the core sequence of the epitope-binding portion of the antibody, while replacing the non-human antibody residue with human antibody sequences. There are four general steps in the humanization of a monoclonal antibody. These are: (1) determination of the nucleotide and predicted amino acid sequences of the original variable domains of the light and heavy chains of the antibody, (2), construction of a humanized antibody or caninized antibody, that is, deciding which framework region of the antibody will be used in the humanization or caninization process, (3 a) actual humanization or caninization methodologies/methods, and (4) transfection and expression of the humanized antibody; see, for example, US Pat. US No. 4816567; 5807715; 5866692 and 6331415.

The epitope-binding site may either contain the entire variable domain combined with the constant domains, or only the complementarity determining regions (CDRs) of such a variable domain grafted into the appropriate framework regions. Epitope binding domains may be wild-type or modified with one or more amino acid substitutions. This rules out the constant region as an immunogen in humans, but leaves the possibility of an immune response to a foreign variable domain (LoBuglio, A.F. et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc. Natl. Acad. Sci (U.S.A.) 86:4220-4224). Another approach focuses not only on providing constant regions of human origin, but also on changing the variable domains in order to change them as accurately as possible to the human form. The variable domains of both heavy and light chains are known to contain three complementarity determining regions (CDRs) that vary depending on the antigens in question and determine binding capacity, flanked by four framework regions (FRs) that are relatively conserved in these species and which presumably provide a supporting scaffold for the CDR. When non-human antibodies are made to a specific antigen, the variable domains can be altered or humanized by grafting the CDRs derived from the non-human antibodies onto the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332:323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; Kettleborough, C.A. et al. (1991) Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation, Protein Engineering 4:773-3783; Maeda, H. et al. (1991) Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity, Human Antibodies Hybridoma 2:124-134; Gorman, S.D. et al. (1991) Reshaping A Therapeutic CD4 Antibody, Proc. Natl. Acad. sci. (U.S.A.) 88:4181-4185; Tempest, P.R. et al. (1991) Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo, Bio/Technology 9:266-271; Co, M.S. et al. (1991) Humanized Antibodies For Antiviral Therapy, Proc. Natl. Acad. sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy, Proc. Natl. Acad. sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) Chi- 14 042568 meric And Humanized Antibodies With Specificity For The CD33 Antigen, J. Immunol. 148:1149-1154. In some embodiments, humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs from mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that differ in sequence from the parent antibody.

Several humanized antibody molecules have been described containing an epitope-binding site derived from a non-human immunoglobulin, including fusion antibodies containing a rodent variable domain or a modified rodent variable domain, and their associated complementarity determining regions (CDRs) fused to constant human domains (see, for example, Winter et al. (1991) Man-made Antibodies, Nature 349:293-299; Lobuglio et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc. Natl Acad Sci (U.S.A.) 86:4220-4224 (1989), Shaw et al (1987) Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen, J. Immunol 138:4534-4538, and Brown et al (1987) Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody, Cancer Res 47:3577-3583). Other sources describe rodent CDRs grafted into a human supportive framework (FR) prior to fusion with the corresponding human antibody constant domain (see, for example, Riechmann L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332:323-327 ; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; and Jones et al. (1986) Replacing The Complementarity-Determinings In A Human Antibody Region With Those From A Mouse , Nature 321:522-525). Another reference describes rodent CDRs maintained by recombinantly venated rodent scaffold regions; see, for example, European Patent Publication No. 519596. These humanized molecules are designed to minimize the adverse immunological response to rodent anti-human protein antibody molecules that limit the duration and efficacy of therapeutic applications of these fragments in human recipients. Other methods for humanizing antibodies that can also be used are disclosed by Daugherty et al. (1991) Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins, Nucl. Acids Res. 19: 2471-2476 and in US Pat. US No. 6180377; 6054297; 5997867; and 5866692.

B. Characteristics of antibody constant domains.

1. Light chain constant domains.

As stated above, each antibody light chain contains a variable domain (VL) and a constant domain (CL).

A preferred CL domain is the kappa CL domain from human IgG. The amino acid sequence of a typical human CL-kappa domain is (SEQ ID NO:1)

RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG

NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

Otherwise, a typical CL domain is the lambda CL domain from human IgG. The amino acid sequence of a typical human lambda CL domain is (SEQ ID NO:2) QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA

GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

2. Heavy chain constant domains.

As indicated above, antibody heavy chains may contain CH1 constant domains, a hinge domain, CH2, and CH3. The CH1 domains of the two heavy chains of the antibody complex with the antibody light chain CL constant region are attached to the CH2 heavy chain domains via an intermediate hinge domain.

An exemplary CH1 domain is the CH1 domain of human IgG1. The amino acid sequence of a representative CH1 domain from human IgG1 is (SEQ ID NO:3) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV

A typical CH1 domain is the CH1 domain of human IgG2. The amino acid sequence of a representative CH1 domain from human IgG2 is (SEQ ID NO:4) ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV

A typical CH1 domain is the CH1 domain of human IgG3. The amino acid sequence of an exemplary human IgG3 CH1 domain is (SEQ ID NO:5) ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRV

An exemplary CH1 domain is the CH1 domain of human IgG4. The amino acid sequence of a representative CH1 domain from human IgG4 is (SEQ ID NO:6) ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

- 15 042568

A typical hinge domain is the hinge domain from human IgG1. The amino acid sequence of a typical hinge domain from human IgG1 is (SEQ ID NO:7):

EPKSCDKTHTCPPCP.

Another exemplary hinge domain is the hinge domain from human IgG2. The amino acid sequence of a representative hinge domain from human IgG2 is (SEQ ID NO:8): ERKCCVECPPCP.

Another exemplary hinge domain is the hinge domain from human IgG3. The amino acid sequence of a typical hinge domain from human IgG3 is (SEQ ID NO:9):

ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK

SCDTPPPCPR CP

Another typical hinge domain is the hinge domain from human IgG4. The amino acid sequence of a typical hinge domain from human IgG4 is (SEQ ID NO:10): ESKYGPPCPSCP. As described herein, the hinge domain of IgG4 may include a stabilizing mutation, such as the S228P substitution. The amino acid sequence of a typical S228P-stabilized hinge domain from human IgG4 is (SEQ ID NO:11): ESKYGPPCPPCP.

The CH2 and CH3 domains of the two heavy chains of an antibody interact to form an Fc domain, which is a region that is recognized by cellular Fc receptors, including, but not limited to, Fc gamma receptors (FcyR). As used herein, the term Fc domain is used to define the C-terminal region of an IgG heavy chain. An Fc domain is assumed to belong to a particular IgG isotype, class, or subclass if its amino acid sequence is most homologous to that isotype relative to other IgG isotypes. In addition to their known diagnostic applications, antibodies have been shown to be useful as therapeutic agents.

As used herein, the residue numbering in the IgG heavy chain constant region corresponds to the EU index numbering as in Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, ^5th ED. PUBLIC HEALTH SERVICE, NH1, MD (1991) (KABAT), expressly incorporated herein by reference. The term EU index, as in Kabat, refers to the numbering of the constant domains of a human IgG1 EU antibody. Amino acids from the variable domains of mature immunoglobulin heavy and light chains are designated by the position of the amino acid in the chain. Kabat described numerous amino acid sequences for antibodies, identified an amino acid consensus sequence for each subgroup, and assigned a residue number to each amino acid, and a CDR was identified as defined by Kabat (it is understood that CDR _H 1 as defined in Chothia, C. & Lesk, AM ((1987 ) Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol. 196: 901-917) starts five residues earlier). The Kabat numbering scheme is extended to antibodies not included in its collection by aligning the antibody in question with one of the consensus sequences in Kabat with respect to conservative amino acids. This method of assigning residue numbers has become standard in the art and readily identifies amino acids at equivalent positions in different antibodies, including hybrid or humanized variants. For example, the amino acid at position 50 of the light chain of a human antibody occupies the equivalent position of the amino acid at position 50 of the light chain of a mouse antibody.

The amino acid sequence of the CH2-CH3 domain of a typical human IgG1 (SEQ ID NO:12) 231 240 250 260 270 280

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

290 300 310 320 330

GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA

340 350 360 370 380

PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430

WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE 440 447

ALHNHYTQKS LSLSPG X according to the EU numbering provided by Kabat, where X is lysine (K) or absent.

The amino acid sequence of the CH2-CH3 domain of a typical human IgG2 is (SEQ ID NO:13)

- 16 042568

231 240 250 260 270 280

APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 320 330

GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380

PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430

WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE 440 447

ALHNHYTQKS LSLSPG X according to the EU numbering provided by Kabat, where X is lysine (K) or absent.

The amino acid sequence of the CH2-CH3 domain of a typical human IgG3 is (SEQ ID NO:14)

231 240 250 260 270 280

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330

GVEVHNAKTK PREQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380

PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430

WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQGNIFSCSVMHE 440 447

ALHNRFTQKS LSLSPGX according to the EU numbering given by Kabat, where X is lysine (K) or absent.

The amino acid sequence of the CH2-CH3 domain of a typical human IgG4 is (SEQ ID NO:15)

231 240 250 260 270 280

APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330

GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380

SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430

WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE 440 447

ALHNHYTQKS LSLSLGX according to the EU numbering given by Kabat, where X is lysine (K) or absent.

Polymorphisms have been observed at several different positions in the antibody constant regions (e.g., at Fc positions including, but not limited to, positions 270, 272, 312, 315, 356, and 358, according to the EU index numbering as given by Kabat), and, thus, there may be slight differences between the sequence shown and sequences in the prior art. Well-known polymorphic forms of human immunoglobulins. Currently, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et al., The Human IgG Subclasses: Molecular Analysis Of Structure, Function And Regulation (Pergamon, Oxford, pp. 4378 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211) . In particular, it is contemplated that the antibodies of the present invention may include any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype, or haplotype of the sequences provided herein. In addition, in some expression systems, the C-terminal amino acid residue (in bold) of the CH3 domain may be post-translationally deleted. Accordingly, the C-terminal residue of the CH3 domain is an optional amino acid residue in the B7-H3 binding molecules (including B7-H3-ADC molecules) of the invention. In particular, the subject of the present invention are B7-H3 binding molecules (including B7-H3-ADC molecules) lacking the C-terminal residue of the CH3 domain. Also specifically included in the present invention are those constructs which contain a C-terminal lysine residue in the CH3 domain.

- 17 042568

In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system results in a wide range of responses, from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis, to immunomodulatory signals such as regulation of lymphocyte proliferation and antibody secretion. All of these interactions are initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cellular receptors on hematopoietic cells and, in particular, to receptors (particularly referred to as the Fc-gamma receptor FcyR, and collectively FcyRs) found on the surfaces of several cell types. immune system (eg, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells). Such receptors have an extracellular portion (which is thus capable of ligating to the Fc domain), a transmembrane portion (which extends across the cell membrane), and a cytoplasmic portion (located within the cell).

The diversity of cellular responses elicited by antibodies and immune complexes is a result of the structural heterogeneity of three Fc receptors: FcyRI (CD64), CD32A (FcyRIIA), FcyRIIB (CD32B), CD16A (FcyRIIIA) and CD16B (FcyRIIIB). FcyRI (CD64), FcyRIIA (CD32A), and FcyRIII (CD16) are activating receptors such that their ligation to the Fc domain activates the immune system or enhances the immune response. In contrast, FcyRIIB (CD32B) is an inhibitory receptor; ligation to the Fc domain inhibits the immune response or attenuates an existing immune response. In addition, the interaction of the Fc domain with the neonatal Fc receptor (FcRn) mediates the recycling of IgG molecules from the endosome to the cell surface and release into the blood. The amino acid sequence of exemplary wild-type Fc domains of IgG1 (SEQ ID NO:12), IgG2 (SEQ ID NO:13), IgG3 (SEQ ID NO:14) and IgG4 (SEQ ID NO:15) is shown above.

CD16 is the common name for the Fc activating receptors, FcyRIIIA (CD16A) and FcyRIIIB (CD16B). CD16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages that bind aggregated but not monomeric human IgG (Peltz, G.A. et al. (1989) Human Fc Gamma RIII: Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG, Proc. Natl. Acad. Sci. (U.S.A.) 86(3): 1013-1017 Bachanova, V. et al. (2014) NK Cells In Therapy Of Cancer, Crit. Rev. Oncog 19(1-2): 133-141; Miller, J. S. (2013) Therapeutic Applications: Natural Killer Cells In The Clinic, Hematology Am. Soc. Hematol. Educ. Program. 2013:247-253; Youinou, P. et al. (2002) Pathogenic Effects Of Anti-Fc Gamma Receptor IIIB (CD16) On Polymorphonuclear Neutrophils In Non-Organ-Specific Autoimmune Diseases Autoimmun Rev. 1(1-2): 13-19; Peipp, M. et al. ( 2002) Bispecific Antibodies Targeting Cancer Cells, Biochem Soc Trans 30(4):507-511). These receptors bind to the Fc portion of IgG antibodies, thereby causing the release of cytokines. If such antibodies are bound to the antigen of foreign cells (eg, tumor cells), then such release mediates the killing of the tumor cell. Because this killing is antibody-dependent, it is called antibody-dependent cell-mediated cytotoxicity (ADCC).

CD32A (FcyRIIA) (Brandsma, A.M. (2015) Fc Receptor Inside-Out Signaling And Possible Impact On Antibody Therapy, Immunol Rev. 268(1):74-87; van Sorge, N.M. et al. (2003) Fcgammar Polymorphisms: Implications Selvaraj, P. et al (2004) Functional Regulation Of Human Neutrophil Fc Gamma Receptors, Immunol Res. 29(13):219-230 ) and CD64 (FcyRI) (Lu, S. et al. (2015) Structural Mechanism Of High Affinity FcyRI recognition Of Immunoglobulin G, Immunol. Rev. 268(1): 192-200; Swisher, J.F. et al. (2015) The Many Faces Of FcyRI: Implications For Therapeutic Antibody Function, Immunol. Rev. 268(1): 160-174; Thepen, T. et al. (2009) Fcgamma Receptor 1 (CD64), A Target Beyond Cancer, Curr. Pharm Rouard, H. et al (1997) Fc Receptors As Targets For Immunotherapy, Int. Rev. Immunol. 16(1-2): 147-185) activate Fc receptors, Des. 15(23):2712-2718; which are expressed on macrophages, neutrophils, eosinophils, etc. endrite cells (and in the case of CD32A, also on platelets and Langerhans cells). In contrast, CD32B (FcyRIIB) is an inhibitory Fc receptor on B lymphocytes (macrophages, neutrophils and eosinophils) (Stopforth, RJ et al. (2016) Regulation of Monoclonal Antibody Immunotherapy by FcyRIIB, J. Clin. Immunol. [2016 Feb 27 Epub], pp. 1-7; Bruhns, P. et al. (2009) Specificity And Affinity Of Human Fcgamma Receptors And Their Polymorphic Variants For Human IgG Subclasses, Blood. 113(16):3716-3725; White, A. L. et al (2014) FcyRIIB As A Key Determinant Of Agonistic Antibody Efficacy, Curr Top Microbiol Immunol 382:355-372 Selvaraj, P. et al (2004) Functional Regulation Of Human Neutrophil Fc Gamma Receptors, Immunol. Res. 29(l-3):219-230).

The ability of different FcyRs to mediate diametrically opposed functions reflects their structural differences, and in particular whether the FcyR possesses an immunoreceptor tyrosine activating motif (ITAM) or an immunoreceptor tyrosine inhibitory motif (ITIM). The recruitment of various cytoplasmic enzymes into these structures dictates the outcome of FcyR-mediated cellular responses. ITAM-containing FcyRs include FcyRI, FcyRIIA, FcyRIIIA and activate the immune system when bound to Fc domains (eg, aggregated Fc domains present in the immune complex). FcyRIIB is the only currently known

- 18 042568 natural ITIM-containing FcyR; it acts by weakening or inhibiting the immune system when bound to aggregated Fc domains. Human neutrophils express the FcyRIIA gene. Clustering of FcyRIIA via immune complexes or specific antibody cross-linking serves to aggregate ITAM with receptor-associated kinases that promote ITAM phosphorylation. Phosphorylation of ITAM serves as a docking site for Syk-kinase, the activation of which leads to the activation of downstream substrates (eg, PI3K). Cellular activation leads to the release of pro-inflammatory mediators. The FcyRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and binds IgG complexes in an indistinguishable manner. The presence of ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. The molecular basis of this inhibition has recently been established. When co-ligated with an activating FcyR, ITIM in FcyRIIB becomes phosphorylated and attracts the SH2 domain of inositol polyphosphate 5'-phosphatase (SHIP), which hydrolyzes the messenger phosphoinositol released by ITAM-containing FcyR mediated tyrosine kinase activation, hence preventing intracellular Ca influx. Thus, FcyRIIB cross-linking attenuates the activating response to FcyR ligation, inhibits cellular response, and stops B cell activation, resulting in cessation of B cell proliferation and antibody secretion.

II. Bispecific antibodies, multispecific diabodies and DART® diabodies.

The ability of an antibody to bind an antigen epitope depends on the presence and amino acid sequence of the antibody's VL and VH domains. The interaction of the light chain and heavy chain of an antibody, and in particular the interaction of its VL and VH domains, forms one of the two epitope-binding sites of a natural antibody, such as IgG. Natural antibodies are only able to bind to one kind of epitope (i.e., they are monospecific), although they can bind multiple copies of that kind (i.e., be bivalent or multivalent).

Antibody functionality can be enhanced by providing multispecific antibody molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen) and/or by providing an antibody molecule that has a higher valency (i.e. e. more than two binding sites) for the same epitope and/or antigen.

To provide molecules with greater potential than natural antibodies, a wide range of recombinant bispecific antibody formats have been developed (see, for example, PCT Publication Nos. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2009/018386, WO 2012/009544, WO 2013/070565), most of which use linker peptides either to connect an additional epitope-binding fragment (e.g. scFv, VL, VH, etc.) to or within the antibody core (IgA, IgD, IgE, IgG, or IgM) or join multiple epitope-binding fragments (eg, two Fab fragments or scFv). Alternative formats use linker peptides to fuse an epitope-binding fragment (e.g. scFv, VL, VH, etc.) to a dimerization domain such as a CH2-CH3 domain or alternative polypeptides (WO 2005/070966, WO 2006/107786AWO 2006/107617A, WO 2007/046893). PCT publications published in WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose a trispecific antibody in which the CL and CH1 domains switch from their respective natural positions and the VL and VH domains have been diversified (WO 2008/ 027236 WO 2010/108127) so that they can bind to more than one antigen. PCT Publication Nos. WO 2013/163427 and WO 2013/119903 disclose the modification of the CH2 domain to contain a fusion protein adduct comprising a binding domain. PCT publications WO 2010/028797, WO 2010028796 and WO 2010/028795 disclose recombinant antibodies whose Fc domains are replaced with additional VL and VH domains to form trivalent binding molecules. PCT Publication Nos. WO 2003/025018 and WO2003012069 disclose recombinant diabodies whose individual chains contain scFv domains. PCT Publication No. WO 2013/006544 discloses multivalent Fab molecules that are synthesized as a single polypeptide chain and then proteolyzed to form heterodimeric structures. PCT Publication Nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/07529.8 1992/022583 and WO 1991/003493 disclose the addition of additional binding domains or functionalities to an antibody or antibody portion (e.g., adding a diabody to an antibody light chain, or adding additional VL and VH domains to the light and heavy chains of an antibody, or adding a heterologous fusion protein, or linking multiple Fab domains to each other).

In addition, the ability to produce diabodies that differ from such natural antibodies capable of binding two or more different kinds of epitopes (i.e., exhibiting bispecificity or multispecificity in addition to the bivalent or multivalent form) is noted in the art (see, for example, Holliger et al (1993) 'Diabodies': Small Bivalent And Bispecific Antibody Fragments, Proc Natl Acad Sci (U.S.A.) 90:6444-6448; US 2004/0058400 (Hollinger et al); uS

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2004/0220388 / WO 02/02781 (Mertens et al); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280(20): 19665-19672; WO 02/02781 (Mertens et al); Olafsen, T. et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, Protein Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001) Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange, Protein Engineering 14(2): 1025-1033; Asano et al. (2004) A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain, Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588; Baeuerle, P.A. et al. (2009) Bispecific T-Cell Engaging Antibodies For Cancer Therapy, Cancer Res. 69(12):4941-4944).

The diabody design is based on an antibody derivative known as a single chain variable domain fragment (scFv). Such molecules are produced by linking the light and/or heavy chain variable domains with a short bridging peptide. Bird et al. (1988) (Single-Chain AntigenBinding Proteins, Science 242:423-426) describes an example of binding peptides that bridge approximately 3.5 nm between the carboxyl terminus of one variable domain and the amino terminus of another variable domain. Linkers with other sequences have been developed and used (Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242:423-426). In turn, linkers can be modified for additional functions such as drug attachment or attachment to solid supports. Single chain variants can be produced either recombinantly or synthetically. An automated synthesizer can be used to generate scFv synthetically. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes an scFv can be introduced into a suitable host cell, either eukaryotic, such as a yeast, plant, insect, or mammalian cell, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by conventional manipulations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification methods known in the art.

Providing bispecific binding molecules (e.g., non-monospecific diabodies) provides a significant advantage over antibodies, including, but not limited to, trans binding capacity sufficient to co-ligate and/or co-localize different cells that express different epitopes and/or cis-binding capacity, sufficient for co-ligation and/or co-localization of different molecules expressed by the same cell. Thus, bispecific binding molecules (eg, non-monospecific diabodies) have a wide range of applications, including therapy and immunodiagnosis. The bispecificity allows greater flexibility in the design and development of the diabody in various applications, allowing increased avidity for multimeric antigens, crosslinking of different antigens, and targeting specific cell types based on the presence of both target antigens. Because of their increased valency, low dissociation rates, and rapid clearance from the circulation (for small sizes, around or below ~50 kDa), diabody molecules known in the art have also shown particular utility in the field of tumor imaging (Fitzgerald et al. ( 1997) Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris, Protein Eng. 10:1221-1225).

The ability to produce bispecific diabodies has led to their use (in trans) for co-ligating two cells together, for example by co-ligating receptors present on the surface of different cells (eg, connecting cytotoxic T cells to tumor cells) (Staerz et al. (1985 ) Hybrid Antibodies Can Target Sites For Attack By T Cells, Nature 314:628-631, and Holliger et al.(1996) Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9:299- 305; Marvin et al (2005) Recombinant Approaches To IgG-Like Bispecific Antibodies, Acta Pharmacol Sin 26:649-658). Alternatively (or additionally) bispecific (or tri- or multispecific) diabodies can be used (in cis) to co-ligate molecules such as receptors etc. that are present on the surface of the same cell. Co-ligation of various cells and/or receptors is useful for modulating effector functions and/or signaling of the immune system. Multispecific molecules (e.g., bispecific diabodies) containing epitope binding sites can be targeted to the surface determinant of any immune cell such as CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc., which are expressed on T-lymphocytes, natural killer (NK) cells, antigen-presenting cells, or other mononuclear cells. In particular, cell surface receptor-targeted epitope-binding sites that are present in immune effector cells are useful for generating multispecific binding molecules capable of mediating retargeted cell killing.

However, the above benefits come at a significant cost. The formation of such non-monospecific diabodies requires the successful assembly of two or more distinct and dissimilar polypeptides.

- 20 042568 (i.e. such formation requires that diabodies are formed by heterodimerization of different types of polypeptide chain). This fact is in contrast to monospecific diabodies, which are formed by homodimerization of identical polypeptide chains. Because at least two heterogeneous polypeptides (i.e., two kinds of polypeptides) must be provided to form a non-monospecific diabody, and because homodimerization of such polypeptides results in the formation of inactive molecules (Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588), the production of such polypeptides must be done in such a way as to prevent covalent linkage between polypeptides of the same species (i.e., to prevent homodimerization) (Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588). Thus, the non-covalent association of such polypeptides has been reported in the art (see, e.g., Olafsen et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows SiteSpecific Conjugation And Radiolabeling For Tumor Targeting Applications, Prot. Engr. Des. Sel 17:21-27; Asano et al. (2004) 'A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain, Abstract 3P-683, J. Biochem. 76(8)02; Takemura, S. et al (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng 13(8):583-588 Lu, D. et al (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J Biol Chem 280(20): 19665-19672).

However, it is recognized in the art that bispecific diabodies composed of non-covalently linked polypeptides are unstable and readily dissociate into non-functional monomers (see, e.g., Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol Chem 280(20): 19665-19672).

Faced with this problem, the art has been able to develop stable, covalently linked, heterodimeric, non-monospecific diabodies called DART® diabodies; see, for example, pub. Pat. USA No. 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publications No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and publ. PCT No. WO 2012/162068; WO2012/018687; WO2010/080538; and Sloan, D.D. et al. (2015) Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells, PLoS Pathog. 11(11):el005233. doi: 10.1371/journal.ppat.1005233; Al Hussaini, M. et al. (2015) Targeting CD 123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®) Platform, Blood pii: blood-201405-575704; Chichili, G.R. et al. (2015) A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates, Sci. Transl. Med. 7(289):289ra82; Moore, P.A. et al. (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma, Blood 117(17):4542-4551; Veri, M.C. et al. (2010) Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold, Arthritis Rheum. 62(7): 1933-1943; Johnson, S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion, J. Mol. Biol. 399(3):436-449). Such diabodies contain two or more covalently complexed polypeptides and involve the creation of one or more cysteine residues in each of the polypeptide compounds used, which allow the formation of disulfide bonds and thereby covalently link one or more pairs of such polypeptide chains to each other. For example, the addition of a cysteine residue to the C-terminus of such constructs has been shown to allow for disulfide bonding between the incorporated polypeptide chains stabilizing the resulting diabody without interfering with the diabody's binding characteristics.

Many variations of such molecules have been described (see, for example, US Pat. No. 2015/0175697, 2014/0255407, 2014/0099318, 2013/0295121, 2010/0174053, 2009/0060910, 2007-0004909, European Publication EP 2714079, EP 2601216, EP 2376109, EP 2158221, EP 1868650 and PCT Public Nos.

Alternative constructs are known in the art for applications in which a tetravalent molecule is preferred but an Fc is not required, including, but not limited to, tetravalent tandem antibodies, also referred to as TandAbs (see, for example, US Pat. No. 2005-0079170 , 2007-0031436, 2010-0099853, 2011-020667 2013-0189263, European patent publications No. № No. EP 1078004, EP 2371866, EP 2361936 and EP 1293514; Publ. RST No. Wo 1999/057150, Wo 2003/025018 and Wo 2013/Wo 2013/Wo 2013/Wo 2013/ 013700), which are formed by the homodimerization of two identical polypeptide chains, each having a VH1, VL2, VH2 and VL2 region.

Recently, trivalent structures have been described comprising two diabody-type binding domains and one non-diabody-type binding domain and an Fc domain (see, for example, PCT Publication Nos. WO 2015/184207 and WO 2015/184203). Such trivalent binding molecules can be used to make monospecific, bispecific, or trispecific molecules. The ability to link three different epitopes provides extended possibilities.

III. B7-H3 human.

Human B7-H3 exists as a 4Ig form and as a 2Ig form. The amino acid sequence of the exemplary human form 4Ig B7-H3 (including the 29 amino acid signal sequence shown underlined) is shown in NCBI NP 001019907 (SEQ ID NO:16, 29 amino acid signal sequence shown underlined)

MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE

PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG

LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ

VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG

RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ

GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH

GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN

AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA

The human 2Ig form B7-H3 amino acid sequence completely encompasses the human form 4Ig B7-H3. The amino acid sequence of a representative form of human 2Ig B7-H3 (including the 29 amino acid signal sequence shown underlined) is shown in NCBI NP 079516 (SEQ ID NO:17) MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC

SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL

AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE

PNKDLRPGDT VTITCSSYRG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG

LFDVHSVLRV VLGANGTYSC LVRNPVLQQD AHGSVTITGQ PMTFPPEALW

VTVGLSVCLI ALLVALAFVC WRKIKQSCEE ENAGAEDQDG EGEGSKTALQ

PLKHSDSKED DGQEIA

In some embodiments, the B7-H3 binding molecules (e.g., scFvs, antibodies, bispecific diabodies, etc.) of the invention are characterized by any one, two, three, four, five, six, seven, eight, or nine of the following criteria:

(1) the ability to immunospecifically bind human B7-H3 endogenously expressed on the surface of a cancer cell;

(2) specifically bind B7-H3 of non-human primates (eg B7-H3 of the cynomolgus monkey);

(3) specifically bind human B7-H3 with an equilibrium binding constant (K _D ) of 1 nM or less;

(4) specifically bind B7-H3 of non-human primates with an equilibrium binding constant (K _D ) of 1 nM or less;

(5) specifically bind human B7-H3 with an association rate (ka) of 1x10 ⁶ M ^-1 min ^-1 or more;

(6) specifically bind B7-H3 of non-human primates with an association rate (ka) of 1x10 ⁶ M ^-1 min ^-1 or more;

(7) specifically bind human B7-H3 with a dissociation rate (kd) of 15 x 10 ^-4 min ^-1 or less;

(8) specifically bind B7-H3 of non-human primates with a dissociation rate (kd) of 15 x 10 ^-4 min ^-1 or less;

(9) has the ability to mediate retargeted cell killing (eg killing cancer cells expressing B7-H3).

As described elsewhere herein, the binding constants of the B7-H3 binding molecule can be determined using surface plasmon resonance, for example, using the BIACORE® assay. The surface plasmon resonance data can be approximated to a 1:1 Langmuir binding model (simultaneously ka kd) and the equilibrium binding constant KD can be calculated from the reaction rate constant ratio kd/ka. Such binding constants can be determined for a monovalent B7-H3 binding molecule (i.e. a molecule containing a single B7-H3 epitope binding site), a bivalent B7-H3 binding molecule (i.e. a molecule containing two B7-H3 -epitope-binding site) or a B7-H3 binding molecule having a higher valence (eg, a molecule containing three, four or more B7-H3 epitope-binding sites).

As used herein, retargeted cell killing refers to the ability of a molecule to mediate the killing of a target cell (eg, cancer cell) by localizing an immune effector cell (eg, T cells, NK cells, etc.) to the location of the target cell by binding epitopes present on the surfaces of such effector and target cells, resulting in the destruction of the target cell. The ability of a B7-H3 binding molecule (eg, a bispecific B7-H3 x CD3 binding molecule) to mediate redirected cell killing activity can be determined using a cytotoxic T-lymphocyte (CTL) assay. Such assays are well known in the art and preferred assays are described below.

The present invention particularly encompasses B7-H3 binding molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) containing anti-B7-H3 variable domains (i.e., VL and/or VH domains ) that immunospecifically bind to an epitope of the human B7-H3 polypeptide. Unless otherwise indicated, all such B7-H3 binding molecules are capable of immunospecific binding to human B7-H3. As used herein, such B7-H3 variable domains are referred to as ''anti-B7-H3-VL'' and anti-B7-H3-VH'', respectively.

IV. Mouse antibodies against human B7-H3 and their humanized derivatives.

From hybridoma cells that were obtained by immunization with cells expressing human B7-H3, the B7-H3 polypeptide or its peptide epitope, four typical anti-B7-H3 antibodies were isolated, designated as mAb-A, mAb-B, mAb-C and mAb-D. The mAbB, mAb-C and mAb-D antibodies were humanized.

The mAb-C and mAb-D antibodies were found to be cross-reactive with B7-H3 of the cynomolgus monkey. Below are the amino acid sequences of the VL and VH domains of mAb-C and mAb-D. In one embodiment, preferred anti-human B7-H3 binding molecules of the present invention possess 1, 2, or all 3 VH domain CDRHs and/or 1, 2, or all 3 VL domain, VH and/or VL domains of a mouse anti-B7-H3 monoclonal mAb-D antibody, chimeric mAb-D monoclonal antibody (chmAb-D), or humanized mAb-C or mAb-D monoclonal antibody (HmAb-C or hmAb-D). Such preferred anti-human B7-H3 binding molecules include bispecific (or multispecific) antibodies, chimeric or humanized antibodies, BiTe, diabodies, etc., and such binding molecules having variant Fc domains. The invention encompasses the use of any mAb-A, mAb-B, mAb-C or mAb-D to form B7-H3 binding molecules and in particular B7-H3-ADC.

A. Mouse anti-human B7-H3 mAb-A.

Shown below is the amino acid sequence of the VL domain of a mouse anti-B7-H3 antibody designated as mAb-A (SEQ ID NO:95) (CDRL residues are shown underlined)

DIAMTQSQKF MSTSVGDRVS VTCKASQNVD TNVAWYQQKP GQSPKALIYS

ASYRYSGVPD RFTGSGSGTD FTLTINNVQS EDLAEYFCQQ YNNYPFTFGS GTKLEIK

Shown below is the amino acid sequence of the VH domain of mAb-A (SEQ ID NO:96) (CDRh residues are underlined)

DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQA PEKGLEWVAY

ISSDSSAIYY ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAMYYCGRGR ENIYYGSRLD YWGQGTTLTV SS

B. Mouse anti-human B7-H3 mAb-B.

Shown below is the amino acid sequence of the VL domain of a mouse anti-B7-H3 antibody designated as mAb-B (SEQ ID NO:97) (CDRL residues are shown underlined)

DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLLIYY

TSRLHSGVPS RFSGSGSGTD YSLTIDNLEQ EDIATYFCQQ GNTLPPTFGG GTKLEIK

Shown below is the amino acid sequence of the VH domain from mAb-B (SEQ ID NO:98) (CDRH residues are shown underlined)

QVQLQQSGAE LARPGASVKL SCKASGYTFT SYWMQWVKQR PGQGLEWIGT

IYPGDGDTRY TQKFKGKATL TADKSSSTAY MQLSSLASED SAVYYCARRG IPRLWYFDVW GAGTTVTVSS

C. Humanized anti-human B7-H3 hmAb-B.

Shown below is the amino acid sequence of the VL domain from hmAb-B (SEQ ID NO:99) (CDRL residues are shown underlined)

DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY

TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG GTKLEIK

In some embodiments, the amino acid sequence of CDRL1 from hmAb-B (RASQDISOTLN) (SEQ ID NO:100) may be replaced with an alternative CDRL1 having the amino acid sequence of RASQMSNYLN (SEQ ID NO:101). Similarly, the amino acid sequence of CDRL2 from hmAb-B (YTSRLHS) (SEQ ID NO:102) can be replaced with an alternative CDRL2 having the amino acid sequence ytsrl^s (SEQ ID NO:103).

Shown below is the amino acid sequence of the VH domain from hmAb-B (SEQ ID NO:104) (CDRH residues are underlined)

QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQWVRQA PGQGLEWMGT

IYPGDGDTRY TQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARRG

IPRLWYFDVW GQGTTVTVSS

- 23 042568

In some embodiments, the amino acid sequence of CDRH2 from hmAb-B (tiypgdgdtrytqkfxg) (SEQ ID NO:105) can be replaced with an alternative CDRH2 having the amino acid sequence: tiypgggdtrytqkf^g (SEQ ID NO:106).

D. Mouse anti-human B7-H3 mAb-C.

Shown below is the amino acid sequence of the VL domain of a mouse anti-B7-H3 antibody designated as mAb-C (SEQ ID NO:18) (CDR _L residues are shown underlined)

DIQMTQSPAS LSVSVGETVT ITCRASESIY SYLAWYQQKQ GKSPQLLVYN

TKTLPEGVPS RFSGSGSGTQ FSLKINSLQP EDFGRYYCQH HYGTPPWTFG GGTNLEIK

Shown below is the amino acid sequence of the VH domain from mAb-C (SEQ ID NO:19) (CDRH residues are shown underlined)

EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT PDKRLEWVAT

INSGGSNTYY PDSLKGRFTI SRDNAKNTLY LQMRSLKSED TAMYYCARHD GGAMDYWGOG TSVTVSS

E. Humanized anti-human B7-H3 hmAb-C.

The variable domains of the anti-B7-H3 mAb-C were humanized. In some cases, alternative humanized variable domains have been designed to optimize binding activity and/or to remove antigenic epitopes and/or to remove potentially labile amino acid residues.

Shown below is the amino acid sequence of the VL domain from hmAb-C (SEQ ID NO:20) (CDR _l residues are shown underlined)

DIQMTQSPSS LSASVGDRVT ITCRASESIY SYLAWYQQKP GKAPKLLVYN

TKTLPEGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH HYGTPPWTFG QGTRLEIK

Shown below is the amino acid sequence of the VH domain from hmAb-C (SEQ ID NO:21) (CDRH residues are shown underlined)

EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLEWVAT INSGGSNTYY PDSLKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHD GGAMDYWGOG TTVTVSS

F. Mouse anti-human B7-H3 mAb-D.

Shown below is the amino acid sequence of the VL domain of a mouse anti-B7-H3 antibody designated as mAb-D (SEQ ID NO:22) (CDR _L residues are shown underlined)

DIVMTQSQKF MSTSVGDRVS VTCKASQNVD TNVAWYQQKQ GHSPEALIYS ASYRYSGVPA RFTGSGSGTD FTLTISNVQS EDLAEYFCQQ YNNYPFTFGG GTKLEIK

The amino acid sequence of the CDR _L 1 domain of mAb-D is (SEQ ID NO:23): KASQNVDTNVA.

The amino acid sequence of the CDR _L 2 domain of mAb-D is (SEQ ID NO:24): SASYRYS.

The amino acid sequence of the CDR _L 3 domain of mAb-D is (SEQ ID NO:25): QQYNNYPFT.

Shown below is the amino acid sequence of the VH domain from mAb-D (SEQ ID NO:26) (residues

CDRH shown underlined)

DVQLAESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQA PEKGLEWVAY

ISSGSGTIYY ADTVKGRFTI SRDNPKNSLF LQMTSLRSED TAMYYCARHG

YRYEGFDYWG QGTTTLTVSS

The amino acid sequence of the CDR _H 1 domain of mAb-D is (SEQ ID NO:27): SFGMH.

The amino acid sequence of the CDR _H 2 domain of mAb-D is (SEQ ID NO:28): YISSGSGTIYYADTVKG.

The amino acid sequence of the CDR _H 3 domain of mAb-D is (SEQ ID

NO:29): HGYRYEGFDY.

G. Humanized anti-human B7-H3 mAb-D.

The variable domains of the anti-B7-H3 antibody mAb-D were humanized. In some cases, alternative humanized variable domains have been designed to optimize binding activity and/or to remove antigenic epitopes and/or to remove potentially labile amino acid residues.

Shown below is the amino acid sequence of the VL domain from hmAb-D (SEQ ID NO:30) (CDR _l residues are shown underlined)

DIQMTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFAEYFCQQ YNNYPFTFGQ GTKLEIK

Shown below is the amino acid sequence of the VH domain from hmAb-D (SEQ ID NO:31) (CDRH residues are shown underlined)

EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHG YRYEGFDYWG QGTTVTVSS

V. Chimeric antigen receptors.

The B7-H3 binding molecules of the present invention may be monospecific single chain molecules such as single chain variable fragments (anti-B7-H3-scFv) or chimeric antigen receptors (anti-B7-H3-CAR)). As discussed above, scFvs are created by linking the light and heavy chain variable domains together with a short bridging peptide. The original CARs usually had an intracellular domain from the CD3 ζ chain, which is the main transmitter of signals from endogenous TCRs. Second generation CARs have additional intracellular signaling domains from various costimulatory protein receptors (eg, CD28, 41BB, ICOS, etc.) attached to the cytoplasmic tail of the CAR in order to provide additional signals to the T cell. Third generation CARs combine multiple signaling domains such as CD3z-CD28-41BB or CD3z-CD28-OX40 to further enhance activity (Tettamanti, S. et al. (2013) Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor, Br J Haematol 161:389-401 Gill, S et al (2014) Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor -Modified T Cells, Blood 123(15): 2343-2354 Mardiros, A. et al.(2013) T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia, Blood 122 :3138-3148 Pizzitola, I. et al (2014) Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo, Leukemia doi:10.1038/leu.2014.62).

The Ahtu-B7-H3-CARs of the present invention contain anti-B7-H3-scFv fused to the intracellular domain of the receptor. The light chain and heavy chain variable domains of ati-B7-H3-scFv are preferably hmAb-C VL (SEQ ID NO:20) and hmAb-C VH (SEQ ID NO:21) or are preferably hmAb-D VL (SEQ ID NO:30) and hVH from mAb-D (SEQ ID NO:31).

The intracellular domain of the anti-B7-H3-CAR of the present invention is preferably selected from the intracellular domain of any of 41BB-CD3Z, b2c-CD3Z, CD28, CD28-4-1BB-CD3Z, CD28-CD3Z, CD28-FcεRIγ, CD28mut-CD3z, CD28 -OX40-CD3z, CD28-OX40-CD3z, CD3z, CD4-CD3z, CD4-FcRRI, CD8-CD3z, FcsRIy, FcRRIyAAIX, heregulin-POS ^L , IL-13-CD3C, or Ly49H-CD3z (Tettamanti, S. et al (2013) Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel Cd 123-Specific Chimeric Antigen Receptor Br. J. Haematol.161:389-401; Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells Blood 123(15): 2343-2354;Mardiros, A. et al.(2013) T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia, Blood 122:3138-3148 Pizzitola, I. et al.(2014) Chimeric Antigen Re receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo Leukemia doi:10.1038/leu.2014.62).

VI. Multispecific B7-H3 binding molecules.

The present invention is also directed to multispecific (eg, bispecific, trispecific, etc.). B7-H3 binding molecules containing an epitope-binding site (preferably containing 1, 2 or all 3 CDR _H domains from the VH domain against B7-H3 according to the invention and/or 1, 2 or all 3 CDRL domains from the VL domain against B7-H3 according to the invention or such an anti-B7H3 VH domain and/or such an anti-B7-H3 VL domain) and further comprising a second epitope-binding site that immunospecifically binds to a second epitope, wherein said second epitope is: (i) another B7-H3 epitope or (ii) ) an epitope of a molecule that is not B7-H3. Such multispecific B7-H3 binding molecules preferably contain a combination of epitope binding sites that recognize multiple antigens unique to the target cell or tissue type. In particular, the present invention relates to multispecific B7-H3 binding molecules that are capable of binding to a B7-H3 epitope and an epitope of a molecule present on the surface of an effector cell, especially a T-lymphocyte, natural killer (NK) or other mononuclear cells. For example, such B7-H3 binding molecules of the present invention can be designed to contain an epitope binding site that immunospecifically binds to CD2, CD3, CD8, CD 16, T cell receptor (TCR), or NKG2D.

One embodiment of the present invention provides bispecific B7-H3 binding molecules that are capable of binding to a first epitope and a second epitope, where such epitopes are not identical to each other. Such bispecific molecules contain VL1/VH1 domains that are capable of binding to a first epitope and VL2/VH2 domains that are capable of binding to a second epitope. The designations VL1 and VH1 indicate, respectively, the light chain variable domain and the heavy chain variable domain that bind the first epitope of such bispecific molecules. Similarly, the designations VL2 and VH2, respectively, indicate the light chain variable domain and the heavy chain variable domain that bind the second epitope of such bispecific molecules. It does not matter whether a particular epitope is designated by the first and second epitopes; and such designations are relevant only to the presence and orientation of the domains of the polypeptide chains of the binding molecules of the present invention. In one embodiment, one of such epitopes is a human B7-H3 epitope and the other is

- 25 042568 another B7-H3 epitope or is an epitope of a molecule that is not B7-H3. In specific embodiments, one such epitope is a human B7-H3 epitope and the other is an epitope of a molecule (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T-lymphocyte, a natural killer (NK) cell or other mononuclear cell. In some embodiments, the bispecific molecule contains more than two epitope binding sites. Such bispecific molecules bind at least one epitope of B7-H3 and at least one epitope of a molecule that is not B7-H3, and may further bind additional epitopes of B7-H3 and/or additional epitopes of a molecule that is not B7-I3.

The present invention specifically relates to bispecific, trispecific, and multispecific B7-H3 binding molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) that possess epitope-binding antibody fragments (e.g., VL and VH domains). ) that allow them to be able to coordinate with at least one epitope of B7-H3 and at least one epitope of a second molecule that is not B7-H3. The selection of the VL and VH domains of the polypeptide domains of such molecules is coordinated such that the polypeptide chains that form such multispecific B7-H3 binding molecules assemble to form at least one functional epitope-binding site that is specific for at least one B7H3 epitope and at least one functional epitope binding site that is specific for at least one epitope of the molecule that is not B7-H3. Preferably, the multispecific B7-H3 binding molecules contain 1, 2 or all 3 VH domain _H CDRs against B7H3 of the invention and/or 1, 2 or all 3 VL domain _L CDRs against B7-H3 of the invention, or such VH domain against B7 -H3 and/or such a VL domain against B7-H3 as provided herein.

A. Bispecific antibodies.

The present invention encompasses bispecific antibodies capable of simultaneously binding to a B7-H3 epitope and an epitope of a molecule that is not B7-H3. In some embodiments, a bispecific antibody capable of simultaneously binding to B7-H3 and a second molecule that is not B7-H3 is prepared using any of the methods described in PCT Publications WO 1998/002463, WO 2005/070966, WO 2006/107786 WO 2007/024715, Wo 2007/075270, Wo 2006/107617, WO 2007/046893, Wo 2007/146968, Wo 2008/003103, Wo 2008/003116, Wo 2008/027236, Wo 2008/024188, Wo 2009/132876, Wo 2009/018386, Wo 2010/028797, Wo 02010028796, Wo 2010/028795, Wo 2010/108127, Wo 2010/136172, Wo 2011/086091, Wo 2011/133886, Wo 2012/009544, WO 2013/003652, Wo 2013/Wo 2013/Wo 2013/Wo 2013/Wo 2013/Wo 2013/Wo 2013 070565, WO 2012/162583, WO 2012/156430, WO 2013/174873 and WO 2014/022540, each of which is incorporated herein by reference in its entirety.

B. Bispecific diabodies lacking Fc domains.

One embodiment of the present invention relates to bispecific diabodies that are capable of binding to a first epitope and a second epitope, wherein the first epitope is a human B7-H3 epitope and the second is an epitope of a molecule that is not B7-I3, preferably a molecule (e.g., CD2, CD3 , CD8, CD16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T lymphocyte, natural killer (NK) cell, or other mononuclear cell. Such diabodies comprise, and most preferably consist of, a first polypeptide chain and a second polypeptide chain whose sequences allow the polypeptide chains to covalently bind to each other to form a covalently associated diabody that is capable of simultaneously binding to the B7-I3 epitope and the second epitope.

The first polypeptide chain of such an embodiment of the bispecific diabodies comprises, in the N-terminal to C-terminal direction: the N-terminus of the VL domain of a monoclonal antibody capable of binding to the first or second epitope (i.e. either VL- _a n _TU -in ₇ -n ₃ - _VL or VL- _enutO π2), the first intermediate spacer peptide (linker 1), the VH domain of the monoclonal antibody capable of binding to the second epitope (if such first polypeptide chain contains VL- _a n _TU - _B7 - n ₃ - _VL ) or B7-H3 (if such first polypeptide chain contains VL- _Enuton 2), a second intermediate spacer peptide (linker 2), optionally containing a cysteine residue, a heterodimerization promoting domain, and a C-terminus (FIG. 1).

The second polypeptide chain of this embodiment of the bispecific diabodies comprises, in the N-terminus to C-terminus direction: N-terminus, a VL domain of a monoclonal antibody capable of binding to the first or second epitope (i.e. either VL- _and n _TU - in ₇ - n ₃ - _VL , or VL- _Enuton 2, which is a VL domain not selected for inclusion in the first diabody polypeptide chain), an intermediate spacer peptide (linker 1), a VH domain of a monoclonal antibody capable of binding either a second epitope (if such a second polypeptide chain contains VL- _{anti.B7 - H3} - _VL ), or B7-H3 (if such second polypeptide chain contains VL- _Enuton 2), a second intermediate spacer peptide (linker 2), optionally containing a cysteine residue, a heterodimerization promoting domain, and C-terminus (Fig. 1).

The VL domain of the first polypeptide chain interacts with the VH domain of the second polypeptide chain to form a first functional epitope binding site that is specific for the first antigen (ie, either B7-H3 or a molecule that includes the second epitope). Similarly, the VL domain

- 26 042568 of the second polypeptide chain interacts with the VH domain of the first polypeptide chain to form a second functional epitope-binding site that is specific for a second antigen (ie either a molecule that includes the second epitope or B7-H3). Thus, the choice of VL and VH domains of the first and second polypeptide chains is coordinated such that the two diabody polypeptide chains together contain VL and VH domains capable of binding to both the B7-H3 epitope and the second epitope (i.e., they together contain VL-anti-B7-H3-VL/VH anti-B7-H3-VH and VL epitope 2/VH epitope 2).

Most preferably, the length of the intermediate spacer peptide (i.e., linker 1 that separates such VL and VH domains) is chosen to substantially or completely prevent the VL and VH domains of the polypeptide chain from linking to each other (i.e., consisting of 0 , 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues of the intermediate linker). Thus, the VL and VH domains of the first polypeptide chain are essentially or completely unable to communicate with each other. Similarly, the VL and VH domains of the second polypeptide chain are essentially or completely unable to communicate with each other. A preferred intermediate spacer peptide (linker 1) has the sequence (SEQ ID NO:32): GGGSGGGG.

The length and composition of the second intermediate spacer peptide (linker 2) is chosen based on the choice of one or more polypeptide domains that promote such dimerization (ie, a domain that promotes heterodimerization). Typically, the second intermediate spacer peptide (linker 2) will contain 3-20 amino acid residues. In particular, when the heterodimerization promoting domain(s) used do not contain a cysteine residue, a cysteine-containing second intermediate spacer peptide (linker 2) is used. The cysteine-containing second intermediate spacer peptide (linker 2) will contain 1, 2, 3 or more cysteines. A preferred cysteine-containing spacer peptide (linker 2) has the sequence GGCGGG (SEQ ID NO:33). Otherwise, linker 2 does not contain cysteine (e.g., GGG, GGGS (SEQ ID NO:34), LGGGSG (SEQ ID NO:35), GGGSGGGGGGG (SEQ ID NO:36), ASTKG (SEQ ID NO:37), LEPKSS (SEQ ID NO:38), APSSS (SEQ ID NO:39), etc.) and a cysteine containing heterodimerization promoting domain as described below. Optionally, both a cysteine-containing linker 2 and a cysteine-containing domain to promote heterodimerization are used.

Heterodimerization promoting domains can be GVEPKSC (SEQ ID NO:40) or VEPKSC (SEQ ID NO:41) or AEPKSC (SEQ ID NO:42) on the same polypeptide chain and GFNRGEC (SEQ ID NO:43) or FNRGEC ( SEQ ID NO: 42) ID NO: 44) on another polypeptide chain (US 2007/0004909).

In a preferred embodiment, the heterodimerization promoting domains will comprise tandemly repetitive helical domains of opposite charge, e.g., E-helix helical domains (SEQ ID NO:45:EVAALEK-EVAALEK-EVAALEK-EVAALEK) whose glutamate residues form a negative charge at pH 7, and K-helix domains (SEQ ID NO:46: KVAALKE-KVAALKE-KVAALKE-KVAALKE) whose lysine residues form a positive charge at pH 7. The presence of such charged domains promotes association between the first and second polypeptides and thus promotes heterodimer formation. . Heterodimerization promoting domains, which include modifications of the E-helix and K-helix sequences described above, may include one or more cysteine residues. The presence of such cysteine residues allows a helix present in one polypeptide chain to covalently bind to an additional helix present in another polypeptide chain, thereby covalently linking the polypeptide chains to each other and increasing the stability of the diabody. Examples of such particularly preferred heterodimerization promoting domains include a modified E-helix having the amino acid sequence EVAACEK-EVAAALEK-EVAALEK-EVAALEK (SEQ ID NO:47) and a modified K-helix having the amino acid sequence KVAACKE-KVAALKE-KVAALKE-KVAALKE ( SEQ ID NO:48).

As disclosed in WO 2012/018687, in order to improve the pharmacokinetic properties of the diabody in vivo, the diabody can be modified to include the polypeptide portion of the serum-bound protein at one or more ends of the diabody. Most preferably, such a serum binding protein polypeptide portion will be positioned at the C-terminus of the diabody polypeptide chain. Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin has several small molecule binding sites that allow it to non-covalently bind to other proteins and thus prolong the serum half-life. Albumin-binding domain 3 (ABD3) of the G protein of G148 Streptococcus strain consists of 46 amino acid residues, forming a stable three-helix knot and having broad albumin-binding specificity (Johansson, M.U. et al. (2002) Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules, J. Biol Chem. more preferably the albumin binding domain 3 (ABD3) of protein G of strain G148 of Streptococcus (SEQ ID NO:49): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

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As disclosed in WO 2012/162068 (herein incorporated by reference), deimmunized variants of SEQ ID NO:49 have the ability to reduce or eliminate class II MHC binding. Based on the results of combinatorial mutations, the following combinations of substitutions are considered to be the preferred substitutions for the formation of such deimmunized ABD: 66D/70S + 71A; 66S/70S+71A; 66S/70S+79A; 64A/65A/71A; 64A/65A/71A + 66S; 64A/65A/71A + 66D; 64A/65A/71A + 66e; 64A/65A/79A + 66S; 64A/65A/79A + 66D; 64A/65A/79A + 66e. ABD variants with L64A, I65A and D79A modifications or N66S, T70S and D79A modifications. Deimmunized ABD variant having the amino acid sequence

LAEAKVLANR ELDKYGVSDY YKNLID ₆₆ NAKS7o A71EGVKALIDE ILAALP (SEQ

IDNO:50), or amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNA6 ₄ A6 ₅ NNAKT VEGVKALIA79E ILAALP (SEQ ID NO :51), or amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNLIS ₆ 6NAKS ₇ o VEGVKALIA79E ILAALP (SEQ

ID NO:52) are particularly preferred as such deimmunized ABDs exhibit substantially wild-type binding while providing reduced binding to MHC class II. Thus, the first polypeptide chain of such a diabody having an ABD contains a third linker (linker 3), preferably located at the C-terminus of the E-helical (or K-helical) domain of such a polypeptide chain, for the purpose of intervening between the E-helical (or K -helical) domain and ABD (which is preferably deimmunized ABD). The preferred sequence for such linker 3 is SEQ ID NO:34: GGGS.

C. Multispecific diabodies containing Fc domains.

One embodiment of the present invention relates to multispecific diabodies capable of simultaneously binding to a B7-H3 epitope and a second epitope (i.e., another B7-H3 epitope or an epitope of a molecule that is not B7-H3) that include an Fc domain. The addition of an IgG CH2-CH3 domain to one or both of the polypeptide chains of an antibody with which complexation of the diabody chains results in the formation of an Fc domain increases the biological half-life and/or changes the valence of the diabody. Such diabodies contain two or more polypeptide chains whose sequences allow the polypeptide chains to covalently bind to each other to form a covalently linked diabody that is capable of simultaneously binding to a B7-H3 epitope and a second epitope. The incorporation of IgG CH2-CH3 domains into both diabody polypeptides will allow the formation of a double-stranded bispecific diabody containing an Fc region (FIG. 2).

Alternatively, incorporation of the CH2-CH3 domains from IgG into only one of the diabody polypeptides would allow for the formation of a more complex four chain bispecific Fc domain containing diabody (FIGS. 3A-3C). In FIG. 3C shows an exemplary four-chain diabody having a light chain constant domain (CL) and a heavy chain constant domain CH1, however, fragments of such domains as well as other polypeptides can be used as an alternative (see, for example, FIGS. 3A and 3B, publ. Pat. US Patent Nos. 2013-0295121, 2010-0174053 and 2009-0060910, European Patent Publication Nos. EP 2714079, EP 2601216, EP 2376109, EP 2158221 and PCT Publication Nos. WO 2012/162068, WO0 2812 WO 2010/080538). For example, instead of the CH1 domain, a peptide having the amino acid sequence GVEPKSC (SEQ ID NO:40), VEPKSC (SEQ ID NO:41) or AEPKSC (SEQ ID NO:42) derived from the human IgG hinge region can be used, and instead of CL domain, the C-terminal 6 amino acids of the human kappa light chain, GFNRGEC (SEQ ID NO:43) or FNRGEC (SEQ ID NO:44) can be used. A representative four chain diabody containing the peptide is shown in FIG. 3A. Alternatively, or in addition, a peptide containing tandem helical domains of opposite charge, such as E-helix helical domains (SEQ ID NO:45: EVAALEK-EVAALEK-EVAALEK-EVAALEK or SEQ ID NO:47: EVAACEK- EVAALEK-EVAALEK-EVAALEK); and K-helix domains (SEQ ID NO:46: KVAALKE-KVAALKE-KVAALKE-KVAALKE or SEQ ID NO:48: KVAAQ^-KVAAL#-KVAALKE-KVAALKE). A representative four-stranded diabody containing a helical domain is shown in FIG. 3b.

The Fc domain containing molecules of the present invention may include additional intermediate spacer peptides (linkers), typically such linkers will be included between the heterodimerization promoting domain (e.g. E-helix or K-helix) and the CH2-CH3 domain and/ or between a CH2-CH3 domain and a variable domain (ie, VH or VL). Typically, additional linkers will contain 3-20 amino acid residues and may optionally contain all or part of the IgG hinge domain (preferably the cysteine-containing part of the IgG hinge domain). Linkers that can be used in the bispecific Fc-domain-containing diabody molecules of the present invention include GGGS (SEQ ID NO:34), LGGGSG (SEQ ID NO:35), GGGSGGGGGGG (SEQ ID NO:36), ASTKG (SEQ ID NO:34) ID NO: 37), LEPKSS (SEQ ID NO:38), APSSS (SEQ ID NO:39), APSSSPME (SEQ ID NO:53), VEPKSADKTHTCPPCP (SEQ ID NO:54), LEPKSADKTHTCPPC (SEQ ID NO:55), DKTHTCPPCP (SEQ ID NO:56), GGC and GGG. LEPCSS (SEQ ID

- 28 042568

NO:38) can be used in place of GGG or GGC for ease of cloning. In addition, amino acids GGG or LEPKSS (SEQ ID NO:38) can immediately follow DKTHTCPPCP (SEQ ID NO:56) to form alternative linkers: GGGDKTHTCPPCP (SEQ ID NO:57); and LEPKSSDKTHTCPPCP (SEQ ID NO:58). The bispecific Fc-domain-containing molecules of the present invention may include an IgG hinge domain in addition to or instead of a linker. Exemplary hinge domains include: EPKSCDKTHTCPPCP (SEQ ID NO:7) from IgG1, ERKCCVECPPCP (SEQ ID NO:8) from IgG2, ESKYGPPCPSCP (SEQ ID NO:10) from IgG4, and ESKYGPPCPPCP (SEQ ID NO:11), a hinge variant from IgG4 including a stabilizing S228P substitution (according to the EU index numbering as specified in Kabat) to reduce the exchange of strands.

As shown in FIG. 3A-3C, the Fc domain-containing diabodies of the invention may include four strands. The first and third polypeptide chains of such a diabody comprise three domains: (i) a VL1 containing domain, (ii) a VH2 containing domain, (iii) a heterodimerization promoting domain, and (iv) a CH2-CH3 containing domain. The second and fourth polypeptide chains contain: (i) a VL2 containing domain, (ii) a VH1 containing domain, and (iii) a heterodimerization promoting domain, wherein the heterodimerization promoting domains promote dimerization of the first/third polypeptide chains with the second/fourth polypeptide chains. chains. The VL and/or VH regions of the third and fourth polypeptide chains, as well as the VL and/or VH domains of the first and second polypeptide chains, may be the same or different so as to provide tetravalent binding that is monospecific, bispecific, or tetraspecific. The designations VL3 and VH3 denote, respectively, the light chain variable domain and the heavy chain variable domain that bind the third epitope of such a diabody. Similarly, the designations VL4 and VH4 denote, respectively, a light chain variable domain and a heavy chain variable domain that bind the fourth epitope of such a diabody. The general structure of the polypeptide chains of representative four-chain bispecific Fc-domain-containing diabodies according to the invention is presented in table. 1.

Table 1

Bispecific ^2nd chain _NH2 -VL2-VH1-HPD-COOH g ⁱ chain NH ₂ -VL1-VH2-HPD-CH2-CH3-COOH ^1st chain NH ₂ -VL1-VH2-HPD-CH2-CH3-COOH ^2nd chain _NH2 -VL2-VH1-HPD-COOH Tetraspecific ^2nd chain _NH2 -VL2-VH1-HPD-COOH g ⁱ chain NH ₂ -VL1-VH2-HPD-CH2-CH3-COOH ^3rd chain NH ₂ -VL3-VH4-HPD-CH2-CH3-COOH ^4th network _NH2 -VL4-VH3-HPD-COOH

HPD = Heterodimerization Promoting Domain.

In a specific embodiment, the antibodies of the present invention are bispecific, tetravalent (ie, have four epitope-binding sites) Fc-containing diabodies that consist of four complete polypeptide chains (FIGS. 3A-3C). The bispecific, tetravalent, Fc-containing diabodies of the invention comprise two B7-H3 immunospecific epitope-binding sites (which may be capable of binding to the same B7-H3 epitope or to different B7-H3 epitopes) and two epitope-binding sites immunospecific to a second molecule (which may be capable of binding to the same epitope of the second molecule or to different epitopes of the second molecule). Preferably, the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T lymphocyte, natural killer (NK ) or other mononuclear cell.

In another embodiment, the Fc-domain-containing diabodies of the present invention may include three polypeptide chains. The first polypeptide of such a diabody comprises three domains: (i) a domain containing VL1, (ii) a domain containing VH2, and (iii) a domain containing a CH2-CH3 sequence. The second polypeptide of such a diabody includes: (i) a VL2 containing domain, (ii) a VH1 containing domain, and (iii) a domain that promotes heterodimerization and covalent bonding with the first diabody polypeptide chain. The third polypeptide of such a diabody includes the sequence CH2-CH3. Thus, the first and second polypeptide chains of such a diabody are combined together to form a VL1/VH1 epitope binding site that is capable of binding to the first antigen (i.e. either B7-H3 or a molecule that includes a second epitope) as well as a VL2 epitope binding site. /VH2 that is capable of binding to a second antigen (ie, either a molecule that includes a second epitope or B7-H3). The first and second polypeptides are linked to each other via a disulfide bond involving cysteine residues in their respective third domains. Notably, the first and third polypeptide chains combine with each other to form an Fc domain that is stabilized via a disulfide bond. Such bispecific diabodies have increased efficacy. In FIG. 4A and 4B show the structures of such diabodies. Such Fc-region-containing diabodies can have one of two orientations (Table 2).

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table 2

First orientation ^3rd chain NH2-CH2-CH3-COOH ^1st chain W-VL 1 - VH2-HPD-CH2-CH3 -COOH ^2nd chain NH2-VL2-VH1-HPD-COOH Second orientation ^3rd chain NH2-CH2-CH3-COOH ^1st chain NH2-CH2-CH3 - VL 1 - VH2-HPD-COOH ^2nd chain NH2-VL2-VH1-HPD-COOH

HPD = Heterodimerization Promoting Domain.

In a specific embodiment, the antibodies of the present invention are bispecific, divalent (ie, have two epitope-binding sites) Fc-containing diabodies that consist of three complete polypeptide chains (FIGS. 4A-4B). The bispecific divalent Fc-containing diabodies of the invention contain one epitope-binding site immunospecific for B7-H3 and one epitope-binding site immunospecific for the second molecule. Preferably, the second molecule is a molecule (e.g., CD2, CD3, CD8, CD 16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T lymphocyte, natural killer (NK) or other mononuclear cells.

In yet another embodiment, Fc-domain-containing diabodies may contain a total of five polypeptide chains. In a specific embodiment, two of these five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such a diabody comprises: (i) a VH1-containing domain, (p) a CH1-containing domain, and (iii) a domain containing a CH2-CH3 sequence. The first polypeptide chain may be an antibody heavy chain that includes VH1 and a heavy chain constant region. The second and fifth polypeptide chains of such a diabody comprise: (i) a VL1-containing domain and (d) a CL-containing domain. The second and/or fifth polypeptide chains of such a diabody may be antibody light chains that contain a VL1 complementary to the VH1 of the first/third polypeptide chain. The first, second and/or fifth polypeptide chains can be isolated from a naturally occurring antibody. Otherwise, they can be engineered recombinantly. The third polypeptide chain of such a diabody comprises: (i) a VH1 containing domain, (i) a CHI containing domain, (iii) a CH2-CH3 containing domain, (iv) a VL2 containing domain, (v) a VH3 containing domain, and (vi) a heterodimerization promoting domain, wherein the heterodimerization promoting domains promote dimerization of the third strand with the fourth strand. The fourth polypeptide of such diabodies includes: (i) a VL3 containing domain, (ii) a VH2 containing domain, and (iii) a domain that promotes heterodimerization and covalent bonding with the third diabody polypeptide chain.

Thus, the first and second, and third and fifth polypeptide chains of such diabodies bind together to form two VL1/VH1 epitope-binding sites capable of binding the first epitope. The third and fourth polypeptide chains of such diabodies are linked together to form a VL2/VH2 epitope binding site that is capable of binding to a second epitope, as well as a VL3/VH3 binding site that is capable of binding to a third epitope. The first and third polypeptides are linked to each other via a disulfide bond comprising cysteine residues in their respective constant regions. Notably, the first and third polypeptide chains are complexed with each other to form an Fc domain. Such multispecific diabodies have increased efficacy. In FIG. 5 shows the structure of such diabodies. It is understood that the VL1/VH1, VL2/VH2, and VL3/VH3 domains may be the same or different in order to allow binding that is monospecific, bispecific, or trispecific. As indicated herein, these domains are preferably chosen to bind a B7-H3 epitope, a second molecule epitope, and optionally a third molecule epitope.

The VL and VH domains of the polypeptide chains are chosen such that VL/VH binding sites specific to the epitope of interest are formed. The VL/VH binding sites formed by the association of polypeptide chains may be the same or different in order to provide univalent binding that is monospecific, bispecific, trispecific, or tetraspecific. In particular, the VL and VH domains may be chosen such that the multivalent diabody may include two binding sites for the first epitope and two binding sites for the second epitope, or three binding sites for the first epitope and one binding site for the second epitope, or two binding site for the first epitope, one binding site for the second epitope and one binding site for the third epitope (as shown in Fig. 5). The general structure of the polypeptide chains of representative five-strand Fc-domain-containing diabodies according to the invention is presented in table. 3.

-30042568

Table 3

Bispecific (2x2) ^2nd chain NH2-VLI-CL-COOH ^1st chain NH2-VH1-CH1-CH2-CH3-COOH ^3rd chain NH2-VHI-CHI-CH2-CH3-VL2-VH2-HPD-COOH ^5th chain NH2-VLI-CL-COOH ^4th chain NH2-VL2-VH2-HPD-COOH Bispecific (3x1) ^2nd chain NH2-VLI-CL-COOH ^1st chain NH2-VH1-CH1-CH2-CH3-COOH ^3rd chain W-VH1 -CHI -CH2-CH3 - VL 1 - VH2-HPD-COOH ^5th chain NH2-VLI-CL-COOH ^4th network NH2-VL2-VHI-HPD-COOH Trispecific (2x1x1) ^2nd chain NH2-VLI-CL-COOH ^1st chain NH ₂ -VH1-CH1-CH2-CH3-COOH ^3rd chain NH2-VH 1 -CH 1-CH2-CH3 - VL2-VH3 -HPD-COOH ^5th chain NH2-VLI-CL-COOH ^4th chain NH2-VL3 - VH2-HPD-COOH

HPD = Heterodimerization Promoting Domain.

In a specific embodiment, the antibodies of the invention are bispecific, tetravalent (i.e., have four epitope-binding sites) Fc-containing diabodies that consist of five complete polypeptide chains having two epitope-binding sites that are immunospecific for B7-H3 (which may be able to bind to the same B7-H3 epitope or different B7-H3 epitopes) and two second molecule-specific epitope-binding sites (which may be capable of binding to the same epitope of the second molecule or to different epitopes of the second molecule). In another embodiment, the bispecific, tetravalent Fc-containing diabodies of the invention comprise three B7-H3 immunospecific epitope-binding sites (which may be capable of binding to the same B7-H3 epitope or to two or three different B7-H3 epitopes) and one epitope-binding site specific to the second molecule. In another embodiment, the bispecific, tetravalent Fc-containing diabodies of the invention comprise one epitope-binding site immunospecific for B7-H3 and three epitope-binding sites specific for a second molecule (which may be capable of binding to the same epitope of the second molecule, or to two or three different epitopes). second molecule). As stated above, the VL and VH domains can be selected to allow trispecific binding. Accordingly, the invention also covers trispecific tetravalent Fc-containing diabodies. The trispecific tetravalent Fc-containing diabodies of the invention contain two epitope-binding sites immunospecific for B7-H3, one epitope-binding site immunospecific for a second molecule, and one epitope-binding site immunospecific for a third molecule. In some embodiments, the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T lymphocyte, a natural killer ( NK) or other mononuclear cell. In some embodiments, the second molecule is CD3 and the third molecule is CD8.

D. Trivalent binding molecules containing Fc domains.

Another embodiment of the present invention relates to trivalent Fc domain-containing binding molecules that are capable of simultaneously binding a first epitope, a second epitope, and a third epitope, wherein at least one of such epitopes is not identical to the other. Such trivalent binding molecules contain three epitope binding sites, two of which are diabody-type binding domains that provide site A binding and site B binding, and one of which is a Fab-type binding domain or scFv-type binding domain that provides a binding site. C (see, for example, FIGS. 6A-6F and PCT application nos: PCT/US15/33O81 and PCT/US15/33076). Thus, such trivalent binding molecules include 'VL1'/'VH1' domains which are capable of binding to the first epitope and 'VL2'/'VH2' domains which are capable of binding to the second epitope and 'VL3' and 'VH3' domains which are capable of bind to the “third” epitope of such a trivalent binding molecule. A “diabody-type binding domain” is a type of epitope-binding site present in a diabody, and in particular a DART® diabody, as described above. Each of the ''Fab-type binding domains'' and ''scFv-type binding domains'' are epitope-binding sites that are formed by the interaction of an immunoglobulin light chain VL domain and an immunoglobulin heavy chain complementary VH domain. Fab-type binding domains differ from diabody-type binding domains because the two polypeptide chains that form a Fab-type binding domain contain only one epitope-binding site, while the two polypeptide chains that form a diabody-type binding domain include at least two epitope binding sites. Similarly, scFv-type binding domains also differ from diabody-type binding domains in that they contain only one epitope-binding site. Thus, the binding houses used in this document

- 31 042568 ns Fab-type and scFv-type differ from the binding domains in the type of diabody.

Typically, the trivalent binding molecules of the present invention will contain four different polypeptide chains (see FIGS. 6A, 6B), however, the molecules may contain fewer or more polypeptide chains, for example, by fusing such polypeptide chains to each other (for example, through peptide bond) or by dividing such polypeptide chains to form additional polypeptide chains or by linking smaller or additional polypeptide chains through disulfide bonds. Fig. 6C-6F illustrate this aspect of the present invention, schematically depicting such molecules having three polypeptide chains. As shown in FIG. 6A-6F, the trivalent binding molecules of the present invention may have alternative orientations in which the diabody-type binding regions are N-terminal (FIGS. 6A, 6C and 6D) or C-terminal (FIGS. 6B, 6E and 6F) relative to the Fc domain. .

In some embodiments, the first polypeptide chain of such trivalent binding molecules of the present invention includes: (i) a domain containing VL1, (ii) a domain containing VH2, (iii) a domain that promotes heterodimerization, and (iv) a domain containing the sequence CH2-CH3 . Domains VL1 and VL2 are located at the N-terminus or C-terminus relative to the domain containing CH2-CH3, as shown in table. 3 (see also FIGS. 6A and 6B). The second polypeptide chain of such embodiments includes: (i) a VL2 containing domain, (ii) a VH1 containing domain, and (iii) a heterodimerization promoting domain. The third polypeptide chain of such embodiments contains: (i) a domain containing VH3, (ii) a domain containing CH1, and (iii) a domain containing a CH2-CH3 sequence. The third polypeptide chain can be an antibody heavy chain that includes VH3 and a heavy chain constant region, or a polypeptide that contains such domains. The fourth polypeptide of such embodiments contains: (i) a domain containing VL3, and (ii) a domain containing CL. The fourth polypeptide chains can be an antibody light chain that contains a VL3 complementary to the VH3 of the third polypeptide chain, or a polypeptide that contains such domains. The third or fourth polypeptide chains can be isolated from naturally occurring antibodies. Alternatively, they may be engineered recombinantly, synthetically, or in other ways.

The light chain variable domain of the first and second polypeptide chains are separated from the heavy chain variable domains of such polypeptide chains by an intermediate spacer peptide that is too short in length to allow their VL1/VH2 domains (or their VL2/VH1) to associate together to form an epitope binding site capable of binding to the first or second epitope. A preferred intermediate spacer peptide (linker 1) for this purpose has the sequence (SEQ ID NO:32): GGGSGGGG. Other domains of trivalent binding molecules can be separated by one or more intermediate spacer peptides (linkers), optionally containing a cysteine residue. In particular, as noted above, such linkers will typically be included between variable domains (i.e. VH or VL) and heterodimerization promoting peptide domains (e.g. E-helical or K-helical) and between heterodimerization promoting peptide domains, (for example, E-helical or K-helical) and CH2-CH3 domains. Exemplary linkers useful for making trivalent binding molecules are listed above and are also presented in PCT Application Nos: PCT/US15/33081 and PCT/US15/33076. Thus, the first and second polypeptide chains of such trivalent binding molecules are linked together to form a VL1/VH1 binding site capable of binding a first epitope as well as a VL2/VH2 binding site that is capable of binding a second epitope. The third and fourth polypeptide chains of these trivalent binding molecules bind together to form a VL3/VH3 binding site that is capable of binding to a third epitope.

As described above, trivalent binding molecules of the present invention may contain three polypeptides. Trivalent binding molecules containing three polypeptide chains can be obtained by linking the N-terminal domains of the fourth polypeptide to the VH3-containing domain of the third polypeptide (eg, using an intermediate spacer peptide (Linker 4)). Otherwise, the third polypeptide chain of the trivalent binding molecule of the invention, containing the following domains, uses: (i) a domain containing VL3, (ii) a domain containing VH3, and (iii) a domain containing a CH2-CH3 sequence in which VL3 and VH3 are separated from each other by an intermediate spacer peptide that is sufficiently long (at least 9 or more amino acid residues) that an epitope-binding site can be formed upon association of these domains. One preferred intermediate spacer peptide for this purpose has the sequence: GGGGGGGGGGGGGGS (SEQ ID NO:59).

It is understood that the VL1/VH1, VL2/VH2 and VL3/VH3 domains of such trivalent binding molecules may be different to allow for monospecific, bispecific or trispecific binding. In particular, the VL and VH domains can be chosen such that the trivalent binding molecule contains two binding sites for the first epitope and one binding site for the second epitope, or one binding site for the first epitope and two binding sites for the second epitope, or one binding site for the first epitope, one binding site for the second epitope, and one binding site for the third epitope.

- 32 042568

However, as indicated herein, these domains are preferably chosen to bind a B7-H3 epitope, a second molecule epitope, and a third molecule epitope. In some embodiments, the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T lymphocyte, natural killer (NK ) or other mononuclear cell. In some embodiments, the third molecule is CD8.

The general structure of the polypeptide chains of representative trivalent binding molecules of the invention is shown in FIG. 6A-6F and in table. 4.

Table 4

Four chains ^1st orientation ^2nd chain NH2-VL2-VH1-HPD-COOH ^1st chain W-VL 1 - VH2-HPD-CH2-CH3 -COOH ^3rd chain W-VH3 -CH 1-CH2-CH3 -COOH ^2nd chain _NH2 -VL3-CL-COOH Four chains 2nd orientation ^2nd chain _NH2 -VL2-VH1-HPD-COOH ^1st chain NH ₂ -CH2-CH3-VL1-VH2-HPD-COOH ^3rd chain NH ₂ -VH3-CH1-CH2-CH3-COOH ^2nd chain _NH2 -VL3-CL-COOH Three chains 1st orientation ^2nd chain _NH2 -VL2-VH1-HPD-COOH ^1st chain NH ₂ -VL1-VH2-HPD-CH2-CH3-COOH ^3rd chain NH ₂ , -VL3-VH3-nrn-CH2-CH3-COOH Three chains ^2nd orientation ^2nd chain _NH2 -VL2-VH1-HPD-COOH ^1st chain NH ₂ -CH2-CH3 - VL 1 - VH2-HPD-COOH ^3rd chain NH ₂ -VL3 - VH3 -HPD-CH2-CH3 -COOH

HPD = heterodimerization promoting domain.

One embodiment of the present invention relates to trivalent binding molecules that include two epitope binding sites for B7-H3 and one epitope binding site for the second molecule. The two epitope binding sites for B7-H3 may bind the same epitope or different epitopes. Another embodiment of the present invention relates to trivalent binding molecules that include one epitope binding site for B7-H3 and two epitope binding sites for the second molecule. The two epitope binding sites for the second molecule may bind the same epitope or different epitopes of the second molecule. Another embodiment of the present invention relates to trispecific trivalent binding molecules, which include one epitope-binding site for B7-H3, one epitope-binding site for the second molecule, and one epitope-binding site for the third molecule. In some embodiments, the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.) present on the surface of an effector cell such as a T lymphocyte, a natural killer cell (NK) or other mononuclear cell. In some embodiments, the second molecule is CD3 and the third molecule is CD8. As indicated above, such trivalent binding molecules may contain three, four, five or more polypeptide chains.

VII. Fc domain modification.

The Fc domain of the Fc domain-containing molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) of the present invention may be either a complete Fc domain (eg, a complete IgG Fc domain) or only a fragment of an Fc domain. Optionally, the Fc domain of the Fc domain-containing molecules of the present invention does not have a C-terminal lysine amino acid residue.

In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system results in a wide range of responses ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation and phagocytosis to immunomodulatory signals such as regulation of lymphocyte proliferation and antibody secretion. All of these interactions are initiated by binding of the Fc domain of antibodies or immune complexes to specialized cell receptors on hematopoietic cells. The diversity of cellular responses caused by antibodies and immune complexes is due to the structural heterogeneity of three Fc receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16). FcyRI (CD64), FcyRIIA (CD32A), and FcyRIII (CD16) are activating (i.e., boosting the immune system) receptors; FcyRIIB (CD32B) is an inhibitory (ie, weakening of the immune system) receptor. In addition, interaction with the neonatal Fc receptor (FcRn) mediates the recycling of IgG molecules from the endosome to the cell surface and release into the blood. Below is the amino acid sequence of a typical IgGl (SEQ ID NO:12), IgG2 (SEQ ID NO:13), IgG3 (SEQ ID NO:14) and IgG4 (SEQ ID NO:15) wild type.

Modification of the Fc domain can result in an altered phenotype, e.g., altered serum half-life, altered stability, altered cell susceptibility

- 33 042568 enzymes or altered effector function. Therefore, it may be desirable to modify, for example, an Fc domain-containing B7-H3 binding molecule with respect to effector function in order to increase the effectiveness of such a molecule in the treatment of cancer. The reduction or exclusion of effector function is desirable in some cases, for example, in the case of antibodies whose mechanism of action is associated with blocking or antagonism, but not with the destruction of cells bearing the target antigen. Increased effector function is generally desirable when directed to unwanted cells such as tumor and foreign cells where FcyRs are expressed at low levels, such as tumor-specific B cells with low levels of FcyRIIB (eg, non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma). Molecules of the invention having such assigned or altered effector functional activity are useful in the treatment and/or prevention of a disease, disorder or infection in which increased efficacy of effector function activity is desired.

Accordingly, in some embodiments, the Fc domain of the Fc domain-containing molecules of the present invention may be an engineered variant Fc domain. Although the Fc domain of the bispecific Fc domain-containing molecules of the present invention may have the ability to bind to one or more Fc receptors (e.g., FcyR (R)), it is more preferred if such a variant Fc domain has altered binding to FcyRIA (CD64) , FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a), or FcyRIIIB (CD16b) (relative to binding exhibited by the wild-type Fc domain), for example, will have increased binding to the activating receptor and/or will have significantly reduced binding or no will have the ability to bind to the inhibitory receptor(s). Thus, the Fc domain of the Fc domain-containing molecules of the present invention may include part or all of the CH2 domain and/or part or all of the CH3 domain of the complete Fc domain, or may include a CH2 variant and/or a CH3 variant (which may include, for example, one or more insertions and/or one or more deletions to the CH2 or CH3 domains of the entire Fc domain). Such Fc domains may contain fragments of a non-Fc polypeptide, or may contain portions of non-natural complete Fc domains, or may contain non-natural orientations of CH2 and/or CH3 domains (such as, for example, two CH2 domains or two CH3 or N-to C-terminus, a CH3 domain linked to a CH2 domain, etc.).

Fc domain modifications identified as altering effector function are known in the art, and include modifications that increase binding to activating receptors (e.g., FcyRIIA (CD16A) and decrease binding to inhibitory receptors (e.g., FcyRIIB (CD32B) (see, e.g., , Stavenhagen, JB et al.(2007) Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors, Cancer Res. 57(18):8882-8890) Table 5 lists exemplary single, double, triple, quadruple and quintuple substitutions (numbering and substitutions relative to the amino acid sequence of SEQ ID NO:12) of a typical modification that increases binding to activating receptors and/or decreases binding to inhibitory receptors.

Table 5

Variations in Preferred Fc Activating Domains.

Variations in one site F243L R292G D270E R292P Y300L P396L Variations in the two sites F243L and R292P F243L and Y300L F243L and P396L R292P and Y300L D270EhP396L R292P and V305I P396L and Q419H P247LHN421K R292P and P396L Y300L and P396L R255LhP396L R292P and P305I K392T and P396L Variations in three sites F243L, P247LHN421K P247L, D270EhN421K F243L, R292P and Y300L R255L, D270EhP396L F243L, R292P and V305I D270E, G316DhR416G F243L, R292P and P396L D270E, K392TiR396E F243L, Y300L and P396L D270E, P396L and Q419H V284M, R292L and K370N R292P, Y300LhP396L Variations at four sites L234F, F243L, R292P and Y300L F243L, P247L, D270E hN421K L234F, F243L, R292P and Y300L F243L, R255L, D270E and P396L L235I, F243L, R292P and Y300L F243L, D270E, G316D and R416G L235Q, F243L, R292P and Y300L F243L, D270E, K392T and P396L P247L, D270E, Y300L HN421K F243L, R292P, Y300L and P396L R255L, D270E, R292G and P396L F243L, R292P, V305I and P396L R255L, D270E, Y300L and P396L F243L, D270E, P396L and Q419H D270E, G316D, P396L and R416G Variations in five sites L235V, F243L, R292P, Y300L and P396L F243L, R292P, V305I, Y300L and P396L L235P, F243L, R292P, Y300L and P396L

Exemplary variants of human IgG1 Fc domains with reduced CD32B binding and/or increased CD16A binding contain F243L, R292P, Y300L, V305I, or P296L substitutions. These amino acid substitutions may be present in the Fc domain of human IgG1 in any combination. In one embodiment, the variant Fc domain from human IgG1 contains F243L, R292P and Y300L substitutions. In a different

- 34 042568 embodiment, the variant Fc domain from human IgG1 contains substitutions F243L, R292P, Y300L, V305I and

P296L.

In some embodiments, it is preferred that the Fc domains of the B7-H3 binding molecules of the present invention show reduced (or virtually no) binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a)) or FcyRIIIB ( CD16b) (relative to binding exhibited by wild-type IgG1 Fc domain (SEQ ID NO:12). In a specific embodiment, the B7-H3 binding molecules of the present invention comprise an IgG Fc domain that exhibits reduced ADCC effector function. In a preferred embodiment, CH2 domains -CH3 of such B7-H3 binding molecules include any 1, 2, 3 or 4 substitutions: L234A, L235A, D265A, N297Q and N297G In another embodiment, the CH2-CI3 domains include substitution N297Q, substitution N297G, substitutions L234A and L235A or substitution D265A, since these mutations abolish binding to FcR Otherwise, the CH2-CI3 domain of the native Fc domain is used, which inherently exhibits reduced (or virtually no) binding to FcRIIIA (C D16a) and/or reduced effector function (relative to binding and effector function exhibited by the Fc domain of wild-type IgG1 (SEQ ID NO:12)). In a specific embodiment, the B7-H3 binding molecules of the present invention comprise an IgG2 Fc domain (SEQ ID NO:13) or an IgG4 Fc domain (SEQ ID NO:4). When an Fc domain from IgG4 is used, the present invention also encompasses the introduction of a stabilizing mutation, such as the S228P substitution in the hinge domain described above (see, for example, SEQ ID NO:11). Since the N297G, N297Q, L234A, L235A, and D265A substitutions abolish effector function, in cases where effector function is desired, these substitutions will preferably not be used.

A preferred IgG1 sequence for the CH2 and CH3 domains of the Fc domain containing molecules of the present invention having reduced or no effector function would include substitutions L234A/L235A (SEQ ID NO:60) apeaaggpsv flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd

where X is lysine (K) or absent.

The serum half-life of proteins containing Fc domains can be increased by increasing the binding affinity of the Fc domain to FcRn. Used in this document, the term half-life means the pharmacokinetic property of the molecule, which is a measure of the average time of survival of molecules after their introduction. The half-life can be expressed as the time required to eliminate 50% of a known amount of a molecule from the body of an object (for example, a human or other mammal) or, for example, from a certain department of it, measured in serum, i.e. half-life in the bloodstream or in other tissues. In general, an increase in the half-life results in an increase in the mean retention time (MRT) in the circulation for the administered molecule.

In some embodiments, the B7-H3 binding molecules of the present invention comprise a variant Fc domain, wherein said variant Fc domain comprises at least one amino acid modification relative to the wild-type Fc domain such that said molecule has an increased half-life (relative to a molecule containing the Fc domain wild type). In some embodiments, the B7-H3 binding molecules of the present invention comprise a variant Fc domain from IgG, wherein said Fc domain includes an amino acid substitution to increase half-life at one or more positions selected from the group consisting of 238, 250, 252, 254 , 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 , 428, 433, 434, 435, and 436. Numerous mutations capable of increasing the half-life of an Fc domain-containing molecule are known in the art and include, for example, M252Y, S254T, T256E, and combinations thereof. For example, see the mutations described in US Pat. US Nos. 6277375, 7083784; 7217797, 8088376; publ. USA No. 2002/0147311; 2007/0148164 and publ. PCT No. WO 98/23289; W02009/058492; and WO 2010/033279, which are incorporated herein by reference in their entirety. Extended half-life B7H3 binding molecules also include those that have variant Fc domains that include substitutions at two or more Fc domain residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 428, 433, 434, 435 and 436. In particular, two or more substitutions selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K and Y436I.

In a specific embodiment, the B7-H3 binding molecule of the present invention has a variant IgG Fc domain comprising the substitutions:

(A) M252Y, S254T and T256E;

(B) M252Y and S254T;

(C) M252Y and T256E;

(D) T250Q and M428L;

- 35 042568 (E) T307Q and N434A;

(F) A378V and N434A;

(G) N434A and Y436I;

(H) V308P and N34A or (i) K288D and H435K.

In a preferred embodiment, the B7-H3 binding molecule of the present invention has a variant IgG Fc domain comprising any 1, 2 or 3 substitutions: M252Y, S254T and T256E. The invention further encompasses B7-H3 binding molecules having variant Fc domains, including:

(A) one or more mutations that alter effector function and/or FcyR; and (B) one or more mutations that prolong the serum half-life.

For certain antibodies, diabodies, and trivalent binding molecules whose Fc domain-containing first and third polypeptide chains are not identical, it is desirable to reduce or prevent homodimerization between the CH2-CH3 domains of the first two polypeptide chains or between the CH2-CH3 domains of the two third polypeptide chains. The CH2 and/or CH3 domains of such polypeptide chains need not be identical in sequence and are advantageously modified to facilitate complex formation between the two polypeptide chains. For example, an amino acid substitution (preferably a substitution with an amino acid containing a bulky side group that forms a ledge, such as tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference prevents interaction with a similarly mutated domain and obliges the mutated domain to pair with the domain in in which a complementary or host mutation has been engineered, i.e. hole (for example, replacement with glycine). Such sets of mutations can be engineered in any pair of polypeptides containing CH2-CH3 domains that form an Fc domain that promotes heterodimerization. Protein engineering techniques that promote heterodimerization rather than homodimerization are well known in the art, particularly in relation to engineering of immunoglobulin-like molecules, and are covered by this document (see, for example, Ridgway et al. (1996) 'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, Protein Engr. 9:617-621, Atwell et al.(1997) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. Mol. Biol.270:26 -35 and Xie et al (2005) A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, J Immunol Methods 296:95-101, each of which is incorporated herein by reference in its entirety).

The preferred overhang is created by changing the Fc domain of IgG to contain the T366W modification. The preferred recess is created by modifying the Fc domain of IgG to contain T366S, L368A and Y407V modifications. In order to facilitate purification of the homodimer of the third recess bearing polypeptide chain from the final bispecific Fc domain containing heterodimeric molecule, the protein A binding site in the recess bearing CH2 and CH3 domains of the third polypeptide strand is preferably mutated by an amino acid substitution at position 435 (H435R). Thus, a third polypeptide chain homodimer bearing a recess will not bind to protein A, while a bispecific heterodimer will retain its ability to bind protein A via the protein A binding site in the first polypeptide chain. In an alternative embodiment, the third recessed polypeptide chain may include amino acid substitutions at positions 434 and 435 (N434A/N435K).

The preferred IgG amino acid sequence for the CH2 and CH3 domains of the first polypeptide chain of the Fc-domain-containing molecule of the present invention will have an overhang sequence (SEQ ID NO:61)

APEAAGGPSV flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd

where X is lysine (K) or absent.

The preferred IgG amino acid sequence for the CH2 and CH3 domains of the second polypeptide chain of an Fc domain-containing molecule of the present invention having two polypeptide chains (or the third polypeptide chain of an Fc domain-containing molecule having three, four, or five polypeptide chains) will have a carrier deepening sequence (SEQ ID NO:62) APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

where X is lysine (K) or absent.

- 36 042568

Of note, the CH2-CH3 domains of SEQ ID NO:61 and SEQ ID NO:62 include a substitution at position 234 to alanine and 235 to alanine and thus form an Fc domain showing reduced (or virtually no) binding to FcyRIA. (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the binding shown by the wild-type Fc domain (SEQ ID NO:12). The invention also covers those CH2-CH3 domains that include wild-type alanine residues, alternative and/or additional substitutions that modify the effector function and/or Fc domain FyR binding activity.The invention also encompasses those CH2-CH3 domains that further include one or more amino acid substitutions that increase half-life. in particular, the invention encompasses such bearing recesses and such bearing ledge CH2-CH3 domains, which further include M252Y/S254T/T256E.

Preferably, the first polypeptide chain will have a CH2-CH3 overhang sequence, such as the sequence of SEQ ID NO:61. However, as will be recognized, a CH2-CH3 recess-bearing domain (e.g., SEQ ID NO:62) may be used in the first polypeptide chain, in which case a CH2-CH3 overhang domain (e.g., SEQ ID NO:61) will be used. in the second polypeptide chain of an Fc domain containing molecule of the present invention having two polypeptide chains (or in the third polypeptide chain of an Fc domain containing molecule having three, four or five polypeptide chains).

In other embodiments, the invention encompasses B7-H3 binding molecules containing CH2 and/or CH3 domains that have been designed to promote heterodimerization rather than homodimerization using mutations known in the art, such as those disclosed in pub. PCT No. WO 2007/110205; WO 2011/143545; W02012/058768; WO 2013/06867, all of which are incorporated herein by reference in their entirety.

VIII. Ability to bind to effector cells.

According to this document, B7-H3 binding molecules of the invention, including B7-H3-ADC molecules, can be designed to contain a combination of epitope binding sites that recognize multiple antigens unique to the target cell or tissue type. In particular, the present invention relates to multispecific B7-H3 binding molecules that are capable of binding to a B7-H3 epitope and an epitope of a molecule present on the surface of an effector cell such as a T-lymphocyte, natural killer (NK) or other mononuclear cell. For example, B7-H3 binding molecules of the present invention may be a construct containing an epitope binding site that immunospecifically binds CD2, CD3, CD8, CD16, T cell receptor (TCR), or NKG2D. The invention also relates to trispecific B7-H3 binding molecules that are capable of binding to a CD3 epitope and a CD8 epitope (see, for example, PCT Publication No. WO 2015/026894).

A. CD2 binding abilities.

In one embodiment, the bispecific, trispecific, or multispecific B7-H3 binding molecules of the invention are capable of binding to a B7-H3 epitope and a CD2 epitope. CD2 is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 enhances NK cell cytotoxicity, possibly as a promoter of NK cell nanotube formation (Mace, E.M. et al. (2014) Cell Biological Steps and Checkpoints in Accessing NK Cell Cytotoxicity, Immunol. Cell. Biol. 92(3):245 -255 Comerci, C. J. et al (2012) CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation, PLoS One 7(10):e47664:1-12). Molecules that specifically bind CD2 include the anti-CD2 antibody Lo-CD2a.

Amino acid sequence of the VH domain from Lo-CD2a (ATCC Accession No: 11423); SEQ ID NO:63) is shown below (CDRH residues are shown underlined)

EVQLQQSGPE LQRPGASVKL SCKASGYIFT EYYMYWVKQR PKQGLELVGR

IDPEDGSIDY VEKFKKKATL TADTSSNTAY MQLSSLTSED TATYFCARGK FNYRFAYWGO GTLVTVSS

Shown below is the amino acid sequence of the VL domain of Lo-CD2a (ATCC Accession No. 11423, SEQ ID NO:64) (CDR _L residues are shown underlined)

DVVLTQTPPT LLATIGQSVS ISCRSSQSLL HSSGNTYLNW LLQRTGQSPQ

PLIYLVSKLE SGVPNRFSGS GSGTDFTLKI SGVEAEDLGV YYCMQFTHYP YTFGAGTKLE LK

B. CD3 binding abilities.

In one embodiment, the bispecific, trispecific, or multispecific B7-H3 binding molecules of the invention are capable of binding to a B7-H3 epitope and a CD3 epitope. CD3 is a T-cell co-receptor consisting of four distinct chains (Wucherpfennig, K.W. et al. (2010) Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling, Cold Spring Harb Perspect Biol 2(4): a005140, pp. 1-14). In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3e chains. These chains bind to a molecule known as the T cell receptor (TCR) in order to generate an activation signal in T lymphocytes. IN

- 37 042568 lack of CD3 TCRs do not assemble properly and degrade (Thomas, S. et al. (2010) Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer, Immunology 129(2): 170-177). CD3 is found bound to the membranes of all mature T cells and is virtually nonexistent on other cell types (see, Janeway, C.A. et al. (2005) In: Immunobiology: The Immune System In Health And Disease, 6th ed. Garland Science Publishing, NY, pp. 214-216 Sun, Z. J. et al (2001) Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3s:y Heterodimer, Cell 105(7) :913-923 Kuhns, M.S. et al (2006) Deconstructing The Form And Function Of The TCR/CD3 Complex, Immunity 2006 Feb;24(2):133-139). Molecules that specifically bind CD3 include the anti-CD3 antibodies CD3 mAb-1 and OKT3. Ahtu-CD3 CD3 mAb-1 is capable of binding to non-human primate protein (eg cynomolgus monkey).

The amino acid sequence of the VH domain from CD3 mAb-1 VH (1) (SEQ ID NO:65) is shown below (CDRH residues are shown underlined)

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR

IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR

HGNFGNSYVS WFAYWGOGTL VTVSS

The amino acid sequence of the alternative VH domain from CD3 mAb-1 VH(2) (SEQ ID NO:66) is shown below (CDRH residues are shown with single underline; differences relative to the VH domain from CD3 MAB-1 VH(1) (SEQ ID NO:65) shown with double underlining).

EVQLVESGGG LVQPGGSLRL SCAASGFTFV TYAMNWVRQA PGKGLEWVGR

IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR

HGNFGNSYVS WFAYWGOGTL VTVSS

Shown below is the amino acid sequence of the VL domain from CD3 mAb-1 (SEQ ID NO:67) (CDRL residues are underlined)

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVOO KPGQAPRGLI

GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF

GGGTKLTVLG

The VH region of CD3 mAb-1 VH(1) (SEQ ID NO:65) can be used with the VL domain of CD3 mAb1 (SEQ ID NO:67) to form a functional CD3 binding molecule according to the present invention. Similarly, the VH domain of CD3 mAb-1 VH(2) (SEQ ID NO:66) can be used with the VL domain of CD3 mAb-1 (SEQ ID NO:67) to form a functional CD3 binding molecule according to the present invention.

As discussed below, to better illustrate the present invention, bispecific B7-H3 x CD3 binding molecules have been prepared. Some B7-H3 x CD3 constructs used the CD3 mAb-1 variant. The CD3 mAb-1 variant (D65G) contains a VL domain from CD3 mAb-1 (SEQ ID NO:67) and a VH domain from CD3 mAb-1 having a D65G substitution (Kabat position 65 corresponding to residue 68 of SEQ ID NO:65 ). Shown below is the amino acid sequence of the VH domain of CD3 mAb-1 (D65G) (SEQ ID NO:68) (CDRH residues shown, substituted position (D65G) shown under double underline) EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA

PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT

EDTAVYYCVR HGNFGNSYVS WFAYWGOGTL VTVSS

Otherwise, an affinity variant of the CD3 mAb-1 may be included.

Variants include a low affinity variant designated CD3 mAb-1 Low and a faster dissociation rate variant designated CD3 mAb-1 Fast. The VL domain of CD3 mAb-1 (SEQ ID NO:67) is shared between CD3 mAb-1 Low and CD3 mAb1 Fast and is shown above. Below are the amino acid sequences of the VH domains of each of CD3 mAb-1 Low and CD3 mAb-1 Fast.

Shown below is the amino acid sequence of the VH domain of the anti-human CD3 mAb-1 Low (SEQ ID NO:69) (CDRH residues are shown underlined, differences relative to the VH domain of CD3 mAb1 VH(1) (SEQ ID NO:65) are shown under double underline)

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA

PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT

EDTAVYYCVR HGNFGNS YVT WFAYWGOGTL VTVSS

Shown below is the amino acid sequence of the VH domain chain from human anti-CD3 mAb-1 Fast (SEQ ID NO:70) (CDR _H residues are shown underlined, differences relative to the VH domain of CD3 mAb-1 VH (1) (SEQ ID NO:65) are shown double underline)

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA

PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT

EDTAVYYCVR HXNFGNSYVT WFAYWGOGTL VTVSS

Another anti-CD3 antibody that can be used is MuromonabCD3 OKT3 antibody (Xu et al. (2000) In Vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol. 200:16-26); Norman, D.J. (1995) Mechanisms Of Action And Overview Of OKT3, Ther. drug monitor. 17(6):615-620; Canafax, D.M. et al. (1987) Monoclonal Antilymphocyte Antibody

- 38 042568 (OKT3) Treatment Of Acute Renal Allograft Rejection Pharmacotherapy 7(4): 121-124; Swinnen, L.J. et al.

(1993) OKT3 Monoclonal Antibodies Induce Interleukin-6 And Interleukin-10: A Possible Cause Of Lymphoproliferative Disorders Associated With Transplantation, Curr. Opin. Nephrol. hypertens. 2(4):670-678).

The amino acid sequence from the VH domain of OKT3 (SEQ ID NO:71) is shown below (CDR _H residues are shown underlined)

QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR

PGOGLEWIGY INPSRGYTNY NQKFKDKATL TTDKSSSTAY MQLSSLTSED

SAVYYCARYY DDHYCLDYWG QGTTLTVSS

Shown below is the amino acid sequence of the VL domain of OKT3 (SEQ ID NO:72) (CDR _l residues are shown underlined)

QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT

SKLASGVPAH FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINR

Additional anti-CD3 antibodies that may be used include, but are not limited to, those described in PCT Publication Nos. WO 2008/119566 and WO 2005/118635.

C. CD8 binding abilities.

In one embodiment, the bispecific, trispecific, or multispecific B7-H3 binding molecules of the invention are capable of binding to a B7-H3 epitope and a CD8 epitope. CD8 is a T cell co-receptor consisting of two distinct chains (Leahy, D.J., (1995) A Structural View of CD4 and CD8, FASEB J., 9:17-25) that is expressed on cytotoxic T cells. Activation of CD8+ T cells has been found to be mediated by co-stimulatory interactions between the antigen:major histocompatibility complex I class (MHC I) molecular complex, which is located on the surface of the target cell, and the complex of CD8 and T-cell receptor, which is located on the surface of CD8+ T- cells (Gao, G., and Jakobsen, B., (2000). Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-Cell Receptor. Immunol Today 21: 630-636). Unlike MHC II molecules, which are expressed only by certain cells of the immune system, MHC I molecules are expressed very widely. Thus, cytotoxic T cells are able to bind to a wide range of cell types. Activated cytotoxic T cells mediate cell death by releasing cytotoxins, perforin, granzymes, and granulisin. Antibodies that specifically bind CD8 include anti-CD8 antibodies OKT8 and TRX2.

Shown below is the amino acid sequence of the VH domain of OKT8 (SEQ ID NO:73) (CDR _H residues are shown underlined)

QVQLLESGPE LLKPGASVKM SCKASGYTFT DYNMHWVKQS HGKSLEWIGY

IYPYTGGTGY NQKFKNKATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS

Shown below is the amino acid sequence of the VL domain of OKT8 (SEQ ID NO:74) (CDR _l residues are shown underlined)

DIVMTQSPAS LAVSLGQRAT ISCRASESVD SYDNSLMHWY QQKPGQPPKV

LIYLASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPY TFGGGTKLEIKR

Shown below is the amino acid sequence of the TRX2 VH domain (SEQ ID NO:75) (CDRH residues are shown underlined)

QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL

IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPH YDGYYHFFDS WGQGTLVTVS S

The amino acid sequence of the TRX2 VL domain (SEQ ID NO:76) is shown below (CDR _l residues are shown underlined)

DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN

TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGOG TKVEIK

D. CD16 binding abilities.

In one embodiment, the multispecific B7-H3 binding molecules of the invention are capable of binding to a B7-H3 epitope and a CD16 epitope. CD16 is the FcyRIIIA receptor. CD16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages that bind aggregated but not monomeric human IgG (Peltz, G.A. et al. (1989) Human Fc Gamma RIII: Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG, Proc. Natl. Acad. Sci. (U.S.A.) 86(3): 1013-1017 Bachanova, V. et al. (2014) NK Cells In Therapy Of Cancer, Crit. Rev. Oncog 19 (1-2): 133-141; Miller, J. S. (2013) Therapeutic Applications: Natural Killer Cells In The Clinic, Hematology Am. Soc. Hematol. Educ. Program 2013: 247-253; Youinou, P. et al.(2002) Pathogenic Effects Of Anti-Fc Gamma Receptor IIIB (CD16) On Polymorphonuclear Neutrophils In Non-OrganSpecific Autoimmune Diseases, Autoimmun Rev. 1(1-2): 13-19;Peipp, M. et al.(2002 ) Bispecific Antibodies Targeting Cancer Cells, Biochem Soc Trans 30(4):507-511). Molecules that specifically bind CD16 include anti-CD16 antibodies 3G8 and A9. Humanized A9 antibodies are described in publ. PCT WO 03/101485.

- 39 042568

Shown below is the amino acid sequence of the VH domain of 3G8 (SEQ ID NO:77) (residues

CDR _H are shown underlined)

QVTLKESGPG ILQPSQTLSL TCSFSGFSLR TSGMGVGWIR QPSGKGLEWL

AHIWWDDDDKR YNPALKSRLT ISKDTSSNQV FLKIASVDTA DTATYYCAQI NPAWFAYWGQ GTLVTVSA

Shown below is the amino acid sequence of the VL domain from 3G8 (SEQ ID NO:78) (CDRL residues are shown underlined)

DTVLTQSPAS LAVSLGQRAT ISCKASQSVD FDGDSFMNWY QQKPGQPPKL

LIYTTSNLES GIPARFSASG SGTDFTLNIH PVEEEDTATY YCQQSNEDPY TFGGGTKLEI K

Shown below is the amino acid sequence of the VH domain of A9 (SEQ ID NO:79) (CDRH residues are shown underlined)

QVQLQQSGAE LVRPGTSVKI SCKASGYTFT NYWLGWVKQR PGHGLEWIGD

IYPGGGYTNY NEKFKGKATV TADTSSRTAY VQVRSLTSED SAVYFCARSA SWYFDVWGAR TTVTVSS

Shown below is the amino acid sequence of the VL domain of A9 (SEQ ID NO:80) (CDRL residues are shown underlined)

DIQAVVTQES ALTTSPGETV TLTCRSNTGT VTTSNYANWV QEKPDHLFTG

LIGHTNNRAP GVPARFSGSL IGDKAALTIT GAQTEDEAIY FCALWYNNHW VFGGGTKLTV L

Additional anti-CD19 antibodies that may be used include, without limitation, those described in PCT publications WO 03/101485; and WO 2006/125668.

E. TCR binding abilities.

In one embodiment, the bispecific, trispecific, or multispecific B7-H3 binding molecules of the invention are capable of binding to a B7-H3 epitope and a T cell receptor (TCR) epitope. The T cell receptor is initially expressed by CD4+ or CD8+ T cells and allows such cells to recognize antigenic peptides that are bound and presented by class I or class II MHC proteins of antigen presenting cells. Recognition of the rMHC complex (peptide-MHC) by the TCR initiates the propagation of a cellular immune response, resulting in cytokine production and lysis of the antigen-presenting cell (see, e.g., Armstrong, KM et al. (2008) Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide-MHC Complexes, Biochem J. 415(Pt 2): 183-196 Willemsen, R. (2008) Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy, Cytometry A. 73(11): 1093-1099; Beier, K. C. et al. (2007) Master Switches Of T-Cell Activation And Differentiation, Eur. Respir. J. 29:804-812; Mallone, R. et al. (2005) Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes, Am. J. Ther. 12(6):534-550). CD3 is a receptor that binds to the TCR (Thomas, S. et al. (2010) Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer, Immunology 129(2): 170-177; Guy, C.S. et al. (2009 ) Organization Of Proximal Signal Initiation At The TCR:CD3 Complex, Immunol. Rev. 232(1):7-21 St. Clair, E.W. (Epub 2009 Oct 12) Novel Targeted Therapies For Autoimmunity, Curr. Opin. Immunol. 21 (6):648-657 Baeuerle, P.A. et al (Epub 2009 Jun 9) Bispecific T-Cell Engaging Antibodies For Cancer Therapy, Cancer Res. 69(12):4941-4944 Smith-Garvin, J.E. et al. (2009) T Cell Activation, Annu Rev Immunol 27:591-619 Renders, L et al (2003) Engineered CD3 Antibodies For Immunosuppression, Clin Exp Immunol 133(3):307-309) .

Molecules that specifically bind to the T cell receptor include the anti-TCR antibody BMA 031 (EP 0403156, Kurrle, R. et al. (1989) BMA 031 - A TCR-Specific Monoclonal Antibody For Clinical Application, Transplant Proc. 21(1 Pt 1): 1017-1019; Nashan, B. et al. (1987) Fine Specificity Of A Panel Of Antibodies Against The TCR/CD3 Complex, Transplant Proc. 19(5):4270-4272; Shearman, C. W. et al. (1991) Construction, Expression, And Biologic Activity Of Murine/Human Chimeric Antibodies With Specificity For The Human α/β T Cell, J. Immunol.146(3):928-935; Shearman, C.W. et al.(1991) Construction , Expression And Characterization of Humanized Antibodies Directed Against The Human α/β T Cell Receptor, J. Immunol. 147(12):4366-4373).

Shown below is the amino acid sequence of the VH domain from BMA 031 (SEQ ID NO:81) (CDRH residues are shown underlined)

QVQLVQSGAE VKKPGASVKV SCKASGYKFT SYVMHWVRQA PGQGLEWIGY

INPYNDVTKY NEKFKGRVTI TADKSTSTAY LQMNSLRSED TAVHYCARGS YYDYDGFVYW GQGTLVTVSS

Shown below is the amino acid sequence of the VL domain from BMA 031 (SEQ ID NO:82) (CDRL residues are shown underlined)

EIVLTQSPAT LSLSPGERAT LSCSATSSVS YMHWYQQKPG KAPKRWIYDT

SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SSNPLTFGQG TKLEIK

F. NKG2D binding capabilities.

In one embodiment, the multispecific B7-H3 binding molecules of the invention are capable of binding to a B7-H3 epitope and an epitope of the NKG2D receptor. The NKG2D receptor is expressed on all human (and other mammalian) natural killer cells (Bauer, S. et al.

- 40 042568 (1999) Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA, Science 285(5428):727-729; Jamieson, A. M. et al. (2002) The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing, Immunity 17(1): 19-29) as well as on all CD8 ⁺ T cells (Groh, V. et al. (2001) Costimulation Of CD8 "e T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells, Nat. Immunol. 2(3):255-260; Jamieson, AM et al. (2002) The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing, Immunity 17(1): 19-29). Such binding ligands, and especially those not expressed on normal cells, include the histocompatibility molecule 60 (H60), the early gene product of inducible retinoic acid-1 (RAE-1), and the murine UL16-binding protein-like transcript 1 (MULT1) ( Raulet DH (2003) Roles Of The NKG2D Immunoreceptor And Its Ligands, Nature Rev. Immunol.3:781-790 Coudert, JD et al.(2005) Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells Blood 106:17111717). Molecules that specifically bind to the NKG2D receptor include anti-NKG2D antibodies KYK-1.0 and KYK-2.0 (Kwong, KY et al. (2008) Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity, J. Mol Biol 384:1143-1156; and PCT/US09/54911).

Shown below is the amino acid sequence of the VH domain of KYK-1.0 (SEQ ID NO:83) (CDR _H residues are shown underlined)

EVQLVESGGG VVQPGGSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAF

IRYDGSNKYY ADSVKGRFTI SRDNSKNTKY LQMNSLRAED TAVYYCAKDR FGYYLDYWGQ GTLVTVSS

Shown below is the amino acid sequence of the VL domain of KYK-1.0 (SEQ ID NO:84) (CDRL residues are underlined)

QPVLTQPSSV SVAPGETARI PCGGDDIETK SVHWYQQKPG QAPVLVIYDD

DDRPSGIPER FFGSNSGNTA TLSISRVEAG DEADYYCQVW DDNNDEWVFG GGTQLTVL

Shown below is the amino acid sequence of the VH domain of KYK-2.0 (SEQ ID NO:85) (CDRH residues are underlined)

QVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAF

IRYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDR GLGDGTYFDY WGQGTTVTVS S

Shown below is the amino acid sequence of the VL domain of KYK-2.0 (SEQ ID NO:86) (CDRL residues are shown underlined)

QSALTQPASV SGSPGQSITI SCSGSSSNIG NNAVNWYQQL PGKAPKLLIY YDDLLPSGVS DRFSGSKSGT SAFLAISGLQ SEDEADYYCA AWDDSLNGPV

FGGGTKLTVL

IX. Multispecific B7-H3 binding molecules A.

B7-H3 x CD3 Bispecific double-stranded diabodies.

The VL and VH regions of the above described B7-H3 binding molecules can be used to construct bispecific B7-H3 x CD3 diabodies consisting of two covalently linked polypeptide chains. To illustrate this aspect of the present invention, the VL and VH domains of the above-described anti-B7-H3 antibody mAb-D were used to construct bispecific B7-H3 x CD3 diabodies consisting of two covalently linked polypeptide chains and containing the murine or humanized VL and VH domains discussed above. from mAb-D. The general structure and amino acid sequences of these B7-H3 x CD3 diabodies are shown below.

The first polypeptide chain of one exemplary bispecific double-chain diabody B7-H3 x CD3 contains in the direction from the N-terminus to the C-terminus: N-terminus; VL domain from an anti-B7-H3 antibody (e.g., mAb-D VL (SEQ ID NO:22) or hmAb-D VL (SEQ ID NO:30); intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO:32 )); VH domain from an anti-CD3 antibody (e.g., CD3 mAb 1 (D65G) (SEQ ID NO:68)); cysteine-containing intermediate spacer peptide (linker 2: GGCGGG (SEQ ID NO:33)); heterodimerization (eg E-helix) (EVAALEKEVAALEK-EVAALEK-EVAALEK (SEQ ID NO:45)); and the C-terminus.

The second polypeptide chain of such an exemplary B7-H3 x CD3 bispecific double-stranded diabody contains, in N-terminal to C-terminal direction: N-terminus; the VL domain of the corresponding anti-CD3 antibody (e.g., the VL domain which, together with the VH domain of the first polypeptide chain, forms the CD3 binding site, e.g., the VL domain of CD3-mAb-1 (SEQ ID NO:67), intermediate spacer peptide (linker : GGGSGGGG (SEQ ID NO:32)), a VH domain corresponding to an anti-B7-H3 antibody (e.g., a VH domain which, in combination with the VL domain of the first chain polypeptide, forms a B7-H3 binding site, e.g., VH from mAb-D ( SEQ ID NO:26) or VH from hmAb-D (SEQ ID NO:31); cysteine-containing intermediate spacer peptide (linker 2: GGCGGG (SEQ ID NO:33)), heterodimerization promoting domain (e.g., K-helix) ( KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:46)) and C-terminus.

According to this document, alternative linkers and/or alternative domains that promote heterodimerization can be used to obtain such diabodies. For example,

- 41 042568 the first polypeptide chain of an alternative exemplary bispecific double-chain diabody B7-H3 x CD3 may contain in the direction from the N-terminus to the C-terminus: N-terminus; a VL domain from an anti-B7-H3 antibody; intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO:32)); a VH domain from an anti-CD3 antibody or corresponding anti-CD3 antibody; intermediate spacer peptide (linker 2: ASTKG (SEQ ID NO:37)); a cysteine-containing domain that promotes heterodimerization (eg, K-helix) (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:46)); and C-terminus. The second polypeptide chain of such an alternative exemplary diabody may contain in the direction from the N-terminus to the C-terminus: N-terminus; the VL domain of the corresponding anti-CD3 antibody; intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO:32)); the VH domain of the corresponding anti-B7-H3 antibody (eg, VH from mAb-D (SEQ ID NO:26) or hVH from mAb-D (SEQ ID NO:31)); intermediate spacer peptide (linker 2: ASTKG (SEQ ID NO:37)); a cysteine-containing domain that promotes heterodimerization (eg, E-helix) (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO:47)); and C-terminus.

1. DART-D1.

A representative BIS-H3 x CD3 bispecific double-stranded diabody was constructed containing the VH and VL domains from hmAb-C (DART-Dr').

Shown below is the amino acid sequence of the first DART-D1 polypeptide chain (SEQ ID NO:87) (VL domain sequence from hmAb-C (SEQ ID NO:20) is underlined)

DIQMTQSPSS LSASVGDRVT ITCRASESIY SYLAWYQQKP GKAPKLLVYN

TKTLPEGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH HYGTPPWTFG

QGTRLEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSTYAM

NWVRQAPGKG LEWVGRIRSK YNNYATYYAD SVKGRFTISR DDSKNSLYLQ

MNSLKTEDTA VYYCVRHGNF GNSYVSWFAY WGQGTLVTVS SGGCGGGEVA

ALEKEVAALE KEVAALEKEV AALEK

The amino acid sequence of the second DART-D1 polypeptide chain (SEQ ID NO:88) is shown below (VH domain sequence from hmAb-C (SEQ ID NO:21) is underlined)

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLVESGGGLV KPGGSLRLSC AASGFTFSSY GMSWVRQAPG KGLEWVATIN SGGSNTYYPD SLKGRFTISR DNAKNSLYLQ MNSLRAEDTA VYYCARHDGG AMDYWGQGTT VTVSSGGCGG GKVAALKEKV AALKEKVAAL KEKVAALKE

2. DART-D2.

A representative BIS-H3 x CD3 bispecific double chain diabody was constructed containing the VH and VL domains from hmAb-D (DART-D2).

Shown below is the amino acid sequence of the first DART-D2 polypeptide chain (SEQ ID NO:89) (VL domain sequence from hmAb-D (SEQ ID NO:30) is underlined)

DIQMTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS

ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFAEYFCQQ YNNYPFTFGQ

GTKLEIKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN

WVRQAPGKGL EWVGRIRSKY NNYATYYADS VKGRFTISRD DSKNSLYLQM

NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS ASTKGEVAAC

EKEVAALEKE VAALEKEVAA LEK

The amino acid sequence of the second DART-D2 polypeptide chain (SEQ ID NO:90) is shown below (VH domain sequence from hmAb-D (SEQ ID NO:31) is underlined)

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF

GGGTKLTVLG GGGSGGGGEV QLVESGGGLV QPGGSLRLSC AASGFTFSSF

GMHWVRQAPG KGLEWVAYIS SGSGTIYYAD TVKGRFTISR DNAKNSLYLQ

MNSLRAEDTA VYYCARHGYR YEGFDYWGQG TTVTVSSAST KGKVAACKEK

V A ALKEK V A A LKEKVAALKE

It should be appreciated in light of the disclosures herein that different domain orientations, VH domains, VL domains, linkers and/or heteromerization promoting domains can be used to generate alternative B7-H3 x CD3 double chain diabodies.

B. B7-H3 x CD3 bispecific three-chain diabodies.

A B7-H3 x CD3 diabody having three chains and an Fc domain was prepared that has one binding site specific for B7-H3 (containing humanized VH and VL domains from hmAb-D) and one binding site specific for CD3 (containing VL and VH domains from CD3 mAb 1 (D65G)). The diabody is referred to as DART-D3.

The first polypeptide chain of an exemplary bispecific three-chain diabody DART-D3 B7H3 x CD3 contains in the direction from the N-terminus to the C-terminus: N-terminus; VL domain from anti-B7-H3 antibody (VL from hmAb-D (SEQ ID NO:30), intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO:32)), VH domain from CD3 mAb 1 (DGCG) ( SEQ ID NO:68), spacer peptide intermediate (linker 2: ASTKG (SEQ ID NO:37)), cysteine-containing heterodimerization-promoting domain (Espiral) (EVAACEK-EVAALEK-EVAAALEK-EVAALEK (SEQ ID NO:47)); intermediate spacer

- 42042568 peptide (linker 3: GGGDKTHTCPPCP (SEQ ID NO:57)); Bulge-bearing CH2-CH3 domain from IgG1 (SEQ ID NO:61); and C-terminus. Polynucleotides encoding this polypeptide chain may encode the C-terminal lysine residue of SEQ ID NO:61 (i.e., X of SEQ ID NO:61), however, as noted above, this lysine residue may be post-translationally deleted in some expression systems. Accordingly, the present invention includes such a first polypeptide chain that contains such a lysine residue (i.e., SEQ ID NO:61, where X is a lysine), as well as a first polypeptide chain that has lost such a lysine residue (i.e., SEQ ID NO:61, where X is absent). The following are the amino acid sequences of such a first polypeptide chain (SEQ ID NO:91) (underlined is the VL domain sequence of hmAb-D (SEQ ID NO:30)) DIQMTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS

ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFAEYFCQQ YNNYPFTFGQ

GTKLEIKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN

WVRQAPGKGL EWVGRIRSKY NNYATYYADS VKGRFTISRD DSKNSLYLQM

NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS ASTKGEVAAC

EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP

PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE

QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR

EPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT

PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS

PGX where X is lysine (K) or absent.

The second polypeptide chain of an exemplary bispecific three-chain diabody DART-D3 B7H3 x CD3 contains in the direction from the N-terminus to the C-terminus: N-terminus; VL domain from CD3 mAb-1 (SEQ ID NO:67); intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO:32)); VH domain from anti-B7-H3 antibody (hVH from mAb-D (SEQ ID NO:31), intermediate spacer peptide (linker 2: ASTKG (SEQ ID NO:37)), cysteine-containing domain (K-helix) promoting heterodimerization (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO:48)) and the C-terminus The amino acid sequence of such a second polypeptide chain (SEQ ID NO:92) is shown below (underlined is the VH domain sequence from hmAb-D (SEQ ID NO: 31))

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI

GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF

GGGTKLTVLG GGGSGGGGEV QLVESGGGLV QPGGSLRLSC AASGFTFSSF

GMHWVRQAPG KGLEWVAYIS SGSGTIYYAD TVKGRFTISR DNAKNSLYLQ

MNSLRAEDTA VYYCARHGYR YEGFDYWGQG TTVTVSSAST KGKVAACKEK VAALKEKVAA LI<EI<VAALI<E

The third polypeptide chain of an exemplary DART-D3 B7H3 x CD3 bispecific three-chain diabody comprises, in N-terminal to C-terminal direction: N-terminus; spacer peptide (DKTHTCPPCP (SEQ ID NO:56)); IgG1 CH2-CH3 recess-bearing domain (SEQ ID NO:62); and C-terminus. Polynucleotides encoding this polypeptide chain may encode the C-terminal lysine residue of SEQ ID NO:62 (i.e. X of SEQ ID NO:62), however, as noted above, this lysine residue may be post-translationally deleted in some expression systems. Accordingly, the present invention includes such a third polypeptide chain which contains such a lysine residue (i.e., SEQ ID NO:62. SEQ ID NO:62 where X is absent). The amino acid sequence of such a third polypeptide chain is shown below (SEQ ID NO:93)

DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED

PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK

CKVSNKALPA

PIEKTISKAK

GQPREPQVYT LPPSREEMTK NQVSLSCAVK

GFYPSDIAVE

WESNGQPENN YKTTPPVLDS

DGSFFLVSKL

TVDKSRWQQG

NVFSCSVMHE ALHNRYTQKS LSLSPGX where X is lysine (K) or absent.

It is understood that in light of the disclosures herein, various domain orientations, VH domains, VL domains, developmental linkers and/or heterodimers can be used to generate alternative B7-H3 x CD3 bispecific three-chain diabodies. In particular, the VH domain and the VL domain from hmAb-C (SEQ ID NO:20-21) can be used.

C. Trivalent binding molecules B7-H3 x CD3 x CD8.

Exemplary trivalent B7-H3 x CD3 x CD8 binding molecules having a single B7-H3 specific binding site (containing the parental and/or humanized anti-B7-H3VL domain and the corresponding anti-B7-H3-VH domain as described above) , one binding site specific for CD3 (comprising e.g. VL domain from CD3 mAb-1 (SEQ ID NO:67) and VH domain from anti-CD3 antibody (e.g. CD3 mAb 1 (D65G) (SEQ ID NO:68) ) and one binding site specific for CD8 (comprising, for example, the VH and VL domains of TRX2 (SEQ ID NOS: 75 and 76, respectively). Such trivalent binding molecules can have two polypeptide chains (see, for example, Fig. 6E and 6F), three polypeptide chains (see, for example, Fig. 6C and 6D), four polypeptide chains (see, for example, Fig. 6A

- 43 042568 and 6B) or five polypeptide chains (see, for example, Fig. 5).

X. Methods of obtaining.

The B7-H3 binding molecules of the present invention are most preferably produced by recombinant expression of nucleotide molecules that encode such polypeptides, as is well known in the art.

The polypeptides of the invention can be readily prepared using solid-phase peptide synthesis (Merrifield, B. (1986) Solid Phase Synthesis Science 232(4748):341-347; Houghten, R.A. (1985) General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of AntigenAntibody Interaction At The Level Of Individual Amino Acids, Proc. Natl. Acad. Sci. (U.S.A.) 82(15):51315135; Century, Mini Rev. Med. Chem. 6(1):310).

Alternatively, antibodies can be produced recombinantly and expressed using any method known in the art. Antibodies can be made recombinant first by isolating antibodies derived from host animals, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in host cells (eg, CHO cells). Another method that can be used is the expression of the antibody sequence in plants (eg tobacco) or transgenic milk. Suitable methods for recombinant expression of antibodies in plants or milk have been disclosed (see, for example, Peeters et al. (2001) Production Of Antibodies And Antibody Fragments In Plants, Vaccine 19:2756; Lonberg, N. et al. (1995) Human Antibodies From Transgenic Mice, Int. Rev. Immunol 13:65-93 and Pollock et al. (1999) Transgenic Milk As A Method For The Production Of Recombinant Antibodies, J. Immunol Methods 231:147-157). Suitable methods for derivatizing antibodies, eg humanized, single chain, etc. are known in the art and have been described above. Alternatively, antibodies can be generated recombinantly using phage display technology (see, for example, US Pat. Immunol 12.433-455).

Vectors containing polynucleotides of interest (e.g., polynucleotides encoding the polypeptide chains of the B7-H3 binding molecules of the present invention) can be introduced into the host cell by any of a number of suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, phosphate calcium, DEAE-dextran or other substances; microparticle bombardment; lipofection; and infection (eg, where the vector is an infectious agent such as vaccinia virus). The choice of vectors or polynucleotides to be administered will often depend on the characteristics of the host cell.

Any host cell capable of overexpressing heterologous DNA can be used to express the polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells.

The invention includes polypeptides containing the amino acid sequence of the B7-H3 binding molecule of the present invention. The polypeptides of the present invention can be obtained by methods known in this field. Polypeptides can be obtained by proteolytic or other degradation of antibodies by recombinant methods (ie, single or fused polypeptides), as described above, or by chemical synthesis. Antibody polypeptides, especially shorter polypeptides of up to about 50 amino acids, are usually prepared by chemical synthesis. Methods for chemical synthesis are known in the art and are commercially available.

The invention includes variants of B7-H3 binding molecules, including functionally equivalent polypeptides that do not significantly affect the properties of such molecules, as well as variants that have increased or decreased activity. Modification of polypeptides is a common practice in this field and does not need to be described in detail in this document. Examples of modified polypeptides include those with conservative amino acid residue substitutions, one or more amino acid deletions or additions that do not significantly adversely affect functionality or use of chemical analogs. Amino acid residues that may be conservatively substituted with each other include, but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as, for example, glycosylation with various sugars, acetylation and phosphorylation. Preferably, the amino acid substitutions will be conservative, ie. the substituted amino acid will have the same chemical properties as the original amino acid. Such conservative substitutions are known in the art and examples have been given above. Amino acid modifications can range from changing or modifying one or more amino acids to a complete reorganization of a site, such as a variable domain. Variable domain changes can alter binding affinity and/or specificity.

- 44 042568

Other modification methods include the use of coupling methods known in the art, including, but not limited to, enzymatic agents, oxidative displacement, and chelation. Modifications can be used, for example, to attach labels for immunoassay, such as attaching radioactive components for radioimmunoassay. The modified polypeptides are prepared using known procedures in the art and can be screened using standard assays known in the art.

The invention encompasses fusion proteins containing one or more anti-B7-H3-VL and/or VH of this invention. In one embodiment, a fusion polypeptide is provided that includes a light chain, a heavy chain, or both a light chain and a heavy chain. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises a light chain variable domain and a variable chain length domain of an antibody produced from a publicly deposited hybridoma. For the purposes of the present invention, an antibody fusion protein contains one or more polypeptide domains that specifically bind to B7-H3 and another amino acid sequence to which it is not attached in the native molecule, such as a heterologous sequence or a homologous sequence from another site.

The present invention particularly encompasses B7-H3 binding molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) conjugated to a diagnostic or therapeutic moiety. For diagnostic purposes, the B7-H3 binding molecule of the invention may be associated with a detectable substance. Such B7-H3 binding molecules are useful for monitoring and/or predicting the development or progression of a disease as part of a clinical trial procedure such as determining the efficacy of a particular therapy. Examples of detectable substances include various enzymes (eg, horseradish peroxidase, beta-galactosidase, etc.), prosthetic groups (eg, avidin/biotin), fluorescent materials (eg, umbelliferone, fluorescein, or phycoerythrin), luminescent materials (eg, luminol ), bioluminescent materials (such as luciferase or aequorin), radioactive materials (such as carbon-14, manganese-54, strontium-85, or zinc-65), positron-active metals, and non-radioactive paramagnetic metal ions. A detectable substance can be linked or conjugated either directly to the B7-H3 binding molecule or indirectly through an intermediate (eg, a linker) using methods known in the art.

For therapeutic purposes, the B7-H3 binding molecules of the invention may be conjugated to a therapeutic moiety, such as a cytotoxin (eg, a cytostatic or cytocidal agent), a therapeutic agent, or a radioactive metal ion, such as an alpha emitter. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells, such as, for example, Pseudomonas aeruginosa exotoxin, diphtheria toxin, botulinum toxin A-F, ricin-abrin, saporin, and cytotoxic fragments of such agents. A therapeutic agent includes any agent having a therapeutic effect for the prophylactic or therapeutic treatment of a disorder. Such therapeutic agents may be chemical therapeutic agents, proteinaceous or polypeptide therapeutic agents, and include therapeutic agents that have the biological activity of interest and/or modify the biological response. Examples of therapeutic agents include alkylating agents, angiogenesis inhibitors, antimitotic agents, hormone therapy agents, and antibodies useful in the treatment of cell proliferative disorders. The therapeutic moiety can be linked or conjugated either directly to the B7-H3 binding molecule or indirectly through an intermediate (eg, a linker) using techniques known in the art.

XI. Drug-antibody conjugates.

The present invention relates to therapeutic antibodies against human B7-H3 (or its B7-H3 binding domains), and in particular to any of the above-described antibodies against human B7-H3 or B7-H3 binding domains, which are conjugated to a drug. agent (B7-H3-ADC molecule). Such B7-H3-ADCs enhance the cytotoxicity of anti-human B7-H3 therapy, especially in the treatment of cancer. As stated above, the B7-H3-ADC molecules of the present invention comprise the formula

Ab-(LM)m-(D) _n where Ab is an antibody that binds to B7-H3, which includes a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL) or is B7-H3 -binding fragment;

D is a cytotoxic drug moiety;

LM is a bond or linker molecule that covalently links Ab and D;

m is an integer from 0 to n and denotes the number of linker molecules in B7-H3ADC;

n is an integer from 1 to 10 and denotes the amount of cytotoxic drug moieties covalently linked to the B7-H3-ADC molecule.

- 45 042568

In preferred embodiments, the B7-H3-ADC will bind to a B7-H3 expressing tumor cell and then be internalized into such a cell via receptor-mediated endocytosis. Once inside the lysosome, B7-H3-ADC will preferentially degrade to thereby cause the release of the cytotoxic drug component inside the cell, leading to cell death. As will be appreciated, the mechanism of action of cell death may vary depending on the class of cytotoxic drug used (eg, disruption of cytokinesis by inhibitors of tubulin polymerization such as maytansines and auristatins, DNA damage by DNA-interacting agents such as calicheamicins and duocarmycins), and others. cancer cells can also be killed when the free drug is released into the tumor environment by a dying cell in a process known as the bystander effect (Panowski, S. et al. (2014) Site-Specific Antibody Drug Conjugates For Cancer Therapy, mAbs 6(1) :34-45; Kovtun, Y. V. et al. (2006) Antibody-Drug Conjugates Designed To Eradicate Tumors With Homogeneous And Heterogeneous Expression Of The Target Antigen, Cancer Res. 66:3214-3221).

The B7-H3-ADC of the present invention may contain an Fc domain, which may be a naturally occurring Fc domain, or may have a sequence that has one or more differences from a naturally occurring Fc domain, and which may be a complete Fc domain, (e.g., a complete Fc domain from IgG) or only part of the complete Fc domain. Such Fc domains may be of any isotype (eg IgG1, IgG2, IgG3 or IgG4). Such an Fc domain may or may not include a C-terminal lysine residue in the CH3 domain. The B7-H3-ADC of the present invention may further include a CH1 domain and/or a hinge domain. If present, the CH1 domain and/or hinge domain can be of any isotype (eg, IgG1, IgG2, IgG3, or IgG4) and will preferably be of the same isotype as the Fc domain of interest.

A. Typical linker molecules of the invention.

The invention thus specifically provides for B7-H3-ADCs in which there is no LM linker molecule (i.e. m = 0) and B7-H3-ADCs which have more than one LM linker molecule (i.e. e. m is an integer from 2 to n, where n is an integer from 2 to 10), wherein each of the LM linker molecules covalently binds the D cytotoxic drug moiety D to an Ab such as B7-H3-ADC.

The invention further provides B7-H3-ADCs whose Ab is covalently linked to more than one LM linker molecule, where all such linker molecules are identical. The cytotoxic D drug moieties that are covalently linked to the Abs of such B7-H3-ADCs may be all the same or may include 2, 3, 4, or more independently different cytotoxic D drug moieties.

In addition, the present invention relates to those B7-H3-ADCs whose Abs are covalently linked to more than one LM linker molecule, where all such linker molecules are not identical and may be independently different. The cytotoxic D drug moieties that are covalently linked to the Ab of such B7-H3-ADC may be all the same or may include 2, 3, 4, or more independently different cytotoxic D drug moieties.

Exemplary humanized VH and VL domains of antibodies that bind to human B7-H3, and exemplary human antibody constant domains that can be included in the B7-H3-ADC of the invention, are given above. As indicated above, the B7-H3-ADC of the invention further comprises at least one cytotoxic drug moiety, which is preferably covalently linked to the side chain atom of the amino acid residue of such VH domain or VL domain and/or constant domain, either directly or via a linker molecule. intercalated between the side chain atom and the drug moiety. The linker molecule may be a non-peptide molecule, or a molecule that includes a non-peptide portion and a peptide portion, or it may be a molecule that consists solely of amino acid residues. The amino acid residues of any such linker molecules may contain natural or non-natural amino acid residues, including D-variants of naturally occurring amino acid residues, p-acetylphenylalanine, selenocysteine, and the like. Optionally or additionally, specific residues having the desired side chain (e.g. -CH2-SH side chain, -CH2-OH side chain, -CH(CH ₂ )-SH side chain, -CH ₂ CH2-S-CH3 side chain, - CH ₂ -C (O) -NH ₂ , side chain -CH ₂ -CH ₂ -C (O) -NH ₂ , side chain -CH ₂ C (O) OH-, side chain CH ₂ -CH ₂ -C ( O) OH-, side chain -CH ₂ -CH ₂ -CH ₂ -Ch ₂ -NH ₂ , side chain -CH2CH ₂ -CH ₂ -NH-C (NH ₂ ) ₂ , imidazole side chain, benzyl side chain, side chain phenol chain, indole side chain, etc.) can be introduced into the B7-H3-ADC construct of the invention.

The linker molecule may be non-cleavable under physiological conditions, for example consisting of a hydrolytically stable moiety, such as a thioether linker or a shielded disulfide linker. Hydrolytically stable linkers are substantially stable in water and do not react with water at useful pH values, including, but not limited to, physiological conditions over an extended period of time. In contrast, hydrolytically unstable or degradable linkers degrade in water or in aqueous solutions, including, for example, blood.

Otherwise, the linker molecule may be cleavable or may contain cleavable

- 46 042568 my part. Examples of such a cleavable portion include an acid labile linker (eg, a 4-(4'-acetylphenoxy)butanoic acid linker that forms a hydrazine bond), a cleavable disulfide linker (which cleaves in a reductive intracellular environment), and a protease cleavable linker. Acid-labile linkers are designed to be stable at pH levels encountered in the blood, but become unstable and degrade when low pH environments are encountered in lysosomes. Protease cleavable linkers are also designed to stabilize in the blood/plasma but rapidly release the free drug inside lysosomes in cancer cells when cleaved by lysosomal enzymes (Panowski, S. et al. (2014) Site-Specific Antibody Drug Conjugates For Cancer Therapy, mAbs 6( 1):34-45). Alternatively, the linker molecule may be an enzyme-cleavable substrate or contain an enzyme-cleavable substrate such as a cleavable peptide (e.g., a cleavable dipeptide such as a valine-citrulline dipeptide para-aminobenzyl alcohol linker (cAC10-mc-vc-PABA) that selectively cleaved by lysosomal enzymes). Suitable cleavable linkers are known in the art, see, for example, de Groot, Franciscus M.H., et al. (2002) Design, Synthesis, and Biological Evaluation of a Dual Tumor-Specific Motive Containing Integrin-Targeted Plasmin-Cleavable Doxorubicin Prodrug, Molecular Cancer Therapeutics, 1: 901-911; Dubowchik et al., (2002) Doxorubicin Immunoconjugates Containing Bivalent, LysosomallyCleavable Dipeptide Linkages, Bioorganic & Medicinal Chemistry Letters 12:1529-1532; Pat. US No. 5547667; 6214345; 7585491; 7754681; 8080250; 8461117 and WO 02/083180.

Enzymatically unstable or degradable linkers can be used. Such linkers are degraded by one or more enzymes. For example, PEG and related polymers may include a degradable linker molecule(s) in the polymer backbone or in a linker group between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linker molecule(s) include, but are not limited to, ester bonds formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active substance, where such ester groups are usually hydrolyzed under physiological conditions to release the biologically active agent. Other hydrolytically degradable linker molecules include, but are not limited to, carbonate bonds; imine bonds formed as a result of the interaction of an amine and an aldehyde; phosphate esters formed by the interaction of alcohol with a phosphate group; hydrazone bonds, which are the reaction product of a hydrazide and an aldehyde; acetal bonds, which are the reaction product of an aldehyde and an alcohol; orthoester bonds, which are the reaction product of formate and alcohol; peptide bonds formed by an amino group, including, without limitation, at the end of a polymer, such as PEG, and the carboxyl group of a peptide; and oligonucleotide bonds formed by the phosphoramidite group, including, but not limited to, at the polymer end and the 5'-hydroxyl group of the oligonucleotide.

In one embodiment, the linker molecule of the present invention may be, or may include, a cleavable linker molecule, V-(W) _k -(X)lA, as described in PCT publication WO 02/083180, which has the formula

Ab - [V-(W) _k -(X)iA] - D where V is an optional split-off component;

(W) _k -(X)lA - elongated self-eliminating spacer system, which is self-eliminated and l,(4+2n)-elimination;

W and X are each an electron cascade spacer al (4+2n) being the same or different;

A is either a spacer group of formula (Y) _m , where Y is a l,(4+2n) electron cascade spacer, or a group of formula U which is a cyclization-elimination spacer;

k, l and m are independently an integer from 0 (inclusive) to 5 (inclusive);

n is an integer from 0 (inclusive) to 10 (inclusive);

provided that: when A is equal to (Y) _m : then k + l + m > 1, and if k + l + m = 1, then n >1;

when A is equal to U, then k + 1 > 1;

W, X and Y are independently selected from compounds having the formula

or the formula

where Q represents -R ⁵ C=CR ⁶ -, S, O, NR ⁵ , -R ⁵ C=N- or -N=CR ⁵ -;

P is NR ⁷ , O or S;

a, b and c are independently an integer from 0 (inclusive) to 5 (inclusive);

- 47 042568

I, F and G are independently selected from compounds having the formula IR®. RY o9 _______ \=^ or \=< ^{n or} —=— / _R 9 / \ where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ independently represent H, C1-6alkyl, C3-20heterocyclyl, C5-20aryl, C1-6alkoxy, hydroxy (OH), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF ₃ , CN, CONH2, SO ₂ Me, CONHMe, cyclic C _1-5 alkylamino, imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SR _x ), tetrazole, carboxy (COOH), carboxylate (COOR _x ), sulfoxy (S(=O) ₂ OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(=O) ₂ R _x ), sulfoxide (S(=O)OH), sulfinate (S (=O)OR _x ), sulfinyl (S(=O)R _x ), phosphonooxy (OP(=O)(OH) ₂ ) and phosphate (OP(=O)(OR _x ) ₂ ) _x , Rx ¹ and Rx ² is independently selected from a C1-6 alkyl group, an O _3-20 heterocyclyl group, or a C _5-20 aryl group, two or more substituents R ¹ , R ² , R 3 , R ⁴ , R ⁵ , R ^{6 ,} R ⁷ , R ⁸ or R ⁹ optionally linked to each other to form one or more aliphatic or aromatic chains iclic structures; U is selected from compounds having the formula

R ³ R* R ⁷ R ² _n L \ III I2 \u003d \ A ^OR -nn - kiLI

R ¹ III a । ।\

R^ R ^c ' R ^a R ¹ R ^{2 λ}

R ³ ^___,R ⁵ R ^H —S— ^OR N— ¹ R ⁷ IL \i

R ^{1 R} R ^a fV RR ^? R ² where a, b and c are independently selected as the integer 0 or 1; provided that a + b + c = 2 or 3;

R ¹ and/or R ² are independently H, C1-6 alkyl, said alkyl being optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF ₃ , CN, CONH2, SO ₂ Me, CONHMe, cyclic O _1-5 alkylamino, imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (CPx), tetrazole, carboxy (COOH), carboxylate (COORx), sulfoxy (S(=O)2OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(=O) ₂ R _x ), sulfoxy (S(=O)OH), sulfinate (S(=O)OR _x ), sulfinyl (S(=O)Rx), phosphonoxy (OP(=O)(OH)2) and phosphate (OP(=O )(ORx)2), where Rx, Rx ¹ and Rx ² are selected from C _1-6 alkyl group, C _3-20 heterocyclic group or C _5-20 aryl group; And

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently H, C1-6alkyl, C3-20heterocyclyl, C5-20aryl, C1-6alkoxy, hydroxy (OH), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF ₃ , CN, CONH2, SO ₂ Me, CONHMe, cyclic C _1-5 alkylamino, imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol ( SH), thioether (SR _x ), tetrazole, carboxy (COOH), carboxylate (COOR _x ), sulfoxy (S(=O) ₂ OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(= O) ₂ R _x ), sulfoxide (S(=O)OH), sulfinate (S(=O)OR _x ), sulfinyl (S(=O)R _x ), phosphonooxy (OP(=O)(OH) ₂ ) and phosphate (OP(=O)(OR _x ) ₂ ), where R _x , Rx ¹ and Rx ² are selected from C 1-6 alkyl group, C _3-20 heterocyclyl group or C _5-20 aryl group and two or more substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , or R ⁸ are optionally bonded to each other to form one or more aliphatic or aromatic ring structures.

Representative molecules include p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl;

p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl;

p-ammocinnamyloxycarbonyl;

p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl;

p-amino-benzyloxycarbonyl-p-aminocinnamyloxycarbonyl;

p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl;

p-aminophenylpentadienyloxycarbonyl;

p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyloxycarbonyl;

p-aminophenylpentadienyloxycarbonyl-aminobenzyloxycarbonyl;

p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyloxycarbonyl;

p-aminobenzyloxycarbonyl (methylamine)ethyl(methylamine)carbonyl;

p-aminocinnamyloxycarbonyl (methylamine)ethyl(methylamine)carbonyl;

p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamine)ethyl(methylamine)carbonyl;

p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl(methylamine)ethyl(methylamine)carbonyl;

p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamine)ethyl(methylamine)carbonyl;

p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamine)ethyl(methylamine)carbonyl;

p-aminobenzyloxycarbonyl-p-aminobenzyl;

p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyl;

p-aminocinnamyl;

p-aminocinnamyloxycarbonyl-p-aminobenzyl;

- 48 042568 p-aminobenzyloxycarbonyl-p-aminocinnamyl;

p-amino-cinnamyloxycarbonyl-p-aminocinnamyl;

p-aminophenylpentadienyl;

p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyl; p-aminophenylpentadienyloxycarbonyl-p-aminobenzyl; p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyl.

In some embodiments, the B7-H3-ADC of the invention contains two, three, four, five, six, seven, eight, nine, or ten cytotoxic drug moieties that may be the same or may independently be the same or different from another B7- cytotoxic drug moiety. H3-ADC. In one embodiment, each such cytotoxic drug moiety is conjugated to an Ab from B7-H3-ADC of the invention via a separate linker molecule. Alternatively, more than one cytotoxic drug moiety may be attached to the B7-H3-ADC Ab of the invention via the same linker molecule.

Cytotoxic drug moieties can be conjugated to the B7-H3-ADC Ab of the invention by methods known in the art (see, for example, Yao, H. et al. (2016) Methods to Design and Synthesize Antibody-Drug Conjugates (ADC), Intl J Molec Sci 17(194): 1-16); Behrens, C.R. et al. (2014) Methods For Site-Specific Drug Conjugation To Antibodies, mAbs 6(1):46-53; Bouchard, H. et al. (2014) Antibody-Drug Conjugates - A New Wave Of Cancer Drugs, Bioorganic & Medicinal Chem. Lett 24:5357-5363). A cysteine thiol group, a lysine, glutamine or arginine amino group, or a glutamate or aspartate carboxyl group can be used to conjugate a linker molecule-cytotoxic drug moiety (LM-D) to an Ab from B7-H3-ADC of the invention. Native antibodies contain multiple lysine conjugation sites and are thus capable of binding multiple conjugated molecules per antibody. Indeed, peptide mapping determined that conjugation occurs at both the heavy and light chains at about 20 different lysine residues (40 lysines per mAb). Thus, more than one million different types of ADC can be generated. Conjugation to cysteine occurs after the reduction of one to four interstrand disulfide bonds, and therefore, in native VL and VH domains, conjugation is limited to eight exposed sulfhydryl groups. However, if desired, additional reactive residues (eg, lysine, cysteine, selenocysteine, etc.) can be introduced into antibodies (eg, within the VL domain and/or VH domain and/or constant domain). For example, one or more naturally occurring amino acid residues may be replaced with a cysteine residue. An unnatural amino acid (eg, p-acetylphenylalanine) can be genetically introduced into an antibody using the stop codon inhibitor amber with a tRNA/aaRS suppressor pair; (See, for example, Behrens C.R., and Liu B. (2014) Methods For Site-Specific Drug Conjugation To Antibodies, mAbs 6(1):46-53. doi:10.4161/mabs.26632; Panowksi, S., et al (2014) Site-Specific Antibody Drug Conjugates For Cancer Therapy, mAbs, 6(1), 34-45, doi:10.4161/mabs.27022 and WO 2008/070593). Alternatively, or additionally, enzymes (eg, glycotransferase) can be used to conjugate the linker molecule-cytotoxic drug moiety (LM-D) to the B7-H3-ADC Ab of the invention. The glycotransferase platform attaches a sugar moiety to a glycosylation site on the antibody (e.g., at position N297 of the Fc domain of a human IgG antibody), which can then serve as a linker molecule of the present invention and conjugate the cytotoxic drug moiety (D) to an Ab from B7-H3-ADC according to the invention. Alternatively, transglutaminase can be used to catalytically form a covalent bond between the free amino group and the glutamine side chain.

Preferred for this purpose is the commercially available transglutaminase from Streptoverticillium mobaraense (mTG) (Pasternack, R. et al. (1998) Bacterial Pro-Transglutaminase From Streptoverticillium. mobaraense Purification, Characterization And Sequence Of The Zymogen, Eur. J. Biochem. 257( 3):570-576 Yokoyama, K. et al (2004) Properties And Applications Of Microbial Transglutaminase, Appl Microbiol Biotechnol 64:447-454). This enzyme does not recognize any naturally occurring glutamine residues in the Fc domain of glycosylated antibodies, but recognizes the tetrapeptide LLQL (SEQ ID NO:94) (Jeger, S. et al. (2010) Site-Specific And Stoichiometric Modification Of Antibodies By Bacterial Transglutaminase, Angew Chem Int. Ed. Engl. 49:9995-9997), which can be introduced into a VL domain and/or a VH domain and/or a constant domain. Such considerations are reviewed by Panowski, S. et al. (2014) Site-Specific Antibody Drug Conjugates For Cancer Therapy, mAbs 6(1):34-45.

B. Exemplary Cytotoxic Drug Components of the Invention.

In some embodiments, the B7-H3-ADC cytotoxic drug moiety of the invention includes a cytotoxin, a radioisotope, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, a DNA molecule, an RNA molecule, an miRNA molecule , an RNAi molecule, a miRNA molecule, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent, or a combination thereof.

1. Cytotoxic medicinal components of tubulizin.

B7-H3-ADC according to the invention may include a cytotoxic drug component

- 49 042568 tubulizin o

Tubulisins are members of a class of natural products isolated from myxobacterial species (Sasse et al. (2000) Tubulysins, New Cytostatic Peptides From Myxobacteria Acting On Microtubuli. Production, Isolation, Physico-Chemical And Biological Properties, J. Antibiot. 53:879-885 ). As cytoskeletal interacting agents, tubulisins are mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (Steinmetz et al. (2004) Isolation, Crystal And Solution Structure Determination, And Biosynthesis Of Tubulysins--Powerful Inhibitors Of Tubulin Polymerization From Myxobacteria, Chem. Int. Ed. 43:4888-4892; Khalil et al. (2006) Mechanism Of Action Of Tubulysin, An Antimitotic Peptide From Myxobacteria, ChemBioChem. 7:678-683; Kaur et al. (2006) Biological Evaluation Of Tubulysin A: A Potential Anticancer And Antiangiogenic Natural Product, Biochem. J. 396: 235-242). Tubulisins are extremely potent cytotoxic molecules whose cell growth inhibitory activity exceeds that of any clinically relevant conventional chemotherapeutic agent, such as epothilones, paclitaxel and vinblastine. In addition, they are effective against multidrug resistant cell lines (Domling, A. et al. (2005) Myxobacterial Epothilones And Tubulysins As Promising Anticancer Agents, Mol. Diversity 9:141-147). These compounds exhibit high cytotoxicity, tested against a panel of cancer lines with IC ₅₀ values in the low picomolar range; thus, they are of interest as anticancer therapy; see, for example, WO 2012/019123, WO 2015/157594. Tubulisin conjugates are disclosed, for example, in US Pat. US No. 7776814. In some embodiments, the tubulisin molecule or its derivative is a prodrug.

2. Cytotoxic medicinal components of auristatin.

Alternatively or additionally, the B7-H3-ADC of the invention may include the cytotoxic drug moiety auristatin, (e.g. MMAE (N-methylvaline-valin-dolaisoleuindolaproin-norephedrine) and MMAF (N-methylvaline-valine-dolaisoleuin-dolaproin-phenylalanine). Dolastatins originally have been discovered as components of the bearded seal Dolabella auricularia and have been modified to give derivatives also known as auristatin (e.g., monomethylauristatin E and F. Dolastatins and auristatins interact with the vinca alkaloid binding site of α-tubulin and block its polymerization. They have been shown to affect microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al., Antimicrob. Agents and Chemother. 45:3580-3584 (2001)) and have antitumor activity (U.S. Pat. No. 5663149, 6884869, 7964566). The auristatin drug moiety can be attached to the antibody through the N-(amino) terminus or C-(carboxy) terminus of the peptide drug moiety (see for example, WO 2002/088172). In some embodiments, the molecule, variant or derivative of auristatin or dolastatin is a prodrug. MMAE can be conjugated to protein by modifying cysteine side chain thiols (Senter, P.D. et al. (2012) The Discovery And Development Of Brentuximab Vedotin For Use In Relapsed Hodgkin Lymphoma And Systemic Anaplastic Large Cell Lymphoma, Nat. Biotechnol. 30:631637; van de Donk, N. W. et al (2012) Brentuximab vedotin, MAbs 4:458-465). This method involves the reduction of one or more disulfide bonds exposed to the solvent of cysteine residues using a reducing agent (for example, dithiothreitol (DTT) or tris-(2carboxyethyl)phosphine (TCEP)) followed by modification of the resulting thiols with a maleimide-containing drug ( see, Behrens, C. R. et al (2014) Methods For Site-Specific Drug Conjugation To Antibodies, mAbs 6(1):46-53).

Scheme 1

A typical cytotoxic drug that can be conjugated in this manner includes a cathepsin B protease cleavage site (VC: valine, citrulline) and a self-cleaving linker (PAB: para-aminobenzyloxycarbonyl) between the maleimide group (MC: maleimidocaproyl) and the cytotoxic drug ( MMAE) (Doronina, SO et al. (2003) Development

Of Potent Monoclonal Antibody Auristatin Conjugates For Cancer Therapy, Nat. Biotechnol. 21:778-784).

Scheme 2. Synthesis of MC-VC-PAB-MMAE

Alternatively, the auristatin-cytotoxic drug moiety may be AcLys-VC-PAB-MMAD (acetylsilinvalinecitrulline-p-aminobenzyloxycarbonylmonomethyldolastatin) which may be conjugated to the NH ₂ group of the side chain of the glutamine residue in the VL domain and/or the VH domain and/ or the constant domain of the Ab portion of the B7-H3-ADC of the invention using the enzyme, microbial transglutaminase, to catalyze a site-specific reaction between the acetylated lysine side chain and the glutamine side chains:

Scheme 3

Alternatively, p-acetylphenylalanine can be incorporated into the VL domain and/or the VH domain and/or the constant domain of the Ab portion of the B7-H3-ADC of the invention and then used to conjugate auristatin F-hydroxyamine to that domain by oxime ligation.

Scheme 4

3. Maytansinoid cytotoxic medicinal components.

The B7-H3-ADC of the invention may alternatively or additionally include a maytansinoid cytotoxic drug moiety, for example, the antibiotic ansamycin, characterized by a 19-membered ansamacrolide structure attached to a chlorine-placed benzene ring of the chromophore. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub Maytenus serrata (US Patent No. 386111). Some microbes have also been found to produce maytansinoids such as maytansinol and maytansinol C-3 esters (US Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogues are disclosed, for example, in US Pat. US Nos. 4,137,230 and 4,248,870. Maytansine drug moieties are attractive drug moieties in drug-antibody conjugates because they are: (i) relatively available for fermentation or chemical modification, derivatization of fermentation products, (ii) amenable to derivatization with functional groups, suitable for conjugation via non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines. Maytansinoid-containing immunoconjugates, methods for their preparation and their therapeutic use are disclosed, for example, in US Pat. US Nos. 5202020 and 5416064 and European patent EP 0425235B1; Liu, C. et al. (1996) Eradication Of Large Colon Tumor Xenografts By Targeted De- 51 042568 livery Of Maytansinoids, Proc. Natl. Acad. sci. (USA) 93: 8618-8623 (described immunoconjugates containing maytansinoid, designated DM1) and Chari, RV et al. (1992) Immunoconjugates Containing

Novel Maytansinoids: Promising Anticancer Drugs, Cancer Research 52:127-131.

Maytansine, DM1 and DM4 are exemplary maytansinoid cytotoxic drug moieties

Maytansine can be conjugated to the Ab portion of the B7-H3-ADC of the invention by reaction with a lysine or glutamine side chain. DM1 and DM4 can be conjugated to the side chain COOH of a glutamate or aspartate residue of the VL domain and/or the VH domain and/or the constant domain of the Ab portion of the B7-H3-ADC of the invention (see, Behrens, C.R. et al. (2014) Methods For Site-Specific Drug Conjugation To Antibodies, mAbs 6(1):46-53; Bouchard, H. et al. (2014) Antibody-Drug Conjugates - A New Wave Of Cancer Drugs, Bioorganic & Medicinal Chem. Lett 24:5357 -5363).

Scheme 5

Trastuzumab emtansine (ado-trastuzumab emtansine, T-DM1, trade name KADCYLA®) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab (HERCEPTIN®) conjugated to the maytansinoid mertansine (DM1); see, for example, LoRusso et al. (2011) Trastuzumab Emtansine: A Unique Antibody-Drug Conjugate In Development For Human Epidermal Growth Factor Receptor 2-Positive Cancer, Clin. Cancer Res. 20:6437-6447. The developed thio-trastuzumab-DM1 ADC is also described in Junutual et al. (2010) Engineered Thio-Trastuzumab-DM1 Conjugate With An Improved Therapeutic Index To Target Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer, Clin, Cancer Res. 16:4769-4778. In some embodiments, the maytansinoid molecule, variant or derivative is a prodrug.

4. Cytotoxic drug fragments of calicheamicin.

The B7-H3-ADC of the invention may alternatively or additionally include the cytotoxic drug moiety calicheamicin

- 52 042568

The calicheamicin-based antibody conjugates described are disulfide variants of the parent trisulfide compound. So far, two coupling strategies have been reported with N-acetyl-c-calicheamicin dimethylhydrazide (CalichDMH): (i) with hydrazide and (ii) with amide coupling (Bouchard, H. et al. (2014) Antibody-Drug Conjugates - A New Wave Of Cancer Drugs, Bioorganic & Medicinal

Chem. Lett 24:5357-5363).

The calicheamicin endiyne anticancer antibiotic family is capable of producing double-stranded DNA breaks at subpicomolar concentrations. The calicheamycins are a class of endyine antibiotics derived from the bacterium Micromonospora echinospora, with calicheamycin γ1 being the most important. Other calicheamins are e1Br, y1Br, a2I, a3I, βH, y1I, and LI (see Lee, M.D. et al. (1989) Calicheamicins, A Novel Family Of Antitumor Antibiotics. 3. Isolation, Purification And Characterization Of Calicheamicins Beta 1Br, Gamma 1Br, Alpha 2I, Alpha 3I, Beta 1I, Gamma 1I And Delta 1I, J. Antibiotics 42(7): 1070-1087). For conjugates of the calicheamicin family, see US Pat. US Nos. 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 577001, and 5877296. Structural analogs of calicheamicin that can be used include, but are not limited to, y1III, a2I, a3I, N-acetyl-γ1I, PSAG, and Θ11 ( Hinman et al.(1993) Preparation And Characterization Of Monoclonal Antibody Conjugates Of The Calicheamicins: A Novel And Potent Family Of Antitumor Antibiotics, Cancer Research 53:3336-3342 (1993), Lode et al.(1998) Targeted Therapy With A Novel Enediyene Antibiotic Calicheamicin Theta(I)1 Effectively Suppresses Growth And Dissemination Of Liver Metastases In A Syngeneic Model Of Murine Neuroblastoma, Cancer Research 58:2925-2928 (1998) In some embodiments, the calicheamicin molecule, variant or derivative is a prodrug.

5. Cytotoxic drug components of pyrrolobenzodiazepine.

The B7-H3-ADC of the invention may alternatively or additionally comprise a pyrrolobenzodiazepine drug component (e.g. natural pyrrolobenzodiazepine and SJG-136, a derivative thereof)

A preferred pyrrolebenzodiazepine drug moiety is vadastuximab thalirin (SGN-CD33A, Seattle Genetics).

Scheme 6

Pyrrolobenzodiazepines (PBDs) are a class of natural products with antibiotic or anticancer activity. In nature, they are produced by actinomycetes. They are DNA alkylating compounds and some are sequence selective. A number of PBDs and their derivatives are known in the art, e.g. PBD dimers (e.g. SJG-136 or SG2000), C2-unsaturated PBD dimers, pyrrolobenzodiazepine dimers containing C2-aryl substitutions (e.g. SG2285) a drug that is activated by hydrolysis ( eg SG2285) and polypyrrole-PBD (eg SG2274). PBDs are further described in WO 2000/012507, WO 2007/039752, WO 2005/110423, WO 2005/085251 and WO 2005/040170 and WO 2014/057119. In some embodiments, the PBD molecule, variant or derivative is a prodrug.

6. Cytotoxic drug components of duocarmycin.

The B7-H3-ADC of the invention may alternatively or additionally include a duocarmycin drug moiety. Duocarmycins are representatives of a number of related natural products first isolated from Streptomyces bacteria and are potent anticancer antibiotics (see Dokter, W. et al. (2014) Preclinical Profile of the HER2-Targeting ADC SYD983/SYD985: Introduction of a New Duocarmycin -Based Linker-Drug Platform, Mol Cancer Ther 13(11):2618-2629 Boger, D.L. et al (1991) Duocarmycins - A New Class Of Sequence Selective DNA Minor Groove Alkylating Agents, Chemtracts: Organic Chemistry 4 (5): 329-349 (1991) Tercel et al (2013) The Cytotoxicity Of Duocarmycin Analogues Is Mediated Through Alkylation Of DNA, Not Aldehyde Dehydrogenase 1: A Comment, Chem Int Ed Engl 52(21) :5442-5446 Boger, D. L. et al (1995) CC-1065 And The Duocarmycins: Unraveling The Keys To A New Class Of Naturally Derived DNA Alkylating Agents, Proc.

- 53 042568

Natl. Acad. sci. (U.S.A.) 92(9):3642-3649; Cacciari, B. et al. (2000) CC-1065 And The Duocarmycins: Recent

Developments, Expert Opinion on Therapeutic Patents 10(12): 1853-1871).

Natural duocarmycins include ducocarmycin A, duocarmycin B1, ducarmycin B1, ducoarmycin B1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065 (PCT Publication No. WO 2010/062171, Martin, D.G. et al. (1980) Structure Of CC-1065 (NSC 298223), A New Antitumor Antibiotic, J. Antibiotics 33:902-903; Boger, D. L. et al. (1995) CC-1065 And The Duocarmycins: Unraveling The Keys To A New Class Of Naturally Derived DNA Alkylating Agents, Proc Natl Acad Sci (U.S.A.) 92:3642-3649).

Duocarmycin SA

Suitable synthetic analogs of duocarmycin include adoselesin, bizelesin, carcelesin (U-80244) and spiro-duocarmycin (DUBA) (Dokter, W. et al. (2014) Preclinical Profile of the HER2-Targeting ADC SYD983/SYD985: Introduction of a New Duocarmycin -Based Linker-Drug Platform Mol Cancer Ther 13(11):2618-2629 Elgersma, R.C. et al (2014) Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985, Mol Pharmaceut 12:18131835)

- 54 042568

Additional synthetic analogues of duocarmycin include those disclosed in publ. PCT No. WO 2010/062171 and in particular those analogues which have the formula

or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein DB is a DNA binding moiety and is selected from the group consisting of

where R is a leaving group;

R ² , R ² ', R ³ , R ³ ', R ⁴ , R ⁴ ', R ¹² and R ¹⁹ are independently selected from H, OH, SH, NH2, N3, NO2, NO, CF3, CN, C(O )NH2, C(O)H, C(O)OH, halogen, R ^a , SR ^a , S(O)R ^a , S(O)2R ^a , S(O)OR ^a , S(O)2OR ^a , OS(O)R ^a , OS(O)2R ^a , OS(O)OR ^a , OS(O)2OR ^a , OR ^a , NHR ^a , N(R ^a )R ^b , +N(R ^a )( R ^b )R ^c , P(O)(OR ^a )(OR ^b ), OP(O)(OR ^a )(OR ^b ), SiR ^a R ^b R ^c , C(O)R ^a , C(O) OR ^a , C(O)N(R ^a )R ^b , OC(O)R ^a , OC(O)OR ^a , OC(O)N(R ^a )R ^b , N(R ^a )C(O) R ^b , N(R ^a )C(O)OR ^b , and N(R ^a )C(O)N(R ^b )R ^c , where R ^a , R ^b , and R ^c are independently selected from H and optionally substituted C 1-3 alkyl or C _1-3 heteroalkyl, or R ³ + R ³ and/or R ⁴ + R ⁴ are independently selected from = O, = S, = NOR ¹⁸ , = C(R ¹⁸ ) R ¹⁸ and = NR ¹⁸ . R ¹⁸ and R ¹⁸ are independently selected from H and optionally substituted C _1-3 alkyl, two or more of R ² , R ² , R ³ , R ³ , R ⁴ , R ⁴ and R ¹² are optionally connected by one or more bonds to the formation of one or more optionally substituted carbocycles and/or heterocycles;

X ² is selected from O, C(R ¹⁴ )(R ¹⁴ ) and NR ¹⁴ where R ¹⁴ and R ¹⁴ have the same meaning as defined for R ⁷ and are independently selected, or R ¹⁴ and R ⁷ are absent, which results in a double bond between atoms destined to carry R ⁷ and R ¹⁴ ;

R ⁵ , R ⁵ ', R ⁶ , R ⁶ ', R ⁷ and R ⁷ ' are independently selected from H, OH, SH, NH2, N3, NO2, NO, CF3, CN, C(O)NH2, C(O )H, C(O)OH, halogen, R ^e , SR ^e , S(O)R ^e , S(O)2R ^e , S(O)OR ^e , S(O)2OR ^e , OS(O)R ^e , OS(O)2R ^e , OS(O)OR ^e , OS(O)2OR ^e , OR ^e , NHR ^e , N(R ^e )R ^f , +N(R ^e )(R ^f )Rg, P (O)(OR ^e )(OR ^f ), OP(O)(OR ^e )(OR ^f ), SiR ^e R ^f Rg, C(O)R ^e , C(O)OR ^e , C(O)N (R ^e )R ^f , OC(O)R ^e , OC(O)OR ^e , OC(O)N(R ^e )R ^f , N(R ^e )C(O)R ^f , N(R ^e ) C(O)OR ^f , N(R ^e )C(O)N(R ^f )R ^g and a water-soluble group, where R ^e , R ^f and R ^g are independently selected from H and optionally substituted (CH2CH ₂ O) _ee CH ₂ CH ₂ X ¹³ R ^e1 , C _1-15 alkyl, C _1-15 heteroαalkyl, C _3-15 cycloalkyl, C ₁₁₅ heterocycloalkyl, C _5-15 aryl or C _1-15 heteroaryl, where it is selected from 1 to 1000, X ¹³ choose from O, S

- 55 042568 and NRf ¹ , and Rf ¹ and R ^e1 are independently selected from H and ^.alkyl, one or more optional substituents in R ^e , Rf and/or R ^g ' are optionally a water-soluble group, two or more of R ^e , Rf and R ^g optionally linked by one or more bonds to form one or more optionally substituted carbocycles and/or heterocycles, or

R ⁵ + R ⁵ and/or R ⁶ + R ⁶ and/or R ⁷ + R ⁷ are independently selected from =O, =S, =NOR ^e3 , =C(R ^e3 )R ^e4 and =NR ^e3 , R ^e3 and R ^e4 is independently selected from H and optionally substituted C _1-3 alkyl or R ⁵ + R ⁶ and/or R ⁶ + R ⁷ and/or R ⁷ + R ¹⁴ resulting in a double bond between the atoms intended to carry R ⁵ + R ⁶ and/or R ⁶ + R ⁷ and/or R ⁷ + R ¹⁴ respectively, two or more of R ⁵ , R ⁵ , R ⁶ , R ⁶ , R ⁷ , R ⁷ , R ¹⁴ and R ¹⁴ optionally linked by one or more bonds to form one or more optionally substituted carbocycles and/or heterocycles;

X ¹ is selected from O, S and NR, where R is selected from H and optionally substituted C ₁ . ₈ alkyl or C ₁ . ₈ heteroalkyl and is not attached to any other substituent;

X ³ is selected from O, S, C(R ¹⁵ )R ¹⁵ ', -C(R ¹⁵ )(R ¹⁵ ')-C(R ¹⁵ '')(R ¹⁵ ''')-, -N(R ¹⁵ )-N(R ¹⁵ ')-, -C(R ¹⁵ )(R ¹⁵ ')-N(R ¹⁵ '')-, -N(R ¹⁵ '')-C(R ¹⁵ )(R ¹⁵ ') -, -C(R ¹⁵ )(R ¹⁵ ')-O-, -OC(R ¹⁵ )(R ¹⁵ ')-, -C(R ¹⁵ )(R ¹⁵ ')-S-, -SC(R ¹⁵ )(R ¹⁵ ')-, -C(R ¹⁵ )=C(R ¹⁵ ')-, =C(R ¹⁵ )-C(R ¹⁵ ')=, -N=C(R ¹⁵ ')-, = NC(R ¹⁵ ')=, -C(R ¹⁵ )=N-, =C(R ¹⁵ )-N=, -N=N-, =NN=, CR ¹⁵ , N, NR ¹⁵ , or DB1 and DB2 -X3-is -X ^3a and X ^3b -, where X ^3a is connected to X ³⁴ , there is a double bond between X ³⁴ and X ⁴ , and X ^3b is linked to X ¹¹ , where X ^3a is independently selected from H and optionally substituted ( CH2CH2O)eeCH2CH2X ¹³ R ^e1 , C1-8 alkyl or C ₁ _ ₈ heteroalkyl and not attached to any other substituent;

X ⁴ is selected from O, S, C (R ¹⁶ ) R ¹⁶ ', NR ¹⁶ , N and CR ¹⁶ ; '

X ⁵ is selected from O, S, C (R ¹⁷ )R ¹⁷ , NOR ¹⁷ and NR ¹⁷ where R ¹⁷ and R ¹⁷ are independently selected from H and optionally substituted C ₁ . ₈ alkyl or C ₁ . ₈ heteroalkyl and is not attached to any other substituent;

X ⁶ is selected from CR ¹¹ , CR ¹¹ (R ¹¹ '), N, NR ¹¹ , O and S;

X ⁷ is selected from CR ⁸ , CR ⁸ (R ⁸ '), N, NR ⁸ , O and S;

X ⁸ is selected from CR ⁹ , CR ⁹ (R ⁹ '), N, NR ⁹ , O and S;

X ⁹ is selected from CR ¹⁰ , CR ¹⁰ (R ¹⁰ '), N, NR ¹⁰ , O and S;

X ¹⁰ is selected from CR ²⁰ , CR ²⁰ (R ²⁰ '), N, NR ²⁰ , O and S;

X ¹¹ is selected from C, CR ²¹ and N or X ¹¹ -X ^3b is selected from CR ²¹ , CR ²¹ (R ²¹ '), N, NR ²¹ , O and S;

X ¹² is selected from C, CR ²² and N;

X ⁶ *, X7*, X8*, X9*, X ¹⁰ * and X ¹¹ * have the same meaning as defined for X ⁶ , X ⁷ ,

X ⁸ , X ⁹ , X ¹⁰ and X ¹¹ , respectively, and independently selected;

X ³⁴ is selected from C, CR ²³ and N;

ring atom X ¹¹ * in DB6 and DB7 is connected to ring atom A so that ring A and ring B in DB6 and DB7 are directly linked by a single bond;

a dotted double bond means that said bond may be a single bond or a non-cumulative, optionally delocalized double bond;

R ⁸ , R ^8' , R ⁹ , R ^9' , R ¹⁰ , R ^10' , R ¹¹ , R ^11' , R ¹⁵ , R 15' , R 15 ^' ' , R 15 ^{'' '} , R ¹⁶ , R ^16' , R ²⁰ , R ^20' , R ²¹ , R ^21' , R ²² and R ²³ are each independently selected from H, OH, SH, NH2, N3, NO2, NO, CF3, CN, C(O)NH2, C(O)H, C(O)OH, halogen, R ^h , SR ^h , S(O)R ^h , S(O)2R ^h , S(O)OR ^h , S(O)2OR ^h , OS( O)R ^h , OS(O)2R ^h , OS(O)OR ^h , OS(OhOR ^h , OR ^h , NHR ^h , N(R ^h )Ri, +N(R ^h )(R')R', P(O)(OR ^h )(OR'), OP(O)(OR ^h )(ORi), SiRhRiRj, C(O)R ^h , C(O)OR ^h , C(O)N(R ^h ) Ri, OC(O)R ^h , OC(O)OR ^h , OC(O)N(R ^h )Ri, N(R ^h )C(O)Ri, N(R ^h )C(O)ORi, N (R ^h )C(O)N(Ri)R' and a water-soluble group, where R ^h , Ri and Rj are independently selected from H and optionally substituted (CH2CH ₂ O) _ee CH ₂ CH ₂ X ¹³ R ^e1 , C ₁ . ₁₅ αalkyl, C _{1 .15} heteroalkyl, C _{3 .15} cycloalkyl, C _{1 .15} heterocycloalkyl, C _5-15 aryl or C _{1 .15} heteroaryl, one or more optional substituents on R ^h , Ri and/or Rj, optionally being water-soluble group, two or more R ^h , Ri and Rj optionally connected to one one or more bonds to form one or more optionally substituted carbocycles and/or heterocycles, or

R ⁸ + R ⁸ and/or R ⁹ + R ⁹ and/or R ¹⁰ + R ¹⁰ and/or R ¹¹ + R ¹¹ and/or R ¹⁵ + R ¹⁵ and/or R ¹⁵ + R ¹⁵ and/or R ¹⁶ + R ¹⁶ and/or R ²⁰ + R ²⁰ and/or R ²¹ + R ²¹ are independently selected from =O, =S, =NOR ^h1 , =C(R ^h1 )R ^h2 and =NR ^h1 , R ^h1 and R ^h2 independently selected from H and optionally substituted C ₁ . ₃ alkyl, two or more R ⁸ , R ⁸ , R ⁹ , R ⁹ , R ¹⁰ , R ¹⁰ , R ¹¹ , R ¹¹ , R ¹⁵ , R ¹⁵ , R ¹⁵ , R ¹⁵ , R ¹⁶ , R ²⁰ , R ²⁰ , R ²¹ , R ²¹ , R ²² and R ²³ are optionally joined by one or more bonds to form one or more optionally substituted carbocycles and/or heterocycles;

R ^8b and R ^9b are independently selected and have the same meaning as R ⁸ except that they cannot be combined with any other substituent;

one of R ⁴ and R ⁴ and one of R ¹⁶ and R ¹⁶ may optionally be joined by one or more bonds to form one or more optionally substituted carbocycles and/or heterocycles;

one of R ⁴ and R ⁴ and one of R ¹⁶ and R ¹⁶ may optionally be attached to one of R ² , R ² , R ³ and R ³ and one of R ⁵ and R ⁵ may optionally be connected by one or more bonds to the formation of one or more optionally substituted carbocycles and/or heterocycles;

a and b are independently selected from 0 and 1;

the DB component does not include a DAI, DA2, DAI', or DA2' component;

- 56 042568 ring B in DB1 is a heterocycle;

if X ³ in DB1 is —X ^3a and X ^3b — and ring B is aromatic, then two vicinal substituents on said ring B are connected to form an optionally substituted carbocycle or heterocycle fused to said ring B;

if X ³ in DB2 is -X ^3a and X ^3b - and ring B is aromatic, then two vicinal substituents on said ring B are connected to form an optionally substituted heterocycle fused to said ring B, an optionally substituted non-aromatic carbocycle fused to said ring B or a substituted aromatic carbocycle which is fused to said ring B and to which at least one substituent is attached which contains a hydroxy group, a primary amino group or a secondary amino group, the primary or secondary amine being not a ring atom in the aromatic ring system or not being part of an amide ;

if ring A in DB2 is a 6-membered aromatic ring, then substituents on ring B do not combine to form a ring fused to ring B;

two vicinal substituents on ring A in DB8 join to form an optionally substituted carbocycle or heterocycle fused to said ring A to form a bicyclic group to which no additional rings are attached; and the A ring in DB9, together with any rings fused to said A ring, contains at least two ring heteroatoms.

The above described linker molecules can be conjugated to a cysteine thiol group using thiol-maleimide chemistry as shown above. In some embodiments, the cytotoxic drug component of duocarmycin is a prodrug. For example, a prodrug, vc-seco-DUBA can be conjugated to a self-eliminating maleimide linker moiety via a cleavable peptide moiety.

Scheme 7

The maleimide linker component of the molecule can be conjugated to the thiol group of a cysteine residue of the VL domain and/or the VH domain and/or the constant domain of the Ab portion of the B7-H3-ADC of the invention. Subsequent proteolytic cleavage of the cleavable peptide component is followed by spontaneous elimination of the self-eliminating fragment, resulting in the release of seco-DUBA, which spontaneously rearranges to form the active drug DUBA.

Scheme 8

he is about (see Dokter, W. et al. (2014) Preclinical Profile of the HER2-Targeting ADC SYD983/SYD985: Introduction of a New Duocarmycin-Based Linker-Drug Platform, Mol. Cancer Ther. 13(11):2618 -2629).

In a preferred method for preparing B7-H3-duocarmycin drug moiety conjugates, the method of Elgersma, R.C. et al. (2014) Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985, Mol. pharmaceuticals. 12: 1813-1835 or document WO 2011/133039. Thus, the thiol-containing group of the VL or VH chain of an antibody or an anti-B7-H3 antibody fragment is conjugated to seco-DUBA or another prodrug via a maleimide linker component, a cleavable peptide component, a self-eliminating component (Scheme 9A).

- 57 042568

Scheme 9A

While the invention has been illustrated with respect to the DUBA prodrug, other prodrugs may alternatively be used, such as CC-1065, as shown in Scheme 9B.

Scheme 9B

Upon cleavage of the cleavable peptide component and elimination of the self-eliminating component, the prodrug component undergoes Winstein spirocyclization to produce the active drug (eg, DUBA from seco-DUBA as shown in Scheme 9C).

Scheme 9C

seco-DUBA is derived from the appropriate DNA alkylating and DNA binding components (e.g. 1,2,9,9a-tetrahydrocyclopropyl[c]benzo[e]indol-4-one backbone as described by Elgersma, R.C. et al. (2014 ) Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2Targeting Antibody-Drug Conjugate SYD985, Mol. Pharmaceut. (±)-N-(tert-Butoxycarbonyl)-CBI, (±)-CBI-CDPI1, and (±)-CBI-CDPI2: CC1065 Functional Agents Incorporating the Equivalent 1,2,9,9a-Tetrahydrocyclopropa[1,2 -c]benz[1,2-e]indol-4one (CBI) Left-Hand Subunit J. Am. Chem. Soc. 111:6461-6463; Boger, D. L. et al. (1992) DNA Alkylation Properties of Enhanced Functional Analogs of CC-1065 Incorporating the 1,2,9,9a-Tetrahydrocyclopropa[1,2c]benz[1,2-e]indol-4-one (CBI) Alkylation Subunit, J. Am. Chem. Soc. 114: 5487-5496).

Scheme 9D illustrates the invention, showing the synthesis of a DNA alkylating fragment for DUBA. Thus, o-tolualdehyde (1) and dimethylsuccinate (2) are reacted to obtain a mixture of acids (3a/3b) via a Stobbe condensation. Circular closure of the mixture of acids can be performed with trifluoroacetic anhydride to give the alcohol (4), which is then protected with benzyl chloride to give the benzyl ester (5). Subsequent hydrolysis of the methyl ester group gives the carboxylic

- 58 042568 acid (6), followed by Curtius rearrangement in a mixture of toluene and tert-butyl alcohol to obtain carbamate (7). Bromination with N-bromosuccinimide gives bromide (8). Bromide (8) is alkylated with (S)-glycidyl nosylate in the presence of potassium tert-butoxide to give epoxide (9). Reaction with n-butyllithium gives a mixture of the desired compound (10) and debrominated rearranged derivative (11). The yield of the target compound (10) is higher when tetrahydrofuran is used as the solvent and the reaction temperature is maintained between -25 and -20°C. Under these conditions, the desired compound (10) and the debrominated, rearranged derivative (11) are obtained in an approximate ratio of 1:1. Treatment with p-toluenesulfonic acid results in the conversion of the debrominated, rearranged derivative (11) to (7), thereby contributing to the reduction of the desired compound (10). Mesylation of the hydroxyl group in (10) followed by chloride displacement using lithium chloride gives the key intermediate (12).

Scheme 9D

Scheme 9E illustrates the invention, showing the synthesis of a DNA binding fragment for DUBA. Thus, the Chichibabin cyclization reaction is allowed to proceed between ethyl bromyruvavat (13) and 5-nitropyridine-2-amine (14), thereby obtaining a nitro compound (15). Reduction of the nitro group with zinc under acidic conditions gives the amine (16). Combination with methyloxymethyl (MOM)-protected 4-hydroxybenzoic acid (17) obtained from methyl 4-hydroxybenzoate by reaction with chloromethyl methyl ether followed by hydrolysis of the ester (see WO 2004/080979) gives ethyl ester (18), which can hydrolyze with sodium hydroxide in aqueous 1,4-dioxane to give acid (19).

Scheme 9E

seco-DUBA is then synthesized from a DNA alkylating unit (12) and a DNA binding fragment (19). The tert-butoxycarbonyl (Boc) protecting group is removed from (12) under acidic conditions to form the amine (20). EDC-mediated coupling of amine (20) and compound (19) gives protected compound (21), which is then completely deprotected in two successive steps (with Pd/C, NH4HCO2, MeOH/THF, 3h, 90%, to give (22) followed by HCl, 1,4-dioxane/water, 1 h, 95% to provide seco-DUBA (23) as its HCl salt (Scheme 9F).

Scheme 9F

Prodrugs of other drugs, such as CC-1065, can be synthesized as described, for example, in WO 2010/062171.

- 59 042568

The prodrug preferably binds to other fragments of the ADC according to scheme 9G. The maleimide linker building block was synthesized starting with a condensation reaction between (24) and 2-(2-aminoethoxy)ethanol (25) to give alcohol (26), which was then converted to reactive carbonate (27) by reaction with 4-nitrophenyl chloroformate. Compound (27) with H-valine-citrulline-PABA (28) prepared according to Dubowchik, G.M. et al. (2002) Cathepsin B-Labile Dipeptide Linkers For Lysosomal Release Of Doxorubicin From Internalizing Immunoconjugates: Model Studies Of Enzymatic Drug Release And Antigen-Specific In Vitro Anticancer Activity, Bioconjugate Chem. 13:855-869) leads to the formation of a linker (29), which is treated with bis(4nitrophenyl) carbonate to give an activated linker (30).

Scheme 9G

As shown in Scheme 9H, seco-DUBA-MOM (22) is modified for conjugation in two steps. Sequential treatment of (22) with 4-nitrophenyl chloroformate and tert-butylmethyl (2(methylamino)ethyl)carbamate (31) gives compound (32). Deprotection of Boc and MOM in (32) with trifluoroacetic acid (TFA) provided (33) as its TFA salt.

Scheme 9H

ADC was synthesized by reacting an activated linker (30) with a cyclization spacer-duocarmycin construct (33) under weakly basic conditions. Under these conditions, the self-elimination of the cyclization spacer and the formation of 3 a were suppressed (Scheme 9I).

Scheme 9I

The process generates an average of two free thiol groups per mAb, resulting in a statistical distribution of B7-H3-ADC with an average drug to antibody ratio (DAR) of about two and low amounts of high molecular weight particles and a residual unconjugated duocarmycin component.

The order of the synthetic steps may be changed as desired. Preferably the method used

- 60 042568 will be that in schemes 9A-9I, as described above.

XII. Applications of the B7-H3 binding molecules of the present invention.

The present invention encompasses compositions, including pharmaceutical compositions, containing B7-H3 binding molecules of the present invention (e.g., antibodies, bispecific antibodies, bispecific diabodies, trivalent binding molecules, B7-H3-ADC, etc.), polypeptides, derived from such molecules, polynucleotides containing sequences encoding such molecules or polypeptides, and other agents as described herein.

According to this document, B7-H3 binding molecules of the present invention, comprising anti-B7-H3-VL and/or VH domains provided herein, have the ability to bind B7-H3 present on the cell surface and induce antibody dependent cell mediated cytotoxicity (ADCC) and/or complementary dependent cytotoxicity (CDC) and/or mediate retargeted cell killing (eg, retargeted T cell cytotoxicity). Without specifying any mechanism of action, the B7-H3-ADC molecules of the present invention are internalized upon binding to B7-H3 expressed by a tumor cell and mediate killing of the tumor cell through the action of a conjugated cytotoxin.

Thus, the B7-H3 binding molecules of the present invention, comprising anti-B7H3-VL and/or VH domains, are capable of treating any disease or condition associated with B7-H3 or characterized by B7-H3 expression. As discussed above, B7-H3 is an oncoembryonic antigen expressed in numerous hematopoietic and solid tumors that is associated with high grade tumors showing less differentiated morphology and correlates with poor clinical outcomes. Thus, without limitation, the B7-H3 binding molecules of the present invention can be used to diagnose or treat cancers, in particular cancers characterized by B7-H3 expression.

Cancers that can be affected by the B7-H3 binding molecules of the present invention include cancers characterized by having a cancer cell selected from the group consisting of an adrenal tumor cell, an AIDS-associated cancer, an alveolar soft tissue sarcoma, an astrocytic tumor, an adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, B-cell neoplasia, breast cancer, carotid tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, ulcerative cell carcinoma, cancer colon, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, skeletal fibrogenesis imperfecta, fibrous bone dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germline tumors cell carcinoma, head and neck cancer, glioblastoma, hemoblastosis, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia (eg, acute myeloid leukemia), liposarcoma/lipomatous tumour, liver cancer, lymphoma, lung cancer (eg, non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma, meningioma, mesothelioma pharyngeal cancer, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer diseases, peripheral nerve sheath tumors, pheochromocytomas, pituitary tumors, prostate cancer, uveal melanoma of the posterior pole, renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcoma, skin cancer, blue-small round cell tumors of children (including neuroblastomas and rhabdomyosarcomas), sarcomas soft tissue, squamous cell carcinoma (eg, squamous cell carcinoma of the head and neck (SCCHN)), gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (eg, metastatic thyroid cancer), and uterine cancer.

In particular, the B7-H3 binding molecule of the present invention can be used to treat adrenal cancer, bladder cancer, breast cancer, colon cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute lymphocytic leukemia , acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkett's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle zone cell lymphoma, marginal zone cell lymphoma, pharyngeal cancer mesothelioma, non-Hodgkin's lymphoma, small lymphocytic lymphoma , multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal carcinoma, blue-small round cell tumors of children (including neuroblastoma and rhabdomyosarcoma), squamous cell carcinoma (eg, head and neck squamous cell carcinoma (SCCHN), cancer testicular, thyroid cancer (eg. ep, metastatic thyroid cancer) and uterine cancer.

The bispecific B7-H3 binding molecules of the present invention complement the cancer therapy provided by B7-H3 by promoting the retargeted killing of tumor cells,

- 61 042568 which express a second specificity of such molecules (eg CD2, CD3, CD8, CD16, T cell receptor (TCR), NKG2D, etc.). Such B7-H3 binding molecules are particularly useful in the treatment of cancer.

In addition to their utility in therapy, the B7-H3 binding molecules of the present invention can be detectably labeled and used for cancer diagnosis or imaging of tumors and tumor cells.

XIII. pharmaceutical compositions.

Compositions of the invention include bulk drug compositions suitable for the manufacture of pharmaceutical compositions (i.e., crude or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to an object or patient) that can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of the B7-H3 binding molecules of the present invention, or a combination of such agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of the B7-H3 binding molecules of the invention and a pharmaceutically acceptable carrier. The invention also encompasses such pharmaceutical compositions which further comprise a second therapeutic antibody (eg, a tumor-specific monoclonal antibody) that is specific for a particular cancer antigen and a pharmaceutically acceptable carrier.

In a particular embodiment, the term pharmaceutically acceptable means approved by a federal or state government regulatory agency or listed in the USP or other generally accepted pharmacopoeia for use in animals, and more particularly in humans. The term carrier refers to the diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic agent is administered. Typically, the ingredients of the compositions of the invention are either supplied separately or mixed together in unit dosage form, for example, in as a dry lyophilized powder or concentrate without water in a sealed container such as an ampoule or sachet indicating the amount of active agent Where the composition is to be administered by infusion, it may be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline. the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

The invention also relates to a pharmaceutical package or kit containing one or more containers filled with a B7-H3 binding molecule of the present invention, alone or with such a pharmaceutically acceptable carrier. In addition, one or more other prophylactic or therapeutic agents useful in the treatment of a disease may also be included in a pharmaceutical package or kit. The invention also relates to a pharmaceutical package or kit containing one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) may be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products that reflects the agency's approval of the manufacture, use, or sale for administration to humans.

The present invention provides kits that can be used in the above methods. The kit may contain any of the B7-H3 binding molecules of the present invention, including B7-H3-ADC. The kit may further contain one or more other prophylactic and/or therapeutic agents useful in the treatment of cancer in one or more containers.

XIV. Methods of administration.

Compositions of the present invention may be provided for the treatment, prevention, and amelioration of one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of a fusion protein or conjugate molecule of the invention, or a pharmaceutical composition comprising a fusion protein or conjugate molecule of the invention. invention. In a preferred aspect, such compositions are substantially pure (ie, substantially free of substances that limit its action or cause undesirable side effects). In a specific embodiment, the subject is an animal, preferably a mammal, such as a non-primate (e.g., bovine, horse, cat, canine, rodent, etc.) or a primate (e.g., a monkey, such as a cynomolgus monkey, a human, etc.). ). In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer the compositions of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or fusion protein, receptor-mediated endocytosis (see, for example, Wu et al., (1987 ) Receptor-Mediated In Vitro Gene Transformation By A Soluble

- 62 042568

DNA Carrier System, J. Biol. Chem. 262: 4429-4432), constructing a nucleic acid as part of a retroviral or other vector, etc.

Methods for administering a molecule of the invention include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (eg, intranasal and oral routes). In a specific embodiment, the B7H3 binding molecules of the present invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g. oral, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agent. The introduction can be systemic or local. Pulmonary administration may be used, for example, by means of an inhaler or nebulizer and a formulation with an aerosolizing agent; see, for example, US Pat. US No. 6019968; 5985320; 5985309; 5934272; 5874064; 5855913; 5290540; and 4880078; and publ. PCT No. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety.

The invention also contemplates that the B7-H3 binding molecule preparations of the present invention are packaged in a hermetically sealed container such as an ampoule or sachet indicating the amount of the molecule. In one embodiment, such molecules are supplied as a dry sterilized lyophilized powder or concentrate without water in a sealed container and can be reconstituted with, for example, water or saline to an appropriate concentration for administration to the subject. Preferably, the B7-H3 binding molecules of the present invention are supplied as a dry sterile lyophilized powder in a sealed container.

Lyophilized preparations of B7-H3 binding molecules of the present invention should be stored at 2 to 8°C in the original container and the molecules should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after recovery. In an alternative embodiment, such molecules are supplied in liquid form in a sealed container indicating the amount and concentration of the molecule, fusion protein, or conjugated molecule. Preferably, such B7-H3 binding molecules, if present in liquid form, are supplied in a sealed container.

The amount of such formulations of the invention that will be effective in treating, preventing, or ameliorating one or more of the symptoms associated with a disorder can be determined by standard clinical methods. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the condition, and should be determined according to the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal test systems.

As used herein, an effective amount of a pharmaceutical composition is an amount sufficient to produce beneficial or desired results, including, but not limited to, clinical results such as reduction in symptoms resulting from a disease, reduction in a symptom of an infection (e.g., viral load, fever, , pain, sepsis, etc.) or a symptom of cancer (such as proliferation, cancer cells, the presence of a tumor, tumor metastases, etc.), thereby improving the quality of life of those suffering from the disease, reducing the dose of other drugs needed for treatment disease, enhancing the effect of another drug, for example, by targeting and/or internalization, thereby delaying the progression of the disease and/or prolonging the survival of individuals.

An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to reduce the proliferation (or effect) of a viral presence, as well as to reduce and/or slow the progress of a viral disease, either directly or indirectly. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of the administration of one or more chemotherapeutic agents, and one agent may be considered to be indicated in an effective amount if, in combination with one or more other agents, the desired result can be achieved or has been achieved. While individual needs vary, it is within the skill of the art to determine the optimal ranges for effective amounts of each component.

For B7-H3 binding molecules encompassed by the invention, the dosage administered to the patient is preferably determined based on the body weight (kg) of the recipient subject. For B7-H3 binding molecules encompassed by the invention, the dosage administered to a patient is typically from about 0.01 μg/kg to about 30 mg/kg or more based on the subject's body weight.

The dosage and frequency of administration of the B7-H3 binding molecule of the present invention may

- 63 042568 be reduced or changed due to increased uptake and tissue penetration of the molecule through modifications such as, for example, lipidation.

The dosage of the B7-H3 binding molecule of the invention administered to a patient may be calculated for use as monotherapy. Alternatively, the molecule may be used in combination with other therapeutic compositions and the dosage administered to the patient is lower than when said molecules are used as monotherapy.

Pharmaceutical compositions of the invention may be administered locally to the area in need of treatment; this can be achieved, for example, without limitation, by local infusion by injection or by means of an implant, said implant being a porous, non-porous or gelatinous material, including membranes such as sialastic membranes or fibers. Preferably, when administering a molecule of the invention, care should be taken to use materials that can absorb the molecule.

Compositions of the invention can be delivered in a vesicle, in particular a liposome (See Langer (1990) New Methods Of Drug Delivery, Science 249:1527-1533); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); LopezBerestein, ibid., pp. 3 17-327).

Treatment of the subject with a therapeutically or prophylactically effective amount of the B7-H3 binding molecule of the present invention may include a single treatment or, preferably, may include a series of treatments. In a preferred example, the subject is treated with such a diabody once a week for 1 to 10 weeks, preferably 2 to 8 weeks, more preferably 3 to 7 weeks, and even more preferably about 4.5 or 6 weeks. Pharmaceutical compositions of the invention can be administered once a day with such administration once a week, twice a week, every other week, once a month, once every six weeks, once every two months, twice a year or once once a year, etc. Alternatively, the pharmaceutical compositions of the invention may be administered twice a day, such administration being once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once a year, etc. Alternatively, the pharmaceutical compositions of the invention may be administered three times a day, with such administration once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once a year, etc. It will also be understood that the effective dose of molecules used for treatment may increase or decrease during a particular treatment.

XV. Embodiments of the invention.

The invention particularly relates to the following embodiments (EA and EB).

E _a 1. Anti-B7-H3 antibody drug conjugate (B7-H3-ADC), which includes the formula

Ab-(LM) _m -(D) _n where Ab is an antibody that binds to B7-H3, which includes a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL) or is B7-H3 -binding fragment;

D is a cytotoxic drug moiety;

LM is a bond or linker molecule that covalently links Ab and D;

m is an integer from 0 to n and denotes the number of linker molecules in B7-H3ADC;

n is an integer from 1 to 10 and denotes the amount of cytotoxic drug moieties covalently linked to the B7-H3-ADC molecule.

EA2. B7-H3-ADC from E _a 1, in which:

(A) (i) said humanized VL domain includes the amino acid sequence of SEQ ID NO:99 and (ii) said humanized VH domain includes the amino acid sequence of SEQ ID NO:104; or (B) (i) said humanized VL domain includes the amino acid sequence of SEQ ID NO:20, and (ii) said humanized VH domain includes the amino acid sequence of SEQ ID NO:21; or (C) (i) said humanized VL domain includes the amino acid sequence of SEQ ID NO:30 and (ii) said humanized VH domain includes the amino acid sequence of SEQ ID NO:31.

E _a 3. The B7-H3-ADC of E _a 1, wherein said humanized VL domain comprises the amino acid sequence of SEQ ID NO:99 and said humanized VH domain includes the amino acid sequence of SEQ ID NO:104.

E _a 4. The B7-H3-ADC of E _a 1, wherein said humanized VL domain comprises the amino acid sequence of SEQ ID NO:20 and said humanized VH domain comprises the amino acid sequence of SEQ ID NO:21.

EA5. B7-H3-ADC by EA1, wherein said humanized VL domain comprises the amino acid sequence of SEQ ID NO:30 and said humanized VH domain includes the amino acid sequence of SEQ ID NO:31.

EA6. B7-H3-ADC according to any of EA1-EA5, wherein Ab is an antibody.

EA7. B7-H3-ADC according to any of EA1-EA5, wherein Ab is an antigen-binding fragment of an antibody.

EA8. B7-H3-ADC according to any one of EA1-EA7, wherein the B7-H3-ADC contains a human IgG Fc domain.

EA9. B7-H3-ADC of EA8, wherein the human IgG is human IgG1, IgG2, IgG3 or IgG4.

E _A 10. B7-H3-ADC to E _A 8 or E _A 9, wherein the Fc domain is a variant Fc domain that includes:

(a) one or more amino acid modifications that reduce the affinity of the variant Fc domain for FcyR; and/or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc domain.

E _A 11. B7-H3-ADC according to E _A 10, in which modifications that reduce the affinity of the variant Fc domain for FcyR include substitution L234A; L235A; or L234A and L235A, where the indicated numbering corresponds to the EU index, as in Kabat.

EA12. B7-H3-ADC according to EA10 or EA11, in which modifications that increase the serum half-life of the variant Fc domain include the substitution M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, where the indicated numbering corresponds to the EU index, as in Kabat.

E _A 13. B7-H3-ADC according to any one of E _A 1-E _A 12, wherein at least one of said LM is a linker molecule.

E _A 14. B7-H3-ADC according to E _A 13, in which the linker molecule LM is a peptide linker.

E _A 15. B7-H3-ADC according to E _A 13, in which the linker molecule LM is a cleavable linker.

E _A 16. B7-H3-ADC according to E _A 15, in which the specified molecule includes the formula

Ab - [V-(W)k-(X)iA] - D where V is the indicated cleavable linker molecule LM, (W) _k -(X) _l -A is an elongated self-eliminating spacer system that is self-eliminated by a l, (4+2n)-eliminations;

W and X are each a spacer of a cascade of electrons a l,(4+2n) being the same or different;

A is either a spacer group of formula (Y)m, where Y is an electron cascade spacer a l,(4+2n), or a group of formula U which is an elimination cyclization spacer;

k, l and m are independently an integer from 0 (inclusive) to 5 (inclusive);

n is an integer from 0 (inclusive) to 10 (inclusive);

provided that when A is (Y) _m : then k + l + m > 1, and if k + l + m = 1, then n >1;

when A equals U: then k + 1 > 1.

W, X and Y are independently selected from compounds having the formula

or formula

where Q represents -R ⁵ C=CR ⁶ -, S, O, NR ⁵ , -R ⁵ C=N- or -N=CR ⁵ -;

P is NR ⁷ , O or S;

a, b and c are independently an integer from 0 (inclusive) to 5 (inclusive);

I, F and G are independently selected from compounds having the formula

Ir ^s _R g _______ \=/ OR or --- ^-

Z / \ where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ independently represent H, C1-6 alkyl, C3-20 heterocyclyl, C 5-20 aryl, C 1-6 alkoxy , hydroxy (OH), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx ¹ Rx ² ), nitro (NO ₂ ), halogen, CF ₃ , CN, CONH ₂ , SO ₂ Me, CONHMe, cyclic C _1-5 alkylamino,

- 65 042568 imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SR _x ), tetrazole, carboxy (COOH), carboxylate (COOR _x ), sulfoxy (S(=O) ₂ OH), sulfonate ( S(=O)2OR _x ), sulfonyl (S(=O)2R _x ), sulfoxide (S(=O)OH), sulfinate (S(=O)OR _x ), sulfinyl (S(=O)R _x ), phosphonooxy (OP(=O)(OH) ₂ ) and phosphate (OP(=O)(OR _x ) ₂ ) _x , R _x 1 and R _x ² are independently selected from C _1-6 alkyl group, C _{3- 20} heterocyclyl group or C _5-20 aryl group, two or more substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ or R ⁹ are optionally bonded to each other to form one or several aliphatic or aromatic ring structures;

U is selected from compounds having the formula

where a, b and c are independently chosen as the integer 0 or 1; provided that a + b + c = 2 or 3;

R ¹ and/or R ² are independently H, C1-6 alkyl, said alkyl being optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF3, CN, CONH2, SO2Me, CONHMe, cyclic C _1-5 alkylamino, imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (CP _x ), tetrazole, carboxy (COOH), carboxylate (COOR _x ), sulfoxy (S(=O) ₂ OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(=O) ₂ R _x ), sulfoxy (S(=O)OH), sulfinate (S(=O)OR _x ), sulfinyl (S(=O)R _x ), phosphonoxy (OP(=O)(OH) ₂ ), and phosphate (OP(= O)(OR _x ) ₂ ), where R _x , R _x 1 and R _x ² are selected from C _1-6 alkyl group, C _3-20 heterocyclic group or C _5-20 aryl group; And

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently H, C1-6alkyl, C3-20heterocyclyl, C5-20aryl, C1-6alkoxy, hydroxy (OH), amino (NH2), monosubstituted amino (NRxH), disubstituted amino (NRx1Rx ² ), nitro (NO2), halogen, CF3, CN, CONH2, SO2Me, CONHMe, cyclic C _1-5 alkylamino, imidazolyl, C _1-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SR _x ), tetrazole, carboxy (COOH), carboxylate (COORx), sulfoxy (S(=O) ₂ OH), sulfonate (S(=O) ₂ OR _x ), sulfonyl (S(=O) ₂ R _x ), sulfoxide (S(=O)OH), sulfinate (S(=O)OR _x ), sulfinyl (S(=O)R _x ), phosphonooxy (OP(=O)(OH) ₂ ) and phosphate ( OP(=O)(OR _x ) ₂ ), where R _x , R _x 1 and R _x ² are selected from C _1-6 alkyl group, C _3-20 heterocyclic group or C _5-20 aryl group and two or more substituents R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , or R ⁸ are optionally bonded to each other to form one or more aliphatic or aromatic ring structures.

E _a 17. B7-H3-ADC according to EA16, wherein the LM linker molecule includes:

(1) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl;

(2) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl;

(3) p-ammocinnamyloxycarbonyl;

(4) p-aminocinnamyloxycarbonyl-p-aminobenzyloxycarbonyl;

(5) p-aminobenzyloxycarbonyl-p-aminocinnamyloxycarbonyl;

(6) p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl;

(7) p-aminophenylpentadienyloxycarbonyl;

(8) p-aminophenylpentadienyloxycarbonyl-p-arninocinnamyloxycarbonyl;

(9) p-aminophenylpentadienyloxycarbonylpaminobenzyloxycarbonyl;

(10) p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyloxycarbonyl;

(11) p-aminobenzyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(12) p-aminocinnamyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(13) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(14) p-aminoindynamicoxycarbonyl-p-aminobenzyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(15) p-aminobenzyloxycarbonyl-p-arninocinnamyloxycarbonyl (methylamino)ethyl(methylamino)carbonyl;

(16) p-aminocinnamyloxycarbonyl-p-aminocinnamyloxycarbonyl(methylamino)ethyl(methylamino)carbonyl;

(17) p-aminobenzyloxycarbonyl-p-aminobenzyl;

(18) p-aminobenzyloxycarbonyl-p-aminobenzyloxycarbonyl-p-aminobenzyl;

(19) p-aminocinnamyl;

(20) p-aminocinnanyloxycarbonyl-p-aminobenzyl;

(21) p-aminobenzyloxycarbonyl-p-aminocinnamyl;

(22) p-amino-cinnamyloxycarbonyl-p-aminocinnamyl;

(23) p-aminophenylpentadienyl;

(24) p-aminophenylpentadienyloxycarbonyl-p-aminocinnamyl;

(25) p-aminophenylpentadienyloxycarbonyl-p-aminobenzyl or

- 66 042568 (26) p-aminophenylpentadienyloxycarbonyl-p-aminophenylpentadienyl.

EA18. A B7-H3-ADC according to any of EA13-EA17, wherein said linker molecule LM is conjugated to an amino acid side chain in the Ab polypeptide chain and links said Ab to said cytotoxic drug moiety D.

EA19. B7-H3-ADC according to any one of EA1-EA18, wherein said cytotoxic drug component D includes a cytotoxin, radioisotope, immunomodulator, cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, DNA, RNA , miRNA RNAi, miRNA, photoactive therapeutic agent, anti-angiogenic agent, pro-apoptotic agent, peptide, lipid, carbohydrate, chelating agent, or combinations thereof.

EA20. B7-H3-ADC according to EA19, wherein said cytotoxic drug component D comprises a cytotoxin and is selected from the group consisting of tubulisin, auristatin, maytansinoid, calicheamicin, pyrrolobenzodiazepine, and duocarmycin.

EA21. The B7-H3-ADC of EA19, wherein said cytotoxic drug moiety D comprises the cytotoxin tubulisin and is selected from the group consisting of tubulisin A, tubulisin B, tubulisin C, and tubulisin D.

EA22. B7-H3-ADC according to EA19, wherein said cytotoxic drug component D comprises auristatin cytotoxin and is selected from the group consisting of MMAE (N-methylvaline-valindolaisoleuin-dolaproin-norephedrine) and MMAF (N-methylvaline-valine-valine-dolaisoleuin-dolaproinphenylalanine) .

EA23. B7-H3-ADC according to EA19, wherein said cytotoxic drug moiety D comprises a maytansinoid cytotoxin and is selected from the group consisting of maytansine, DM1 and DM4.

EA24. B7-H3-ADC according to EA19, wherein said cytotoxic drug moiety D comprises the cytotoxin calicheamicin and is selected from the group consisting of calicheamycin G1, calicheamicin e1Br, calicheamycin y1Br, calicheamycin a2I, calicheamycin a3I, calicheamycin βΠ, calicheamycin γΠ, and calicheamycin A1I.

EA25. B7-H3-ADC according to EA19, wherein said cytotoxic drug moiety D comprises pyrrolobenzodiazepine cytotoxin and is selected from the group consisting of vadastuximab thalirin, SJG-136, SG2000, SG2285 and SG2274.

EA26. B7-H3-ADC according to EA19, wherein said cytotoxic drug component D comprises the cytotoxin duocarmycin and is selected from the group consisting of duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC1065, adoselesin, bizelesin, carzelesin (U-80244) and spiro-duocarmycin (DUBA).

E _A 27. B7-H3-ADC according to any one of E _A 1-E _A 26, wherein the linker molecule LM is covalently linked to said Ab by reduced interchain disulfides.

EA28. A pharmaceutical composition that includes an effective amount of B7-H3-ADC according to any of EA1-EA27 and a pharmaceutically acceptable carrier, excipient or diluent.

EA29. The use of B7-H3-ADC according to any of EA1-EA27 or pharmaceutical composition EA28 in the treatment of a disease or condition associated with B7-H3 expression or characterized by B7-H3 expression.

EA30. The use of EA29 wherein said disease or condition associated with or characterized by B7-H3 expression is cancer.

EA31. The use of EA30, wherein said cancer is characterized by the presence of a cancer cell selected from the group consisting of an adrenal tumor cell, AIDS-associated cancer, soft tissue alveolar sarcoma, astrocytic tumor, adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, B-cell neoplasia, breast cancer, carotid tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, ulcerative cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumors, ependymomas, Ewing tumors, extraskeletal myxoid chondrosarcoma, bone fibrogenesis imperfecta, fibrous bone dysplasia, cancer of the gallbladder or bile duct, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, glioblastoma, hemoblastosis, hepatocellular carcinoma carcinomas, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (eg, acute myeloid leukemia), liposarcoma/lipoma malignant tumor, liver cancer, lymphoma, lung cancer (eg, non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma, meningiomas , mesothelioma pharyngeal cancer, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcoma, skin cancer, blue-small round cell tumors of children (including neuroblastomas and rhabdomyosarcomas), soft tissue sarcomas, squamous cell carcinoma (eg, squamous cell th head and neck cancer (SCCHN)), gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (eg, metastatic thyroid cancer), and uterine cancer.

EA31. Use of EA30, wherein said cancer is selected from the group consisting of adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkett's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle zone cell lymphoma, marginal zone cell lymphoma, pharyngeal cancer mesothelioma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal carcinoma, blue-small round cell tumors of children (including neuroblastoma and rhabdomyosarcoma), squamous cell cancer (eg, head and neck squamous cell carcinoma (SCCHN), testicular cancer, thyroid cancer ( e.g. metastatic thyroid cancer) and cancer m atki.

EB1. A B7-H3 binding molecule that includes a light chain variable domain (VL), which includes a CDRL1 domain, a CDRL2 domain, and a CDRL3 domain, and a heavy chain variable domain (VH), which includes a CDRH1 domain, a CDRH2 domain, and a CDRH3 domain, where:

(1) the specified CDRH1 domain includes the amino acid sequence of SEQ ID NO:27;

(2) said CDRH2 domain includes the amino acid sequence of SEQ ID NO:28; and (3) said CDRH3 domain includes the amino acid sequence of SEQ ID NO:29.

EB2. An EB1 B7-H3 binding molecule that includes said VL domain that includes a CDRL1 domain, a CDRL2 domain, and a CDRL3 domain, and said VH domain that includes a CDRH1 domain, a CDRH2 domain, and a CDRH3 domain, where:

(1) the specified CDRL1 domain includes the amino acid sequence of SEQ ID NO:23;

(2) said CDRL2 domain includes the amino acid sequence of SEQ ID NO:24; and (3) said CDRL3 domain includes the amino acid sequence of SEQ ID NO:25.

EB3. A B7-H3 binding EB1 molecule that includes said VL domain, which includes a CDRL1 domain, a CDRL2 domain, and a CDRL3 domain, and said VH domain, which includes a CDRH1 domain, a CDRH2 domain, and a CDRH3 domain, said domains being selected from the group consisting of:

(1) CDRH1 domain, includes the amino acid sequence of SEQ ID NO:27;

(2) CDRH2 domain, includes the amino acid sequence of SEQ ID NO:28;

(3) CDRH3 domain, includes the amino acid sequence of SEQ ID NO:29;

(4) CDRL1 domain, includes the amino acid sequence of SEQ ID NO:23;

(5) CDRL2 domain, includes the amino acid sequence of SEQ ID NO:24; and (6) CDRL3 domain, includes the amino acid sequence of SEQ ID NO:25. EB4. B7-H3 binding molecule of any of EB1-EB3, wherein said VH domain comprises the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:31.

E _B 5. A B7-H3 binding molecule of any of EB1-EB4, wherein said VL domain comprises the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:30.

E _B 6. A B7-H3 binding molecule that includes a VL domain and a VH domain, wherein said VL domain includes the amino acid sequence of SEQ ID NO:20.

E _B 7. A B7-H3 binding molecule that includes a VL domain and a VH domain, wherein said VH domain includes the amino acid sequence of SEQ ID NO:21.

E _B 8. A B7-H3 binding molecule that includes a VL domain and a VH domain, wherein said VL domain contains the amino acid of SEQ ID NO:20 and said VH domain includes the amino acid sequence of SEQ ID NO:21.

E _B 9. A B7-H3 binding molecule of any of EB1-EB8, wherein said molecule is an antibody or antigen-binding fragment thereof.

EB10. B7-H3 binding molecule of any of EB1-EB8, wherein said molecule is:

(a) a bispecific antibody; or (b) a diabody, said diabody being a covalently linked complex that contains two, three, four or five polypeptide chains; or (c) a trivalent binding molecule, wherein the trivalent binding molecule is a covalently linked complex that contains three, four, five or more polypeptide chains.

EB11. A B7-H3 binding molecule of any of EB1-EB10, wherein said molecule includes an Fc domain.

EB12. An EB10 B7-H3 binding molecule, wherein said molecule is a diabody and includes an albumin binding domain (ABD).

EB13. EB11 B7-H3 binding molecule, where said Fc domain is a variant domain

- 68 042568

Fc which includes:

(a) one or more amino acid modifications that reduce the affinity of the variant Fc domain for FcyR; and/or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc domain.

EB14. B7-H3-binding molecule EB13, where these modifications, which reduce the affinity of the variant Fc domain for FcyR, include substitution L234A; L235A; or L234A and L235A, where the indicated numbering corresponds to the EU index, as in Kabat.

EB15. A B7-H3 binding molecule of any of EB13 or EB14, wherein said modifications that improve the serum half-life of the variant Fc domain include substitution M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E or K288D and H435K, where the indicated numbering corresponds to the EU index, as in Kabat.

EB16. A B7-H3 binding molecule of any of EB1-EB15, wherein said molecule is bispecific and contains two epitope-binding sites capable of immunospecific binding to a B7-H3 epitope and two epitope-binding sites capable of immunospecific binding to an epitope of a molecule present on the surface of an effector cell.

EB17. A B7-H3 binding molecule of any of EB1-EB15, wherein said molecule is bispecific and contains one epitope-binding site capable of immunospecific binding to a B7-H3 epitope and one epitope-binding site capable of immunospecific binding to an epitope of the molecule present on the surface of the effector cell .

EB18. A B7-H3 binding molecule of any of EB1-EB15, wherein said molecule is trispecific and includes:

(a) one epitope-binding site capable of immunospecific binding to a B7-H3 epitope;

(b) one epitope-binding site capable of immunospecific binding to the epitope of the first molecule present on the surface of the effector cell; and (c) one epitope-binding site capable of immunospecific binding to an epitope of a second molecule present on the surface of the effector cell.

EB19. A B7-H3 binding molecule of any one of EB1-EB8, wherein said molecule is capable of simultaneously binding to B7-H3 and a molecule present on the surface of the effector cell.

EB20. A B7-H3 binding molecule of any of EB16-EB18, wherein said molecule present on the surface of the effector cell is CD2, CD3, CD8, TCR, or NKG2D.

EB21. A B7-H3 binding molecule of any of EB16-EB20, wherein the effector cell is a cytotoxic T cell or a natural killer (NK) cell.

EB22. The B7-H3 binding molecule of any of EB16-EB21, wherein said molecule present on the surface of the effector cell is CD3.

EB23. An EB18 B7-H3 binding molecule, wherein said first molecule present on the surface of the effector cell is CD3 and said second molecule present on the surface of the effector cell is CD8.

EB24. A B7-H3 binding molecule of any of EB16-EB23, wherein said molecule mediates coordinated binding of a B7-H3 expressing cell and a cytotoxic T cell.

EB25. A pharmaceutical composition that comprises an effective amount of a B7-H3 binding molecule of any of E _B 1 -E _B 24 and a pharmaceutically acceptable carrier, excipient or diluent.

EB26. The use of a B7-H3 binding molecule of any of EB1-EB24 or an EB26 pharmaceutical composition in the treatment of a disease or condition associated with B7-H3 expression or characterized by B7-H3 expression.

EB27. Use of EB26, wherein said disease or condition associated with B7-H3 expression or characterized by B7-H3 expression is cancer.

EB28. The use of EB27, wherein said cancer is characterized by the presence of a cancer cell selected from the group consisting of an adrenal tumor cell, AIDS-associated cancer, soft tissue alveolar sarcoma, astrocytic tumor, adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, B-cell neoplasia, breast cancer, carotid tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, ulcerative cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumors, ependymomas, Ewing tumors, extraskeletal myxoid chondrosarcoma, bone fibrogenesis imperfecta, fibrous bone dysplasia, cancer of the gallbladder or bile duct, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, glioblastoma, hemoblastosis, hepatocellular carcinoma cynomas, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (eg, acute myeloid leukemia), liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer (eg, non-small cell lung cancer (NSCLC)), medulloblastoma , melanomas, meningiomas, mesothelioma of pharyngeal cancer, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma , pituitary tumors, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcoma, skin cancer, blue-small round cell tumors of children (including neuroblastomas and rhabdomyosarcomas), soft tissue sarcomas, squamous cell carcinoma (eg, squamous cell th head and neck cancer (SCCHN)), gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (eg, metastatic thyroid cancer), and uterine cancer.

EB29. The use of EB27, wherein said cancer is selected from the group consisting of adrenal cancer, bladder cancer, breast cancer, colon cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkett's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle zone cell lymphoma, marginal zone cell lymphoma, pharyngeal cancer mesothelioma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, multiple myeloma, melanoma , ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, blue-small round cell tumors of children (including neuroblastoma and rhabdomyosarcoma), squamous cell cancer (eg, head and neck squamous cell carcinoma (SCCHN), testicular cancer, thyroid cancer (e.g. metastatic thyroid cancer) glands) and uterine cancer.

Examples

Now that the invention as a whole has been described, it will become more clear with the help of the following examples. The following examples illustrate various ways of compositions in diagnostic or therapeutic methods of the invention. The examples are intended to illustrate, but in no way limit the scope of the invention.

Example 1 Preparation, humanization and characterization of anti-B7-H3 antibodies.

Monoclonal antibodies were generated by immunizing mice with viable human fetal progenitor cells or tumor initiating/cancer stem-like cells (CSLC) as previously described (Loo et al. (2007) The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo Mol Cancer Ther; 6: 856-65). Screening of IHC with tumor-specific mAbs identified a panel of anti-B7-H3 (CD276) reactive mAbs with highly differentiated binding in tumor tissue relative to normal tissue. The subset of anti-B7-H3 antibodies that were efficiently internalized were identified using the internalization assay performed in the 5-day assay using saporin-conjugated anti-mouse Fab at 1:1 or 10:1 Fab-ZAP: according to with manufacturer's protocol (Advanced Targeting Systems). As shown in FIG. 7, a number of anti-B7-H3 antibodies, including anti-B7-H3 antibodies designated mAb-C and mAb-D, were effectively internalized.

The mouse anti-B7-H3 mAbs described above: mAb-B, mAb-C, and mAb-D are used to form humanized VL and VH domains in which the _L and CDRH of their domains are fused to human framework domains. The humanized VH and VL domains are then used to generate humanized light chains having the kappa light chain constant region (i.e., SEQ ID NO:1) and an IgG1 CH1 domain, a hinge domain, and an Fc domain (i.e., SEQ ID NO: 3, 7, 12). The humanized antibodies were designated hmAb-B, hmAb-C and hmAb-D.

The amino acid sequences of the humanized VL and VH domains are shown above. It should be noted that hmAb-B CDRs can be modified to provide alternative humanized VL and VH domains as described above. The amino acid sequence of all humanized hmAb-C and hmAb-D light and heavy chains is shown above.

The binding kinetics of the humanized antibodies were examined using a Biacore assay in which a soluble human or cynomolgus monkey B7-H3 (4Ig) His-tagged fusion protein (shB7H3-His or scB7-H3-His, respectively) was passed through immobilized antibodies. Briefly, each humanized antibody was captured in immobilized goat anti-human Fc Fab ₂ and incubated with shB7-H3-His or scB7-H3-his (6.25-100 nM) and binding kinetics determined by Biacore assay. Calculated k _a , kd and K _D from these studies using the approximation of bivalent binding are presented in table. 6. The results show that the humanized antibodies bind human and cynomolgus B7-H3 in a range of affinities.

- 70 042568

Tissue cross-reactivity of the humanized antibodies was examined by immunohistochemistry (IHC). In table. 7 shows the results of several IHC studies performed on normal human tissues, human tumor tissues, human cancer cell lines, and CHO cell lines expressing or not expressing B7-H3 using humanized antibodies against

B7-H3 at the indicated antibody concentrations. The criteria for evaluating these studies are given in Table. 8.

Table 6

Human Javanese macaque Antibody K _a (xlO ⁴ ) Kd(xlO- ⁴ ) _KD (hM) K _a (x10 ⁴ ) K _d (xio- ⁴ ) _KD (hM) hmAb-B 11.0 0.12 o,n 7.1 2.9 4.1 hmAb-C 16 34 21.3 6.4 31 48.4 hmAb-D 3.4 22 62.9 1.35 77 592.3

Table 7

Textile Sample hmAb-B 0.313 µg/ml hmAb-C 0.625 µg/ml hmAb-D 2.5 µg/ml Colon MG06CHTN-94F epi 2+ (c) episodic lamina propria 1+ cells (c) very rare Liver ILS11103A hepatocytes 3+ (t) from episodic to frequent hepatocytes 2-3 + (m) rare to episodic Hepatocytes 1+ (r) rare Bud ILS11119D epi 1+ (c) rare Pancreas TLS10266 fibril 3+ (c) rare endo 1+ (s, w) very rare Lung MG06CHTN-85-A- 2 pneumocytes 1+ (c) episodic Heart MG06CHTN-76B endo 2+ (c) rare to episodic endo 1+ (c) very rare Leather MG03- St.Agn-50B epi 2+ (c) episodic squamous epi 1+ (s, t) rare adrenal MG04St.Agn-22B-A 4+ (m, s) frequent epi 3+ (m, s), episodic to frequent epi 2+ (s, t) rare to episodic SA of the head and neck (Squamous cell) VNM00340- D03 2 2 1 VNM00302- D01 3 2 1 ILS7068-D04 h 2 1 ILS2073-D01 3 3 2 lung SA (NSCLC) ILS7115-C 3 2 1 ILS7253-C 2 1 1 TLS2153-G 3 2 1 ILS-11149-C 2 1(BV) 1 Hs700T ABC= 2.1е6 91812 4+ (s, w) frequent 4+ (m > s) frequent 2-3+ (t) NCI-H1703 ABC = 8.1e5 033115-1 3-4 + (c) frequent 2-3 + (s, w), episodic to frequent ± CHO+B7H3C13 ABC = 4.9e6 32113 2+ (c) frequent 3-4 + (s, w) frequent 3+ (t, s) CHO + V7НЗС12 ABC = 2.2 e5 31813 3+ (c) frequent 1-2 + (c) rare to episodic ± CHO - B7-NC 060414-2

BV:

c: cytoplasm w: membrane epi: epithelium T: tumor blood vessel

Table 8

Criteria for scoring normal tissue: Tumor scoring criteria: - negative 0 . _ no staining (negative): ± ambiguous 1-100% specifically stained cells with , _z _ staining intensity 1+ or 1-20% 1 (weak): , ^g specifically stained cells with staining intensity 2+ 1+ weak 2+ moderate staining intensity 2+ in 21-79% _ _z _ of specifically stained cells or 2 (moderate): ^rr _ . _n0/ staining intensity 3+ in 1-49% of specifically stained cells 3+ strong 4+ very strong staining intensity 2+ in 80-100% _ _z _ specifically stained cells or 3 (strong): ^{t g} _ _{n0 /} ^{v 7} staining intensity 3+ in > 50% of specifically stained cells.

These results show that all humanized antibodies exhibit binding to numerous B7-H3 positive tumor cells. hmAb-B exhibits the highest tumor reactivity under the conditions tested, but also exhibits normal tissue reactivity to hepatocytes in the liver and adrenal tissue. hmAb-C shows some reduced reactivity in tumor compared to hmAb-B, but also shows significantly less reactivity with normal liver hepatocytes as well as reactivity on less independent samples. hmAb-D has a general reduced reactivity to tumor and normal tissues. The antibodies show comparable cross-reactivity with cynomolgus monkey tissues, although hmAb-D binds at a lower intensity in these IHC studies. To minimize target toxicity, hmAb-C and hmAb-D may be preferred for making the B7-H3-ADC molecules of the invention.

Example 2 Preparation of B7-H3-ADC.

The mouse anti-B7-H3 mAbs described above: mAb-A, mAb-B, mAb-C, and mAb-D were used to generate chimeric antibodies in which the VL domain of such antibodies was combined with a human kappa light chain constant region (SEQ ID NO: 1) and in which the VH domain of such antibodies is combined with the CH1 Hinge-CH2-CH3 constant region from human IgG1 (SEQ ID NOS: 3, 7 and 12, respectively). Chimerized antibodies (chmAb-A, chmAb-B, chmAb-C, and chmAb-D) were converted to B7-H3-ADC by cysteine conjugation in its B7-H3 binding domain with a cleavable linker-71 042568 payload auristatin E vc- MMAE (Concortis Biosystems) as discussed above.

Example 3 B7-H3-ADCs demonstrate effective in vitro activity.

In order to demonstrate the antitumor activity of the B7-H3-ADC of the present invention, the above-described B7-H3-ADC (MMAE) was incubated at concentrations of 1 to 100,000 pM with B7-H3 expressing JIMT-1 breast cancer cells, breast cancer cells MDA-MB-468, A375.52 melanoma cells, Calu-6 non-small cell lung cancer cells, NCI-H1703 non-small cell lung cancer cells, NCI-H1975 non-small cell lung cancer cells, PA-1 ovarian cancer cells, Hs700T pancreatic cancer cells, cells prostate cancer DU145 or B7-H3-negative Raji B-cell lymphoma cells. Cytotoxicity in vitro was quantified after 7 days. Briefly, B7-H3-ADC and controls were diluted and placed in microtiter plates, 5000 cells were added to each well and incubated at 37°C for 4-7 days. Alamar blue reagent (eg, BioRad/ThermoFisher/Invitrogen) was added to the plates and data was read according to the manufacturer's protocol. The number of antibody binding sites present in these cells was determined using the Bangs QFACS™ kit.

Cytotoxicity curves from this study are presented in FIG. 8A-8J. The values of IC ₅₀ were determined and are shown in table. 9. The results of these studies show that each of the internalized anti-B7-H3 antibodies tested exhibited dose-dependent cytotoxicity in vitro against B7-H3 expressing tumor cells. The antibodies showed a range of activity. The relative activity in these assays was: chmAb-C > chmAb-B > chmAb-D > chmAb-A.

Table 9

B7-HZADC cell line Mammary cancer Melanoma Non-small cell lung cancer ovarian cancer Pancreas cancer prostate cancer lmt- 1 MDAMB468 A375.52 Calu- NCI111703 NCI111975 RA-1 Hs700T DU145 Antibody binding sites per cell (x 10') And | 4.2 | 7.5 | 8.5 | 8.1 4.8 |b,1 |21 |2,4 1CDU( pivij chmAb- A B7- NZ ADC 9100 8095 703 995 1517 26976 8326 607 20153 chmAb- In B7- NC ADC 221 352 153 59 90 31 555 159 3770 chmAb- From B7- NZ ADC 124 201 267 thirty 43 16 409 109 465 chmAb- D B7- NC ADC 735 1383 887 171 219 162 1795 303 2587

Example 4 B7-H3-ADC shows potent activity in vivo.

To further demonstrate the antitumor activity of B7-H3-ADC of the present invention, the chmAb-B B7-H3-ADC, chmAb-C B7-H3-ADC and/or chmAb-D B7-H3-ADC (MMAE) molecules described above were evaluated for in vivo toxicity in a CD1 nude mouse model using various tumor cell lines. Briefly, ~5x10 ⁶ tumor cells (1:1 suspended in medium and MATRIGEL®) were implanted subcutaneously in the flank of nude CD1 mice (Charles River Laboratories). When the tumors reached a volume of approximately 150 mm ³ , mice were randomized and B7H3-ADC or vehicle control was injected intraperitoneally. In these studies, a single dose of B7-H3-ADC or vehicle control (qdx1) was administered. Tumors were measured twice a week by orthogonal measurements with electronic calipers, with tumor volumes calculated as: (length x width x height)/2. Tumor volume was determined (relative to control) (T/C).

Finding that the tumor volume of the treated animals decreased to <5 mm ³ during the study period was considered a complete response (CR).

In vivo activity against MDA-MB-468 breast cancer tumor cells.

The results of this study in relation to tumor cells of breast cancer MDAMB-468 are presented in table. 10 and in FIG. 9 and show the responsiveness of MDA-MB468 tumor cells.

Table 10

Treatment (Starting dose on day 30) Dose (mg/kg) T/C CR Answer chmAb-B B7-NC ADC 10 4 6/7 high activity chmAb-C B7-NC ADC 10 20 4/7 high activity chmAb-D B7-H3 ADC 10 8 1/7 high activity

In vivo activity against NCI-H1703 non-small cell lung cancer cells.

The results of this study on subcutaneously implanted NKI-H1703 non-small cell lung cancer cells are shown in Table 1. 11 and in FIG. 10A-10C and show the responsiveness of NCI-H1703 tumor cells.

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Table 11

Treatment (Starting dose on day 52) Dose (mg/kg) t/s CR Answer chmAb-B B7-H3-ADC 10 28 5/7 highly active 3 22 3/7 highly active 1 74 0/7 Active chmAb-C B7-H3-ADC 10 0 6/7 highly active 3 eleven 5/7 highly active 1 70 0/7 Active chmAb-D B7-H3-ADC 10 32 5/7 highly active 3 4 6/7 highly active 1 76 0/7 Active

In vivo activity against PA-1 ovarian cancer tumor cells.

The results of this study on subcutaneously implanted ovarian cancer cells RA-1 are presented in table. 12 and in FIG. 11A-11C and show the responsiveness of PA-1 tumor cells.

Table 12

Treatment (Starting dose on day 42) Dose (mg/kg) t/s CR Answer chmAb-B B7-H3-ADC 10 0 6/7 highly active 3 65 0/7 Active 1 105 0/7 not active chmAb-C B7-H3-ADC 10 37 3/7 highly active 3 76 1/7 Active 1 93 0/7 not active chmAb-D B7-H3-ADC 10 AND 7/7 highly active 3 57 1/7 active agent 1 FROM 0/7 not active

In vivo activity against Calu-6 non-small cell lung cancer cells.

The results of this study on subcutaneously implanted tumor cells of non-small cell lung cancer Calu-6 are presented in table. 13 and in FIG. 12A-12C and show the responsiveness of Calu-6 tumor cells.

Table 13

Treatment (Starting dose on day 20) Dose (mg/kg) t/s CR Answer chmAb-B B7-H3-ADC 10 15 3/7 highly active 3 35 0/7 Active 1 64 0/7 Active chmAb-C B7-H3-ADC 10 1 3/7 highly active 3 87 0/7 not active 1 68 0/7 Active chmAb-D B7-H3-ADC 10 39 2/7 highly active 3 43 0/7 Active 1 54 0/7 Active

In vivo activity against A375.S2 melanoma tumor cells.

The results of this study on subcutaneously implanted A375.S2 melanoma tumor cells are presented in Table 1. 14 and in FIG. 13A-13C and show the responsiveness of A375.S2 melanoma cells.

Table 14

Treatment (Starting dose on day 20) Dose (mg/kg) t/s CR Answer chmAb-B B7-H3-ADC 10 3 2/7 highly active 3 13 0/7 highly active 1 65 0/7 Active chmAb-C B7-H3-ADC 10 4 1/7 highly active 23 0/7 highly active 1 70 0/7 Active chmAb-D B7-H3-ADC 10 26 0/7 highly active 3 7 0/7 highly active 1 80 0/7 highly active

The results of these studies show that each of the B7-H3-ADCs tested exhibited significant dose-dependent in vivo antitumor activity against B7-H3 positive tumors in xenograft mouse models of breast, lung and ovarian cancer and melanoma.

The pharmacokinetics of the above B7-H3-ADC (MMAE) molecules were evaluated in tumor-free CD1 nude mice by intraperitoneal administration of such molecules at a single dose of 5 mg/kg. Blood samples were collected over 10 days and a sandwich ELISA was performed on serum to quantify total antibodies and intact B7-H3-ADC concentrations.

Representative results from this study, for chmAb-B B7-H3 ADC, chmAb-C B7-H3 ADC, and chmAb-D B7-H3 ADC, are shown in FIG. 14A-14C and in the table. 15 and show that the B7-H3-ADC molecules were very stable with a half-life of approximately 2.2-3.6 days. The half-life of the conjugates was comparable to that of the unconjugated molecules, demonstrating that the B7-H3-ADC molecules are highly stable in mice.

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Table 15

B7-H3-ADC Generic anti-B7-H3 antibody Intact B7-H3-ADC* Tl/2 (watch) AUC (h * ng / ml) Т1/2 (hours) AUC (h * ng / ml) chmAb-B B7-NC ADC 114.1 4796235 58.9 4032575 chmAb-C B7-NC ADC 75.9 2698831 52.6 2201893 chmAb-D B7-H3 ADC 177.2 5162024 87.3 3502158

* MMAE conjugate.

Example 5 B7-H3-ADC with cleavable linker duocarmycin component.

A B7-H3-ADC (hmAb-C B7-H3-ADC) was constructed having a typical duocarmycin moiety (DUBA) linked to the amino acid residue of the Ab moiety via a cleavable linker conjugated to an antibody via reduced interchain disulfides as described above (see schemes 9A-9I) and Elgersma, R.C. et al. (2014) Design, Synthesis, and Evaluation of LinkerDuocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985, Mol. pharmaceuticals. 12:1813-1835 (see also WO 02/083180; WO 2010/062171; WO 2011/133039; WO 2015/104359; and WO 2015/185142). The average drug/antibody ratio (DAR) is about 2-4, typically about 2.7. It is understood that the exact DAR may vary for each drug. The order of the synthetic steps may be changed as desired. Preferably, the method used is described in Schemes 9A-9I as described above and the linker-DUBA is conjugated to the antibody via reduced interchain disulfides.

Example 6 B7-H3-ADC with cleavable duocarmycin linker retains biological activity.

The above hmAb-C B7-H3-ADC (having a typical duocarmycin component (DUBA) linked to the amino acid residue of the Ab part via a cleavable linker) (hmAb-C-DUBA) was incubated with cells for 7 days and viability was determined using assay with alamar blue as described above. As shown in FIG. 15A-15C, the hmAb-C-DUBA construct retained biological activity as evidenced by its cytotoxic activity on B7-H3 positive tumor cells. Similar results were observed for the duocarmycin-associated chmAb-C described above (chmAb-C-DUBA).

In this study and additional studies described below, a molecule that binds unbound antigen (CD20) conjugated to DUBA (Ctrl-DUBA) was used as the binding control ADC to account for non-specific activity in vivo due to the rodent-specific carboxyesterase CES1c. present in rodent plasma.

Example 7 B7-H3-ADC demonstrates potent antitumor activity in vivo.

A multi-dose study was conducted to evaluate the efficacy of the molecule in vivo. Calu-6, non-small cell lung carcinoma cells, were implanted subcutaneously into groups of mice (n = 5) essentially as described above, which then received doses of hmAb-C-DUBA (1 x 3.3 mg/kg x 3 or 6 mg /kg x 3) on days 24, 31, 38 and 45 (shown by arrows) after inoculation and animals were assessed for tumor volume (essentially as described above) up to day 62. As shown in FIG. 16, all three tested doses of hmAb-C-DUBA were effective in reducing or eliminating tumor volume. Calu-6 demonstrated an IHC score of 2+ and antibody binding sites per cell (ABC) are shown in Table 1. 9.

In a second in vivo study (performed essentially as described above), Calu-6 non-small cell lung carcinoma cells were subcutaneously implanted in groups of mice (n = 7), which then received a single dose of hmAb-C-DUBA or Ctrl-DUBA ( 3 or 10 mg/kg) on the 20th day (shown by the arrow). In table. 16 and 17 show the results and show that administration of hmAb-C-DUBA significantly reduces tumor volume.

Table 16

Treatment Dose - QW (mg/kg) Tumor Volume Treatment/Control % Complete remission hmAb-C-DUBA 10 8 0/7 hmAb-C-DUBA 41 0/7 Ctrl-DUBA 10 71 0/7 Ctrl-DUBA 3 71 0/7

In a third in vivo study (performed essentially as described above), PA-1 ovarian carcinoma cells were subcutaneously implanted in groups of mice (n = 6), which then received a single dose of hmAb-C-DUBA or Ctrl-DUBA (1 mg/day). kg, 6 mg/kg, or 10 mg/kg) on day 25 (arrow). In table. 17 and in FIG. 18 shows the results and shows that administration of hmAb-C-DUBA significantly reduced tumor volume and resulted in complete remission in up to half of the treated animals.

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Table 17

Treatment Dose - QW (mg/kg) Volume Treatment/Tumor Control% Complete remission hmAb-C-DUBA 10 AND 3/6 hmAb-C-DUBA 6 9 2/6 hmAb-C-DUBA 3 57 1/6 Ctrl-DUBA 10 84 0/6 Ctrl-DUBA 6 89 0/6 Ctrl-DUBA 3 111 0/6

Potent in vivo activity is also observed against A375.S2 melanoma cells. Such cells were implanted subcutaneously into groups of mice (n = 7) (essentially as described above), which then received a single dose of hmAb-C-DUBA or Ctrl-DUBA (1 or 3 mg/kg) on day 25 (arrowed) . In table. 18 and in FIG. 19 shows the results and shows that administration of hmAb-C-DUBA significantly reduced tumor volume and resulted in complete remission in 5/7 treated animals at the higher dose.

Table 18

Treatment Dose - QW (mg/kg) Tumor Volume Treatment/Control % Complete remission hmAb-C-DUBA 3 1 5/7 hmAb-C-DUBA 1 16 1/7 Ctrl-DUBA 3 33 0/7 Ctrl-DUBA 1 70 1/7

Potent in vivo activity was observed against MDA-MB468 breast carcinoma cells. Such cells were implanted into the mammary fat pad of groups of mice (n = 5) (essentially as described above), which then received either a single dose of hmAb-C-DUBA or Ctrl-DUBA (3 or 6 mg/kg) per day 70 or three doses of hmAb-C-DUBA or Ctrl-DUBA (3 mg/kg (arrows)). Animals were assessed for tumor volume (essentially as described above) for up to 110 days. MDA-MB468 cells showed an IHC 2+ score and ABC is shown in Table 1. 9. The results are presented in table. 19 and in FIG. 20A-20D. In FIG. 20A shows results for vehicle, hmAb-C-DUBA or CtrlDUBA at 6 mg/kg (single dose). In FIG. 20B shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 3 mg/kg (single dose). In FIG. 20C shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 3 mg/kg (three doses). In FIG. 20D shows all results in one graph. The data show that administration of hmAb-C-DUBA significantly reduced tumor volume and achieved complete remission in 4/5 treated animals at the higher dose, and that administration of repeated doses significantly improved treatment outcome.

Table 19

Treatment Dose - QW (mg/kg) Tumor Volume Treatment/Control % Complete remission hmAb-C-DUBA 6 1 4/5 hmAb-C-DUBA 3 51 1/5 hmAb-C-DUBA 3x3 doses 2 3/5 Ctrl-DUBA 6 41 0/5 Ctrl-DUBA 3 43 0/5 Ctrl-DUBA 3x3 doses 53 0/5

In a further study, xenografts of PA-1 ovarian carcinoma cells (~5x10 ⁶ suspended 1:1 in medium and MATRIGEL®) were subcutaneously injected into groups of mice, which then received a dose of hmAbC-DUBA or Ctrl-DUBA (single dose of 3, 6, or 10 mg/kg) on day 24 post-inoculation or two doses of 10 mg/kg hmAb-C-DUBA (on days 24 and 24 28 post-inoculation) or four doses of 6 mg/kg hmAb-C-DUBA (on days 24, 28, 31 and 35 after inoculation). Animals were assessed for tumor volume up to 70 days (essentially as described above). PA-1 cells showed an IHC 2+ score, and ABC is presented in tab. 9. FIG. 21A-21D summarize the results. In FIG. 21A shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 10 mg/kg (single or double dose). In FIG. 21B shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 6 mg/kg (single or quadruple dose). In FIG. 21C shows results for vehicle, hmAb-C-DUBA or Ctrl-DUBA at 3 mg/kg (single doses). In FIG. 21D shows all results in one graph. The data show that administration of hmAb-C-DUBA significantly reduced tumor volume in treated animals.

Example 8 Pharmacokinetics of B7-H3-ADC.

The pharmacokinetics of the above-described chmAb-C-DUBA was investigated using a log/line plot of total IgG or an intact ADC curve in mice (n=3) each receiving a single intravenous dose of chmAb-C-DUBA (5 mg/kg). The results are shown in FIG. 22.

The pharmacokinetics of hmAb-C-DUBA was studied using a log/line plot of total IgG or an intact ADC curve in cynomolgus monkeys each receiving a single intravenous dose of hmAb-C-DUBA (1 mg/kg (1 male, 1 female), 3 mg /kg (1 male, 1 female), 10 mg/kg (1 male, 1 female) or 27 mg/kg (2 males, 2 females)). The results are shown in FIG. 23A (total IgG) and 23B (intact ADC).

In these studies, total IgG was determined using ELISA. Briefly, serum samples, standards and controls were fixed on microtiter plates coated with goat anti-human IgG (H + L). After washing, plates were incubated with peroxidase-conjugated goat anti-human IgG Fc. After washing, staining with the substrate 3, 3, 5, 5'-tetramethylbenzidine (TMB) was allowed to develop in the plates, the reaction was stopped by phosphorus

Claims (36)

phoric acid and the plates were read at 405 nm. Total IgG in test samples was calculated from a standard curve. Intact ADC was also determined using ELISA. Briefly, the mouse anti-duocarmycin mAb was immobilized on microtiter plates. After washing, plates were incubated with peroxidase-conjugated goat anti-human IgG Fc. After washing, the stain with 3,3,5,5'-tetramethylbenzidine (TMB) substrate was allowed to develop in the plates, the reaction was stopped with phosphoric acid and the plates were read at 405 nm. Intact ADC in test samples was calculated from the standard curve. Pharmacokinetic parameters for the 5 mg/kg dose in mice and the 3 and 10 mg/kg doses in the cynomolgus monkey were obtained by comparing these data and summing them up in Table 1. 20 (where AUC Last is the area under the curve from start to end of observation). Mouse exposure to intact ADC is limited due to the rodent-specific carboxyesterase CES1c. These data indicate a large therapeutic index in the preclinical setting. Table 20 View dose (mg/kg) Т1/2 (h) Stax (ng/ml) AUC Last (h * μg/ml) mouse 5 ND 5909 45 cynomolgus monkeys 3 62.7 113484 3798 cynomolgus monkey 10 57.3 330983 17978 Example 9 Characterization of anti-B7-H3 diatel. Bispecific double and triple chain B7-H3 x CD3 diabodies were evaluated to determine their ability to mediate retargeted cell killing and/or release of cytokines from target cells expressing on the surface of B7-H3 cells. Retargeted killing of cells was investigated using the analysis of cytotoxic T-lymphocytes (CTL). Briefly, a B7-H3 x CD3 bispecific antibody (or a negative control diabody that binds an irrelevant antigen in place of B7-H3) was incubated for 24 h with effector pan T cells and target B7-H3 expressing tumor cells at an effector:cell ratio. -target 10:1. Percent cytotoxicity (ie, cell death) was determined by measuring the release of lactate dehydrogenase (LDH) into the medium by damaged cells. The release of cytokines was investigated using a similar format. Briefly, B7-H3 x CD3 bispecific antibodies (or negative control diabodies lacking a B7-H3 binding site) were incubated for 24 h with PBMC effector cells alone or in the presence of target tumor cells (e.g. SK-MES-1 lung carcinoma cells) at an effector:target ratio of 10:1 or 30:1, and the release of cytokines IFNy, TNF-α, and IL-10 was determined. The assay shows the ability of the B7-H3 x CD3 bispecific diabodies to mediate retargeted cell killing and cytokine release. All publications and patents mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually designated for inclusion by reference in their entirety. Although the invention has been described in connection with its specific embodiments, it should be understood that it is capable of further modifications, and this application is intended to cover any changes, applications or adaptations according to the invention, following, in general, the principles of the invention and including such deviations from the present disclosure. , which are known or common practice in the field to which the invention relates, and which can be applied to the main features set forth above. CLAIM 1. A B7-H3 binding molecule, where said B7-H3 binding molecule is an antibody, a diabody, or an antigen-binding fragment thereof that is capable of binding B7-H3, where said B7-H3 binding molecule contains: (i) a humanized light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO:20; and (ii) a humanized heavy chain variable region (VH) domain comprising the amino acid sequence of SEQ ID NO:21. 2. A B7-H3 binding molecule according to claim 1, which also contains an Fc domain from human IgG. 3. The B7-H3 binding molecule according to claim 2, wherein said human IgG is human IgG1, human IgG2, human IgG3, or human IgG4. 4. The B7-H3 binding molecule according to claim 2, wherein said human IgG is human IgG1. 5. A B7-H3 binding molecule according to any one of claims 2-4, wherein said Fc domain is a modified Fc domain that contains: (a) one or more amino acid modifications that reduce the affinity of the modified Fc domain for FcyR, wherein the amino acid modifications that reduce the affinity of the modified Fc domain for FcyR include L234A substitutions; L235A or L234A and L235A, where the indicated numbering corresponds to the EU index, as in Kabat; and/or (b) one or more modifications that increase the serum half-life of the modified Fc domain, wherein the modifications that increase the serum half-life of the modified Fc domain comprise M252Y substitutions; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E or K288D and H435K, where the indicated numbering corresponds to the EU index, as in Kabat. 6. A B7-H3 binding molecule according to any one of claims 1 to 5, wherein said B7H3 binding molecule is an antibody or an epitope-binding fragment thereof. 7. Anti-B7-H3 antibody drug conjugate (B7-H3-ADC) which has the formula Ab-(LM) _m -(D) _n , where Ab is a humanized B7-H3 antibody or epitope-binding fragment thereof and contains: (i) a humanized light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO:20; and (ii) a humanized heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO:21; D is a cytotoxic drug moiety; LM is at least one bond or linker molecule that covalently links Ab and D; m is an integer from 0 to n and denotes the amount of LM in B7-H3-ADC; n is an integer from 1 to 10 and denotes the amount of cytotoxic drug moieties covalently linked to the B7-H3-ADC molecule. 8. The conjugate according to claim 7, characterized in that at least one of said LM components is a linker molecule. 9. The conjugate according to claim 8, wherein the LM linker molecule is a peptide linker. 10. The conjugate of claim 9, wherein the peptide linker is a valine-citrulline dipeptide linker. 11. The conjugate according to claim 9 or 10, characterized in that the LM linker molecule further comprises a para-aminobenzyloxycarbonyl self-eliminating spacer between the peptide linker and D. 12. A conjugate according to claims 9 to 11, characterized in that the LM linker molecule further comprises a maleimide linker component between the peptide linker and Ab. 13. The conjugate according to any one of claims 7-12, wherein said cytotoxic drug component D contains a cytotoxin, radioisotope, immunomodulator, cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, DNA, RNA , miRNA, RNAi, miRNA, photoactive therapeutic agent, anti-angiogenic agent, pro-apoptotic agent, peptide, lipid, carbohydrate, chelating agent, or combinations thereof. 14. The conjugate of claim 13, wherein said cytotoxic drug component D comprises a cytotoxin and is selected from the group consisting of tubulisin, auristatin, maytansinoid, calicheamicin, pyrrolobenzodiazepine, duocarmycin, Pseudomonas exotoxin, Diptheria toxin, botulinum toxin A, botulinum toxin B, botulinum toxin toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F, ricin, abrin, saporin and their cytotoxic fragments. 15. The conjugate of claim 13, wherein said cytotoxic drug moiety D comprises tubulisin cytotoxin and is selected from the group consisting of tubulisin A, tubulisin B, tubulisin C, and tubulisin D. 16. The conjugate of claim 13 wherein said cytotoxic drug component D comprises the cytotoxin auristatin and is selected from the group consisting of MMAE (N-methylvaline-valindolaisoleuindolaproin-norephedrine) and MMAF (N-methylvaline-valine-dolaisoleuin-dolaproin-phenylalanine). ). 17. The conjugate of claim 13 wherein said cytotoxic drug moiety D comprises a maytansinoid cytotoxin and is selected from the group consisting of maytansine, DM1 and DM4. 18. The conjugate of claim 13, wherein said cytotoxic drug moiety D comprises calicheamicin cytotoxin and is selected from the group consisting of calicheamicin γ1, calicheamicin B1Br, calicheamycin γ1Βη, calicheamicin α2Ι, calicheamicin α3Ι, calicheamycin β 1I, calicheamicin γ1Ι, and calicheamicin β 1I, calicheamycin γ1Ι, and calicheamicin γ1Ι. 19. The conjugate of claim 13 wherein said cytotoxic drug moiety D comprises pyrrolobenzodiazepine cytotoxin and is selected from the group consisting of vadastuximab thalirin, SJG-136, SG2000, SG2285 and SG2274. 20. The conjugate of claim 13, wherein said cytotoxin is a duocarmycin cytotoxin. 21. The conjugate of claim 20 wherein the duocarmycin cytotoxin is selected from the group consisting of duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duo- - 77 042568 carmycin SA, CC-1065, adoselesin, bizelesin, carcelesin (U-80244), seco-duocarmycin and spiroduocarmycin (DUBA). 22. The conjugate of claim 21 wherein said cytotoxic drug moiety D comprises secoduocarmycin. 23. A B7-H3 binding molecule according to any one of claims 1-6, or a conjugate according to any one of claims 7-22, wherein said B7-H3 binding molecule contains a human IgG kappa chain constant region domain. 24. A B7-H3 binding molecule or a conjugate according to claim 23, wherein the human IgG kappa chain constant region domain comprises the amino acid sequence of SEQ ID NO:1. 25. A conjugate according to any one of claims 7-24, wherein Ab is said humanized B7H3 antibody and said antibody contains: (i) a light chain comprising a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO:20 and the CL kappa domain of SEQ ID NO:1; and (ii) a heavy chain comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:21, a CH1 domain of SEQ ID NO:3, a hinge domain of SEQ ID NO:7, and an Fc domain comprising a CH2- CHS sequence SEQ ID NO:12; said D contains seco-duocarmycin; and said LM contains a linker molecule containing a maleimide linker group, a valine-citrulline dipeptide linker and an aminobenzyloxycarbonyl group. 26. A pharmaceutical composition that comprises a B7-H3 binding molecule according to any one of claims 1-6 or a conjugate according to any one of claims 7-25 and a pharmaceutically acceptable carrier, excipient or diluent. 27. The use of a B7-H3 binding molecule, conjugate or pharmaceutical composition according to any one of claims 1 to 26 as a medicament for the treatment of a disease or condition associated with B7-H3 expression or characterized by B7-H3 expression. 28. Use according to claim 27, characterized in that said disease or condition associated with B7-H3 expression or characterized by B7-H3 expression is cancer. 29. Use according to claim 28, wherein said cancer is selected from the group consisting of adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkett's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, pharyngeal cancer mesothelioma, non-Hodgkin's lymphoma, small cell lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal carcinoma, blue-small round cell tumors of children (including neuroblastoma and rhabdomyosarcoma), squamous cell carcinoma, head and neck squamous cell carcinoma (SCCHN), cancer testicular, thyroid cancer, metastatic shield cancer ovarian gland and uterine cancer. 30. Use according to claim 28, wherein said cancer is selected from the group consisting of acute myeloid leukemia, non-small cell lung cancer (NSCLC), neuroblastoma, head and neck squamous cell carcinoma (SCCHN), prostate cancer, and metastatic thyroid cancer. 31. Use according to claim 28, wherein said cancer is prostate cancer. 32. Use according to claim 28, wherein said cancer is breast cancer. 33. Use according to claim 28, wherein said cancer is non-small cell lung cancer (NSCLC). 34. The use of a B7-H3 binding molecule, conjugate or pharmaceutical composition according to any one of paragraphs. 1-26 in the preparation of a medicament for the treatment of a disease or condition associated with B7-H3 expression or characterized by B7-H3 expression. 35. Use according to claim 34, characterized in that said disease or condition associated with B7-H3 expression or characterized by B7-H3 expression is cancer. 36. Use according to claim 35, wherein said cancer is selected from the group consisting of adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkett's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, pharyngeal cancer mesothelioma, non-Hodgkin's lymphoma, small cell lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, blue-small round cell tumors of children (including neuroblastoma and rhabdomyosarcoma), squamous cell cancer (eg, head and neck squamous cell carcinoma (SCCHN) , testicular cancer, thyroid cancer (eg, metas static thyroid cancer) and uterine cancer. -