B7-H3 antibody drug conjugates

Abstract

The present disclosure relates to B7-H3-ADCs comprising a human B7-H3 binding domain of a humanized anti-human B7-H3 antibody, the humanized anti-human B7-H3 antibody being conjugated to at least one camptothecin drug moiety. The present disclosure also relates to pharmaceutical compositions comprising such B7-H3-ADCs, and to methods of using any such B7-H3-ADCs in the treatment of cancer and other diseases and conditions.

Description

Technical Field

[0001] This disclosure relates to B7-H3 antibody-drug conjugates (“B7-H3-ADCs”) comprising a human B7-H3 binding domain of a humanized anti-human B7-H3 antibody conjugated to at least one pharmaceutical moiety. This disclosure also relates to pharmaceutical compositions comprising such B7-H3-ADCs, and to methods of treating cancer and other diseases and conditions associated with or characterized by B7-H3 expression using any such B7-H3-ADC. Background Technology I.B7 Superfamily and B7-H3

[0002] B7-H3 is a member of the B7-CD28 superfamily and is expressed on antigen-presenting cells. B7-H3 is unique in that the predominant human form contains two extracellular tandem IgV-IgC domains (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 initially thought to contain only two Ig domains (IgV-IgC, see, e.g., NCBI sequence NP_079516), four immunoglobulin extracellular domain variants ("4Ig-B7-H3") have been identified, and it has been found to be the protein in the more common human form (Sharpe, AH et al., (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2:116-126; see also, e.g., NCBI sequence NP_001019907). B7-H3 mRNA expression has been found in heart, kidney, testis, lung, liver, pancreas, prostate, colon, and osteoblasts (Collins, M. et al., (2005) "The B7 Family Of Immune-Regulatory Ligands," Genome Biol. 6:223.1-223.7). At the protein level, B7-H3 has been found in human liver, lungs, bladder, testes, prostate, mammary glands, placenta, and lymphoid organs (Hofmeyer, K. et al., (2008) "The Contrasting Role Of B7-H3", Proc. Natl. Acad. Sci. (USA) 105(30): 10277-10278).

[0003] Although B7-H3 is not expressed on resting B or T cells, monocytes, or dendritic cells, it is induced by IFN-γ on dendritic cells and by GM-CSF on monocytes (Sharpe, AH 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 T cell co-stimulation and co-inhibition (Hofmeyer, K et al., (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278; Martin-Orozco, N. et al., (2007) "Inhibitory Costimulation And Anti-Tumor Immunity," Semin. Cancer Biol. 17(4):288-298). B7-H3 binds to unidentified receptors to mediate co-suppression of T cells. In addition, B7-H3 is an inhibitor of NK cells and osteoblasts through interaction with unknown receptors (Hofmeyer, K. et al., (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (USA)105(30):10277-10278). II. Tumors expressing B7-H3

[0004] B7-H3 is expressed in a variety of cancer cells (e.g., neuroblastoma, gastric cancer, ovarian cancer, non-small cell lung cancer, etc., see, for example, Modak, S., et al., (2001) "Monoclonal antibody 8H9 targets a novel cellsurface antigen expressed by a wide spectrum of human solid tumors," CancerRes 61:4048-54) as well as cultured cancer stem-like cells. Several independent studies have shown that human malignant tumor cells exhibit significantly increased expression of B7-H3 protein, and that this increased expression is associated with increased disease severity (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., (2013) "B7-H3 Mediated Tumor Immunology: Friend Or Foe?," Int. J. Cancer 134(12):2764-2771), indicating that B7-H3 is used by tumors as an immune evasion pathway (Hofmeyer, K. et al., (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).

[0005] The role of B7-H3 in suppressing the immune system and the increased expression of B7-H3 in human tumors suggest that this molecule could serve as a therapeutic target for cancer treatment. The potential uses of anti-B7-H3 antibodies and other molecules that regulate B7-H3 expression in the treatment of tumors and / or upregulation of immune responses have been proposed (see, Loo, D. et al., (2012) "Development of an Fc-Enhanced Anti–B7-H3 Monoclonal Antibody with Potent Antitumor Activity," ClinCancer Res; 18: 3834-3845; Ahmed, M. et al., (2015) "Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loopof B7-H3," J. Biol. Chem. 290: 30018-30029; 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); see also U.S. Patent Nos. 7,279,567, 7,527,969, 7,718,774, 8,779,098, 8,802,091; U.S. Patent Publication Nos. 2002 / 0168762; 2008 / 0081346, 2008 / 0116219, 2013 / 0078234, 2015 / 027483 8; PCT Publication Nos. WO2009 / 073533; WO2008 / 066691, WO2006 / 016276, WO2008 / 116219, WO2001 / 094413, WO2002 / 32375, WO2004 / 093894, WO2006 / 016276, WO2008 / 116219 and WO2011 / 109400. Summary of the Invention

[0006] This disclosure relates to B7-H3 antibody-drug conjugates (“B7-H3-ADCs”) comprising a human B7-H3 binding domain of a humanized anti-human B7-H3 antibody conjugated to at least one pharmaceutical moiety. This disclosure relates to pharmaceutical compositions comprising such B7-H3-ADCs, and to methods of treating cancer and other diseases and conditions using any such B7-H3-ADC.

[0007] In some aspects, this disclosure provides an anti-B7-H3 antibody-drug conjugate (B7-H3-ADC) comprising the formula: Ab-(LM) m -(D) n , in: Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) Contains CDRs in its variable light chain (VL) domain. L 1. Sequence RASESIYSYLA (SEQ ID NO:16), CDR L 2. Sequence NTKTLPE (SEQ ID NO:17) and CDR L The sequence QHHYGTPPWT (SEQ ID NO:18) and (ii) Contains CDRs within its variable heavy chain (VH) domain. H 1. Sequence SYGMS (SEQ ID NO:19), CDR H 2. Sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20) and CDR H 3 sequences HDGGAMDY (SEQ ID NO:21) or HEGGAMDY (SEQ ID NO:26); D represents the cytotoxic drug component; LM is a linker molecule that covalently connects Ab and D; m is an integer between 1 and n and represents the number of linker molecules in the B7-H3-ADC; and n is an integer between 1 and 10 and represents the number of cytotoxic camptothecin moieties covalently linked to the B7-H3-ADC molecule.

[0008] In some aspects, this disclosure provides an anti-B7-H3 antibody-drug conjugate (B7-H3-ADC) comprising the formula: Ab-(LM) m -(D) n , in: Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (iii) Contains CDRs in its variable light chain (VL) domain. L 1. Sequence RASESIYSYLA (SEQ ID NO:16), CDR L 2. Sequence NTKTLPE (SEQ ID NO:17) and CDR L The sequence QHHYGTPPWT (SEQ ID NO:18) and (iv) Contains CDRs in its variable heavy chain (VH) domain. H 1. Sequence SYGMS (SEQ ID NO:19), CDR H 2. Sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20) and CDR H 3 sequences HDGGAMDY (SEQ ID NO:21) or HEGGAMDY (SEQ ID NO:26); D is the camptothecin portion; LM is a linker molecule that covalently connects Ab and D; m is an integer between 1 and n and represents the number of linker molecules in the B7-H3-ADC; and n is an integer between 1 and 10 and represents the number of cytotoxic camptothecin moieties covalently linked to the B7-H3-ADC molecule.

[0009] In some respects, this disclosure further provides such a B7-H3-ADC, wherein Ab comprises: a) Contains a humanized VL domain containing the amino acid sequence SEQ ID NO:12, and b) Contains a humanized VH domain containing the amino acid sequence SEQ ID NO:14 or SEQ ID NO:22.

[0010] In some aspects, this disclosure further provides such a B7-H3-ADC, wherein Ab is an antibody. In some aspects, this disclosure further provides such a B7-H3-ADC, wherein Ab is an antigen-binding fragment of an antibody.

[0011] In some aspects, this disclosure further provides such a B7-H3-ADC, wherein the Ab includes the Fc domain of human IgG. In some aspects, this disclosure further provides such a B7-H3-ADC, wherein the human IgG is human IgG1, IgG2, IgG3, or IgG4.

[0012] In some respects, this disclosure further provides such a B7-H3-ADC, wherein the Fc domain is a variant Fc domain comprising: (a) One or more amino acid modifications that reduce the affinity of the variant Fc domain for FcγR; and / or (b) One or more amino acid modifications that enhance the serum half-life of the variant Fc domain.

[0013] In some aspects, this disclosure further provides such B7-H3-ADCs wherein modifications that reduce the affinity of the variant Fc domain for FcγR include the following substitutions: L234A; L235A; or L234A and L235A, wherein the numbers are EU index numbers in Kabat. In some aspects, this disclosure further provides such B7-H3-ADCs wherein modifications that enhance the serum half-life of the variant Fc domain include the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T, and T256E; or K288D and H435K, wherein the numbers are EU index numbers in Kabat.

[0014] In some aspects, this disclosure further provides such a B7-H3-ADC, wherein the LM includes a peptide linker. In some aspects, this disclosure further provides such a B7-H3-ADC, wherein the LM includes a cleavable linker.

[0015] In some respects, this disclosure further provides such a B7-H3-ADC, wherein the LM comprises formula (4a) or (4b) or a salt thereof: in: a is either 0 or 1 independently; b is either 0 or 1 independently; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1 to 150; g is 0 or 1; i is 0 or 1; D represents the cytotoxic drug component; Q 1 It is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclic triazole group, or a cycloalkenyl group; Q 1 Functional groups attached to antibodies; Sp 1 Sp 2 Sp 3 and Sp 4Independently select from the following groups: C1-C of straight chains or branches. 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkenyl groups, C8-C 200 Cycloynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imide groups and C9-C 200 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynynyl group, alkylarylyl group, arylalkylene group, arylalkenyl group, and arylynylynyl group are optionally substituted by one or more heteroatoms and optionally spaced by one or more heteroatoms, wherein the one or more heteroatoms are selected from O, S, and NR. 3 The group, where R 3 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted; Z 1 It is Q 1 or SP 3 Connect to SP 2 O or C(O) or N(R) 1 () linking group; Z 2 Is it D or SP? 4 Connect to SP 1 、N(R 1 ), O or C(O) linking groups; Z 1 and Z 2 Choose independently from the following groups: -O-, -S-, -NR 2 -、-N=N-、-C(O)-、-C(O)NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 -NR 2 -C(O)-、-NR 2 -C(O)-O-、-NR 2 -C(O)-NR 2-、-S-C(O)-、-S-C(O)-O-、-S-C(O)-NR 2 -、-S(O)-、-S(O)2-、-O-S(O)2-、-O-S(O)2-O-、-O-S(O)2-NR 2 -、-O-S(O)-、-O-S(O)-O-、-O-S(O)-NR 2 -、-O-NR 2 -C(O)-、-O-NR 2 -C(O)-O-、-O-NR 2 -C(O)-NR 2 -、-NR 2 -O-C(O)-、-NR 2 -O-C(O)-O-、-NR 2 -O-C(O)-NR 2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-、-O-NR 2 -C(S)-NR 2 -、-NR 2 -O-C(S)-、-NR 2 -O-C(S)-O-、-NR 2 -O-C(S)-NR 2 -、-O-C(S)-、-O-C(S)-O-、-O-C(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-、-NR 2 -C(S)-NR 2 -、-S-S(O)2-、-S-S(O)2-O-、-S-S(O)2-NR 2 -、-NR 2 -O-S(O)-、-NR 2 -O-S(O)-O-、-NR 2 -O-S(O)-NR 2 -、-NR 2 -O-S(O)2-、-NR 2 -O-S(O)2-O-、-NR 2 -O-S(O)2-NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR2 -S(O)2-O-、-O-NR 2 -S(O)2-NR 2 -、-O-NR 2 -S(O)2-、-OP(O)(R 2 )2-、-SP(O)(R 2 )2-、-NR 2 -P(O)(R 2 )2- and combinations of two or more of them, where R 2 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, ynyl groups, and cycloalkyl groups are optionally substituted; and R 1 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, the C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 The (hetero)arylalkyl group is optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, said one or more heteroatoms being selected from O, S, and NR. 3 , where R 3 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 It is D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 Sp 2Sp 3 Sp 4 Z 1 Z 2 D, Q 1 b, c, d, e, g, and i are as defined above.

[0016] In some respects, this disclosure further provides such a B7-H3-ADC, wherein the LM comprises formula (4a) or (4b) or a salt thereof: in: a is either 0 or 1 independently; b is either 0 or 1 independently; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1 to 150; g is 0 or 1; i is 0 or 1; D is the cytotoxic camptothecin moiety; Q 1 It is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclic triazole group, or a cycloalkenyl group; Q 1 Functional groups that attach to antibodies; Sp 1 Sp 2 Sp 3 and Sp 4 Independently select from the following groups: straight chain or branched chain C1-C 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkenyl groups, C8-C 200 Cycloynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imide groups and C9-C 200 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynynyl group, alkylarylyl group, arylalkylene group, arylalkenyl group, and arylynylynyl group are optionally substituted by one or more heteroatoms and optionally spaced by one or more heteroatoms, wherein the one or more heteroatoms are selected from O, S, and NR. 3 The group, where R 3Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted; Z 1 It is Q 1 or SP 3 Connect to SP 2 O or C(O) or N(R) 1 () linking group; Z 2 Is it D or SP? 4 Connect to SP 1 、N(R 1 ), O or C(O) linking groups; Z 1 and Z 2 Choose independently from the following groups: -O-, -S-, -NR 2 -、-N=N-、-C(O)-、-C(O)NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 -NR 2 -C(O)-、-NR 2 -C(O)-O-、-NR 2 -C(O)-NR 2 -, -SC(O)-, -SC(O)-O-, -SC(O)-NR 2 -, -S(O)-, -S(O)2-, -OS(O)2-, -OS(O)2-O-, -OS(O)2-NR 2 -, -OS(O)-, -OS(O)-O-, -OS(O)-NR 2 -、-O-NR 2 -C(O)-、-O-NR 2 -C(O)-O-、-O-NR 2 -C(O)-NR 2 -、-NR 2 -OC(O)-、-NR 2 -OC(O)-O-、-NR 2 -OC(O)-NR 2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-、-O-NR 2-C(S)-NR 2 -、-NR 2 -OC(S)-、-NR 2 -OC(S)-O-、-NR 2 -OC(S)-NR 2 -, -OC(S)-, -OC(S)-O-, -OC(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-、-NR 2 -C(S)-NR 2 -, -SS(O)2-, -SS(O)2-O-, -SS(O)2-NR 2 -、-NR 2 -OS(O)-、-NR 2 -OS(O)-O-、-NR 2 -OS(O)-NR 2 -、-NR 2 -OS(O)2-、-NR 2 -OS(O)2-O-、-NR 2 -OS(O)2-NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O)2-O-、-O-NR 2 -S(O)2-NR 2 -、-O-NR 2 -S(O)2-、-OP(O)(R 2 )2-、-SP(O)(R 2 )2-、-NR 2 -P(O)(R 2 )2- and combinations of two or more of them, where R 2 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, ynyl groups, and cycloalkyl groups are optionally substituted; and R 1 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, the C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 The (hetero)arylalkyl group is optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, said one or more heteroatoms being selected from O, S, and NR. 3 , where R 3 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 It is D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 Sp 2 Sp 3 Sp 4 Z 1 Z 2 D, Q 1 b, c, d, e, g, and i are as defined above.

[0017] In some respects, this disclosure further provides such a B7-H3-ADC, wherein Sp 1 Sp 2 Sp 3 and Sp 4 If it exists, independently select a group consisting of the following: straight chains or branches C1-C. 20 An alkylene group, wherein the alkylene group is optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, the one or more heteroatoms being selected from O, S, and NR. 3 The group consisting of R 3 The group consisting of hydrogen and C1-C4 alkyl groups is selected independently.

[0018] In some aspects, this disclosure further provides such a B7-H3-ADC, wherein the LM includes a valine-alanine (Val-Ala) amino acid linker. In some aspects, this disclosure further provides such a B7-H3-ADC, wherein the Val-Ala linker is a Val-Ala-PABC linker.

[0019] In some respects, this disclosure further provides such B7-H3-ADCs, wherein the camptothecin portion is selected from the group consisting of: SN-38 (S-10-hydroxycamptothecin), topotecan (HYCAMPTIN; (S)-9-N,N-dimethylaminoethyl-10-hydroxycamptothecin), 9-aminocamptothecin (9-amino-20(S)-camptothecin), 9-nitrocamptothecin (also known as rubitecan), letopotecan (7-(4-methylpiperazinemethylene)-10,11-ethylenedioxy-20(S)-camptothecin), exatecan, karenitecin, and holocorostachycin.

[0020] In some respects, this disclosure further provides such a B7-H3-ADC, wherein the camptothecin portion is ixotecan.

[0021] In some respects, this disclosure further provides such a B7-H3-ADC, wherein LM and D together comprise: .

[0022] In some respects, this disclosure further provides an anti-B7-H3 antibody-drug conjugate (B7-H3-ADC) comprising the formula: Ab-(LM) m -(D) n , in: Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) Contains CDRs in its variable light chain (VL) domain. L 1. Sequence RASESIYSYLA (SEQ ID NO:16), CDR L 2. Sequence NTKTLPE (SEQ ID NO:17) and CDR L The sequence QHHYGTPPWT (SEQ ID NO:18) and (ii) Contains CDRs within its variable heavy chain (VH) domain. H 1. Sequence SYGMS (SEQ ID NO:19), CDR H2. Sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20) and CDR H 3 sequences HDGGAMDY (SEQ ID NO:21) or HEGGAMDY (SEQ ID NO:26); LM and D together include: m is an integer between 0 and n and represents the number of linker molecules in B7-H3-ADC; and n is an integer between 1 and 10 and represents the number of cytotoxic camptothecin moieties covalently linked to the B7-H3-ADC molecule.

[0023] In some respects, this disclosure further provides such B7-H3-ADC molecules, wherein the Ab includes a humanized VL domain comprising the amino acid sequence SEQ ID NO:12, and a humanized VH domain comprising the amino acid sequence SEQ ID NO:14 or SEQ ID NO:22.

[0024] In some respects, this disclosure further provides pharmaceutical compositions comprising an effective amount of B7-H3-ADC and a pharmaceutically acceptable carrier, excipient, or diluent.

[0025] In some respects, this disclosure further provides the use of B7-H3-ADC or pharmaceutical compositions in the treatment of diseases or conditions associated with or characterized by B7-H3 expression.

[0026] In some respects, this disclosure further provides a method for treating a disease or condition associated with or characterized by B7-H3 expression, said method comprising administering a B7-H3-ADC or a pharmaceutical composition to a subject.

[0027] In some respects, this disclosure further provides for such uses or methods, wherein a disease or condition associated with or characterized by B7-H3 expression is cancer.

[0028] In some respects, this disclosure further provides for such uses or methods, wherein the cancer is selected from the group consisting of: adrenal tumors, AIDS-related cancers, alveolar soft tissue sarcomas, astrocytic tumors, adrenal carcinoma, bladder cancer, bone cancer, brain and spinal cord cancers, metastatic brain tumors, B-cell carcinomas, breast cancers, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, fibroproliferative small round cell tumors, ependymoma, Ewing's tumor, extraosseous myxoid chondrosarcoma, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, glioblastoma, hematologic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, renal cancer, leukemia (e.g., acute myeloid leukemia), liposarcoma / malignant tumors. Lipomas, liver cancer, lymphoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), medulloblastoma, melanoma, meningioma, mesothelioma, laryngeal cancer, multiple endocrine tumors, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumors, childhood cancers, peripheral nerve sheath tumors, pheochromocytoma, pituitary adenoma, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumors in children (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck (SCCHN)), gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (e.g., metastatic thyroid cancer), and uterine cancer.

[0029] In some respects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is selected from the group consisting of: adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, renal cancer, non-small cell lung cancer (NSCLC), acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, mesothelioma, laryngeal cancer, non-Hodgkin's lymphoma. Lymphoma, small lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, platinum-resistant ovarian cancer (PROC), pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), skin cancer, renal cell carcinoma, small cell lung cancer (SCLC), extensive-stage small cell lung cancer (ES-SCLC), childhood small round blue cell tumors (including neuroblastoma and rhabdomyosarcoma), squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN), testicular cancer, thyroid cancer (e.g., metastatic thyroid cancer), and uterine cancer.

[0030] In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is melanoma. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is lung cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is head and neck squamous cell carcinoma. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is pancreatic cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is prostate cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is small cell lung cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is small cell ovarian cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is small cell colorectal cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is esophageal squamous cell carcinoma. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is non-small cell lung cancer (NSCLC). In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is bladder cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is sarcoma. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is endometrial cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC). In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is breast cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is ovarian cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is cervical cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is colorectal cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is gastric cancer or gastroesophageal junction cancer. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is clear cell renal cell carcinoma. In some aspects, this disclosure further provides such uses or methods for treating cancer, wherein the cancer is hepatocellular carcinoma. Brief description of the attached diagram

[0031] Figure 1This is a graph showing the percentage of cell survival versus ADC concentration in an in vitro cytotoxicity study of A375.S2 human melanoma cells expressing B7-H3, as described in Example 1.

[0032] Figures 2A-2E The graph shows the percentage of cytotoxicity versus monoclonal antibody concentration in ADCC assays of unconjugated MGA017, conjugated MGC018, and conjugated MGC026 in LnCAP cells (2A), Hs700T cells (2B), JIMT1 cells (2C), Calu-6 cells (2D), and A498 cells (2E) as described in Example 2. Figures 3A-3B The graph shows the change of resonance units (RU) over time in surface plasmon resonance (SPR) assays of MGC026 and MGA017 with histidine-labeled human or cynomolgus monkey B7H3 (4Ig) extracellular domain protein (3A) and histidine-labeled human CD16A extracellular domain protein (3B), as described in Example 3. Figure 3C-3D The graph shows the change of resonance units (RU) over time in surface plasmon resonance (SPR) measurements of MGA017 (3C) and MGA017 (D96E) (3D) with recombinant B7-H3 as described in Example 3.

[0033] Figure 4 A graph showing the change in average tumor volume over time as described in Example 4 is presented, demonstrating the antitumor activity of MGC026 and MGC018 against Calu-6 lung adenocarcinoma tumor cells.

[0034] Figure 5 A graph showing the change in mean tumor volume over time is presented, demonstrating the antitumor activity of MGC026 and MGC018 against Calu-6 lung adenocarcinoma tumor cells at the minimum effective dose, as described in Example 4.

[0035] Figure 6 A graph showing the change in mean tumor volume over time is presented, demonstrating the antitumor activity of MGC026 and MGC018 against A375.S2 melanoma tumor cells, as described in Example 4.

[0036] Figures 7A-7C A graph showing the change in mean tumor volume over time is presented, which demonstrates the antitumor activity of MGC026 and MGC018 against A375.S2 melanoma tumor cells at doses of 3 mg / kg (7A), 1 mg / kg (7B), and 0.3 mg / kg (7C), as described in Example 4.

[0037] Figure 8A graph showing the change in average tumor volume over time demonstrates the antitumor activity of MGC026 against FaDu pharyngeal and head and neck squamous cell carcinoma (“HNSCC”) tumor cells, as described in Example 4.

[0038] Figure 9 A graph showing the change in mean tumor volume over time demonstrates the antitumor activity of MGC026 against Hs700T pancreatic adenocarcinoma tumor cells, as described in Example 4.

[0039] Figure 10 A graph showing the change in average tumor volume over time demonstrates the antitumor activity of MGC026 against 22Rv1 prostate cancer cells, as described in Example 4.

[0040] Figure 11A-11C A graph showing the change in mean tumor volume over time is presented, which shows the antitumor activity of MGC026 and DS-mAb-Dxd (an ADC with an M30-H1-L4 antibody that binds to B7-H3 (which is conjugated with derutecan (Dxd))) against Calu-6 tumor cells at doses of 3 mg / kg (11A), 1 mg / kg (11B), and 0.3 mg / kg (11C), as described in Example 5.

[0041] Figures 12A-12C A graph showing the change in mean tumor volume over time is presented, which demonstrates the antitumor activity of MGC026 and DS-mAb-Dxd against A375.S2 melanoma tumor cells at doses of 3 mg / kg (12A), 1 mg / kg (12B), and 0.3 mg / kg (12C), as described in Example 5.

[0042] Figure 13 A graph showing the change in mean tumor volume over time is displayed, demonstrating the antitumor activity of MGC026 against tumor fragments derived from patients with small cell lung cancer (SCLC) of Model 1, as described in Example 9.

[0043] Figure 14 A graph showing the change in mean tumor volume over time is presented, demonstrating the antitumor activity of MGC026 against tumor fragments derived from small cell lung cancer (SCLC) patients of Model 2, as described in Example 9.

[0044] Figure 15 A graph showing the change in mean tumor volume over time is presented, demonstrating the antitumor activity of MGC026 against tumor fragments derived from small cell lung cancer (SCLC) patients of Model 3, as described in Example 9.

[0045] Figure 16A graph showing the change in average tumor volume over time demonstrates the antitumor activity of MGC026 against tumor fragments derived from ovarian cancer patients, as described in Example 9.

[0046] Figure 17 A graph showing the change in average tumor volume over time demonstrates the antitumor activity of MGC026 against tumor fragments derived from melanoma patients, as described in Example 9.

[0047] Figure 18 A graph showing the change in mean tumor volume over time demonstrates the antitumor activity of MGC026 against tumor fragments derived from colorectal cancer patients, as described in Example 9.

[0048] Figure 19 A graph showing the change in mean tumor volume over time demonstrates the antitumor activity of MGC026 against tumor fragments derived from patients with head and neck squamous cell carcinoma (SCCHN), as described in Example 9. Invention Details

[0049] This disclosure relates to B7-H3-ADCs comprising a human B7-H3 binding domain of a humanized anti-human B7-H3 antibody conjugated to at least one pharmaceutical moiety. This disclosure also relates to pharmaceutical compositions comprising such B7-H3-ADCs, and to methods of treating cancer and other diseases and conditions using any such B7-H3-ADC. For example, certain B7-H3-ADCs and their use in cancer treatment are described in PCT Publication No. WO 2017 / 180813 (which is expressly incorporated herein by reference). I. Antibodies and their binding domains

[0050] The antibodies disclosed herein are immunoglobulin molecules capable of specifically binding to targets (e.g., carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in a variable domain of an immunoglobulin molecule. Therefore, the B7-H3-ADC of this disclosure includes antibodies that bind to B7-H3. As used herein, the terms "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelified antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), intracellular antibodies, and any of the epitope-binding fragments described above. In particular, the term "antibody" includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing epitope-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies can “immune-specifically bind” to polypeptides or proteins or non-protein molecules (or can bind to such molecules in an “immune-specific manner)” because such molecules possess specific domains or portions or conformations (“epitopes”). Molecules containing epitopes can be immunogenic, causing them to elicit an antibody response in animals; such molecules are called “antigens.”

[0051] As used herein, an antibody, biantibody, or other epitope-binding molecule is said to "immune-specifically" bind to a region (i.e., epitope) of another molecule if, relative to an alternative epitope, it reacts or associates with the epitope of another molecule more frequently, more rapidly, for a longer duration, and / or with higher affinity. For example, an antibody that is immune-specifically bound to a viral epitope is an antibody that binds to a viral epitope with higher affinity, more readily, and / or for a longer duration compared to its immune-specific binding to other viral or non-viral epitopes. Reading this definition, it should also be understood that, for example, an antibody (or part or epitope) that is immune-specifically bound to a first target may or may not specifically or preferentially bind to a second target. Therefore, "immune-specific binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, the term "binding" refers to "immune-specific" binding. If such binding exhibits the specificity of receptor binding to their respective ligands, the two molecules are said to be able to bind to each other in a "physiologically specific" manner.

[0052] The term "monoclonal antibody" refers to a group of homogeneous antibodies, wherein a monoclonal antibody includes amino acids (naturally or non-naturally present) that participate in the selective binding of antigens. Monoclonal antibodies are highly specific, targeting a single epitope (or antigenic site). The term "monoclonal antibody" encompasses not only complete 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 antibody portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified conformations of immunoglobulin molecules containing antigen recognition sites with the desired specificity and ability to bind antigens. It is not intended to limit the source of the antibody or the method of its preparation (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments described above under the definition of "antibody." Methods for preparing monoclonal antibodies are known in the art. One approach is the method described in 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 developed in mice, rats, or rabbits. Antibodies are generated by immunizing the animal with an immunogenic amount of cells, cell extracts, or protein preparations containing the desired epitope. Immunogens can be, but are not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissues. Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immune-specific to the desired pathogenic epitope can be generated by sequencing and recombining using any method known in the art. In one aspect, such antibodies are sequenced, and the polynucleotide sequence is then cloned into a vector for expression or proliferation. The sequence encoding the antibody of interest can be maintained in a vector within host cells, and the host cells can then be amplified and frozen for future use. The polynucleotide sequences of such antibodies can be used for genetic manipulation to produce monospecific or multispecific (e.g., bispecific, trispecific, and tetraspecific) molecules, as well as affinity-optimized chimeric antibodies, humanized antibodies, and / or canine antibodies to improve antibody affinity or other properties. The general principle of humanizing antibodies involves retaining the basic sequence of the antigen-binding portion of the antibody while exchanging the non-human portion of the antibody with the human antibody sequence.

[0053] Natural antibodies (such as IgG antibodies) are composed of two “light chains” and 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 naturally occurring immunoglobulins (such as IgG) is a tetramer with two light chains and two heavy chains, typically expressed as a glycoprotein of about 150,000 Da. The amino-terminal (“N-terminal”) portion of each chain includes a variable domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal (“C-terminal”) portion of each chain defines the constant region, where the light chain has a single constant domain and the heavy chain typically has three constant domains and a hinge domain. Therefore, the light chain structure of the IgG molecule is n-VL-CL-c, and the heavy chain structure of IgG is n-VH-CH1-H-CH2-CH3-c (where n and c represent the N-terminus and C-terminus of the polypeptide, respectively). A. Characteristics of antibody variable domains

[0054] The variable domains of an IgG molecule consist of a complementarity-determining region (“CDR”) (containing residues that contact the epitope) and non-CDR segments (called frame segments (“FR”)). These frame segments typically maintain the structure and define the position of the CDR loop to allow this contact (although some frame residues may also contact the antigen). Therefore, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c. The amino acid sequence of the CDR determines whether the antibody can bind to a specific epitope. The interaction between the antibody light chain and the antibody heavy chain, particularly the interaction between their VL and VH domains, forms the antibody's epitope binding site.

[0055] The amino acids of the variable domains in the mature heavy and light chains of immunoglobulins are named according to their positions within the chain. Kabat (Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, NH1, MD (1991)) describes multiple amino acid sequences of antibodies, identifies the consensus amino acid sequence for each subgroup, assigns residue numbers to each amino acid, and identifies CDRs and FRs as defined by Kabat (which is easily understood, as CDRs are defined by Chothia, C. & Lesk, AM ((1987), "Canonical structures for the hypervariable regions of immunoglobulins," J. Mol. Biol. 196:901-917)). H 1. Starting from the first five residues. By referencing conserved amino acids and comparing the antibody in question with one of the consensus sequences in Kabat, the Kabat numbering scheme can be extended to antibodies not included in its outline. This method of assigning residue numbers has become standard in the field and can readily identify amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of the light chain of a human antibody occupies the same position as the amino acid at position 50 of the light chain of a mouse antibody. Therefore, the start and end positions of the CDR within the VL and VH domains are well defined and can be determined by examining the sequences of the VL and VH domains (see, for example, Martin, CR (2010), "Protein Sequence and Structure Analysis of Antibody Variable Domains," In: Antibody Engineering Vol. 2 (Kontermann, R. and Dübel, S. (eds.), Springer-Verlag Berlin Heidelberg, Chapter 3 (pages 33-51)).

[0056] The polypeptides that are (or can act as) the first, second, and third CDRs of the antibody light chain are named CDRs in this paper. L 1. Structural Domain, CDR L 2. Structural Domains and CDRs L 3. Domains. Similarly, peptides that are (or can act as) the first, second, and third CDRs of the antibody heavy chain are named CDRs in this paper. H 1. Structural Domain, CDRH 2. Structural Domains and CDRs H 3. Structural Domains. Therefore, the term CDR... L 1. Structural Domain, CDR L 2. Structural Domains, CDR L 3. Structural Domains, CDR H 1. Structural Domain, CDR H 2. Structural Domains and CDRs H The 3-domain relates to a polypeptide that, when incorporated into a protein, enables that protein to bind to a specific epitope, whether such a protein is an antibody with both light and heavy chains, a biantibody, a single-chain binding molecule (e.g., scFv, BiTe, etc.), or another type of protein. Therefore, as used herein, the term "epitope-binding fragment" refers to a molecular fragment capable of immune-specific binding to an epitope. An epitope-binding fragment may contain any 1, 2, 3, 4, or 5 CDR domains of an antibody, or may contain all 6 CDR domains of an antibody, and although capable of immune-specific binding to such epitopes, may exhibit different immune specificity, affinity, or selectivity for such epitopes than such antibodies. However, an epitope-binding fragment preferably contains all 6 CDR domains of such antibodies. An antibody's epitope-binding fragment may be a single polypeptide chain (e.g., scFv), or may contain two or more polypeptide chains, each having an N-terminus and a C-terminus (e.g., biantibodies, Fab fragments, Fab2 fragments, etc.). Unless otherwise specified, the domain order of the protein molecules described herein is "N-terminal to C-terminus".

[0057] This disclosure specifically covers single-chain variable domain fragments ("scFv") comprising humanized anti-B7-H3-VL and / or VH domains. Single-chain variable domain fragments comprise VL and VH domains linked together using short "linker" peptides. Such linkers can be modified to provide additional functionality, such as allowing attachment of drugs or allowing attachment to solid supports. Single-chain variants can be recombinantly or synthetically produced. For the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, which can be a eukaryotic cell, such as yeast, plant, insect, or mammalian cell, or a prokaryotic cell, such as *Escherichia coli*. The polynucleotide encoding the scFv of interest can be prepared by conventional procedures, such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0058] This disclosure specifically covers binding molecules (including antibodies and biantibodies) comprising the VL and / or VH domains of humanized antibodies. The term "humanized" antibody refers to a chimeric molecule typically prepared using recombinant techniques, having an epitope binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The polynucleotide sequence of the variable domains of such antibodies can be used for genetic manipulation to produce such derivatives and improve the affinity or other properties of such antibodies. It is known that both the heavy and light chain variable domains contain three complementarity-determining regions (CDRs) that vary in response to the antigen discussed and determine binding capacity, flanked by four frame regions (FRs) that are relatively conserved in a given species and presumably provide scaffolding for the CDRs. When preparing non-human antibodies against a specific antigen, the variable domains can be "reshaped" or "humanized." A general principle for humanizing antibodies is to retain the basic sequence of the antibody epitope binding portion while exchanging the non-human portion of the antibody with the human antibody sequence. Humanization of monoclonal antibodies involves four general steps. These steps are: (1) determining the nucleotide and predicted amino acid sequences of the variable domains of the light and heavy chains of the starting antibody; (2) designing the humanized or canine antibody, i.e., deciding which antibody framework region to use in the humanization or canine conversion process; (3) the actual humanization or canine conversion method / technique; and (4) transfection and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

[0059] Numerous humanized antibody molecules containing epitope binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies with rodent or modified rodent variable domains and their associated complementarity-determining regions (CDRs) fused with human constant domains (see, for example, Lobuglio et al., (1989) "Mouse / HumanChimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989)). Other references describe rodent CDRs grafted into human supporting frame regions (FRs) prior to fusion with appropriate human antibody constant domains (see, for example, Riechmann, L. et al., (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; and Jones et al., (1986) "Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse," Nature 321:522-525). Another reference describes rodent CDRs supported by recombinant veneer rodent frame regions. See, for example, European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize unwanted immune responses to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of these portions in human recipients. 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 U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; and 5,866,692 disclose other methods for utilizing humanized antibodies. In some aspects, humanized antibodies retain all CDR sequences (e.g., humanized mouse antibodies, which contain all six CDRs derived from mouse antibodies).In other respects, humanized antibodies have one or more CDRs (one, two, three, four, five or six) that are different in sequence from the original antibody. B. Characteristics of antibody constant domains 1. Constant structural domains of light chains

[0060] As noted above, each light chain of an antibody contains a variable domain ("VL") and a constant domain ("CL").

[0061] The representative CL domain is the human IgG CLκ domain. The amino acid sequence of the human CLκ domain is (SEQ ID NO:1): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[0062] Another representative CL domain is the human IgG CLλ domain. The amino acid sequence of the human CLλ domain is (SEQ ID NO:2): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQSNNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS 2. Constant structural domains of heavy chains

[0063] As noted above, the heavy chain of an antibody may include a CH1, hinge domain, and CH2 and CH3 constant domains. The CH1 domains of the two heavy chains of the antibody complex with the CL constant region of the antibody light chain and are attached to the CH2 domain of the heavy chain via an intervening hinge domain.

[0064] The representative CH1 domain is the human IgG1 CH1 domain. The amino acid sequence of the human IgG1 CH1 domain is (SEQ ID NO:3): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV

[0065] Another representative CH1 domain is the human IgG4 CH1 domain. The amino acid sequence of the human IgG4 CH1 domain is (SEQ ID NO:4): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

[0066] The representative hinge domain is the human IgG1 hinge domain. The amino acid sequence of the human IgG1 hinge domain is (SEQ ID NO:5): EPKSCDKTHTCPPCP.

[0067] Another representative hinge domain is the human IgG4 hinge domain. The amino acid sequence of the human IgG4 hinge domain is (SEQ ID NO:6): ESKYGPPCPSCP. The IgG4 hinge domain can include stable mutations, such as the S228P substitution. The amino acid sequence of the S228P stable human IgG4 hinge domain is (SEQ ID NO:7): ESKYGPPCPPCP.

[0068] The CH2 and CH3 domains of the two heavy chains of an antibody interact to form an "Fc domain," which is a domain recognized by cellular Fc receptors, including but not limited to the Fc γ receptor (FcγR). As used herein, the term "Fc domain" is used to define the C-terminal region of the IgG heavy chain. An Fc domain is said to possess that specific IgG isotype, class, or subclass if its amino acid sequence is most homologous to other IgG isotypes. In addition to their known diagnostic uses, antibodies have also shown usefulness as therapeutic agents.

[0069] The amino acid sequence of the CH2-CH3 domain of representative human IgG1 is (SEQ ID NO:8): As described in Kabat, the EU index number is used, where X is lysine (K) or is absent.

[0070] The amino acid sequence of the CH2-CH3 domain of representative human IgG4 is (SEQ ID NO:9): As described in Kabat, the EU index number is used, where X is lysine (K) or is absent.

[0071] Throughout this specification, the residue numbers in the constant region of the IgG heavy chain are those of the EU index from Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, NH1, MD (1991) (which is explicitly incorporated herein by reference). The term “EU index in Kabat” refers to the numbering of the constant domain of the human IgG1 EU antibody.

[0072] Polymorphisms have been observed at many different locations in antibody constant regions (e.g., Fc positions, including but not limited to EU index numbers 270, 272, 312, 315, 356, and 358 as described in Kabat), and therefore the presented sequences may differ slightly from prior art sequences. Polymorphic forms of human immunoglobulins have been well characterized. 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. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). In particular, the antibodies of this disclosure can be incorporated into any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to allotypes, isoallotypes, or haplotypes of the sequences provided herein. Furthermore, in some expression systems, the C-terminal amino acid residue of the CH3 domain (bold above) can be removed post-translationally. Therefore, the C-terminal residue of the CH3 domain is optional. This disclosure specifically covers B7-H3-ADC lacking the C-terminal residue of the CH3 domain. This disclosure also specifically covers such constructs including the C-terminal lysine residue of the CH3 domain.

[0073] This disclosure specifically covers B7-H3-ADCs that include anti-B7-H3 variable domains (i.e., VL and / or VH domains) and that bind immunospecifically to epitopes of human B7-H3 peptides. Such B7-H3-ADCs are capable of immunospecifically 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. II. Anti-B7-H3 antibody mAb-A

[0074] A representative anti-B7-H3 antibody (designated "mAb-A") was isolated from hybridoma cells generated by immunization with cells expressing human B7-H3 (which possess the B7-H3 polypeptide or its peptide epitope). Antibody mAb-A is humanized.

[0075] Antibody mAb-A has been found to be cross-reactive with B7-H3 from cynomolgus monkeys. The amino acid sequences of the VL and VH domains of mAb-A are provided below. The B7-H3-ADC has all three CDRs of the VH domain. H All three CDRs of the VL domain L , and the entire VH and VL domains of the optionally humanized monoclonal antibody mAb-A (“hmAb-A”). A. Mouse anti-B7-H3 antibody mAb-A

[0076] The amino acid sequence (SEQ ID NO:10) of the VL domain of mouse anti-B7-H3 antibody mAb-A is shown below (CDR). L Residues are shown underlined: DIQMTQSPAS LSVSVGETVT ITC RASESIY SYLA WYQQKQ GKSPQLLVY N TKTLPE GVPSRFSGSGSGTQ FSLKINSLQP EDFGRYYC QH HYGTPPWT FG GGTNLEIK

[0077] The amino acid sequence (SEQ ID NO:11) of the VH domain of anti-B7-H3 antibody mAb-A is shown below (CDR). H Residues are shown underlined: EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMS WVRQT PDKRLEWVA T INSGGSNTYY PDSLKGRFTI SRDNAKNTLY LQMRSLKSED TAMYYCAR HD GGAMDY WGQG TSVTVSS B. Humanized anti-B7-H3 antibody hmAb-A

[0078] The variable domain of the anti-B7-H3 antibody mAb-A is humanized to generate humanized mAb-A (“hmAb-A”). In some cases, optional humanized variable domains are generated to optimize binding activity and / or remove antigenic epitopes and / or remove potentially unstable amino acid residues.

[0079] The amino acid sequence of the VL domain of hmAb-A (SEQ ID NO:12) is shown below (CDR). L Residues are shown underlined: DIQMTQSPSS LSASVGDRVT ITC RASESIY SYLA WYQQKP GKAPKLLVY N TKTLPE GVPSRFSGSGSGTD FTLTISSLQP EDFATYYC QH HYGTPPWT FG QGTRLEIK

[0080] The amino acid sequence (SEQ ID NO:13) of the light chain of hmAb-A containing the VL domain and CLκ domain of hmAb-A is shown below: DIQMTQSPSS LSASVGDRVT ITCRASESIY SYLAWYQQKP GKAPKLLVYN TKTLPEGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQH HYGTPPWTFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGTASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQGLSSPVTKSF NRGEC

[0081] In sequence SEQ ID NO:13, amino acid residues 1-108 correspond to the VL domain of hmAb-A (SEQ ID NO:12), and amino acid residues 109-215 correspond to the κ constant region of the light chain (SEQ ID NO:1).

[0082] The amino acid sequence of the VH domain of hmAb-A (SEQ ID NO:14) is shown below (CDR). H Residues are shown underlined: EVQLVESGGGLVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HD GGAMDY WGQG TTVTVSS

[0083] The amino acid sequence (SEQ ID NO:15) of the heavy chain containing the VH domain of hmAb-A and the CH1-H-CH2-CH3 domain of IgG1 is shown below: EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLEWVAT INSGGSNTYYPDSLKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHD GGAMDYWGQG TTVTVSSAST KGPSVFPLAPSSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQY N STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKGQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X Where X is lysine (K) or is absent.

[0084] In SEQ ID NO:15, amino acids 1-117 correspond to the VH domain of hmAb-A (SEQ ID NO:14), amino acid residues 118-215 correspond to the CH1 domain of IgG1 (SEQ ID NO:3), amino acid residues 216-230 correspond to the hinge domain of IgG1 (SEQ ID NO:5), and amino acid residues 231-447 correspond to the CH2-CH3 domain of IgG1 (SEQ ID NO:8). An N-linked glycosylation site is present at Kabat position 296 (underlined). The C-terminal residue "X" is lysine (K) or absent.

[0085] CDR L The amino acid sequence of domain 1 is (SEQ ID NO:16): RASESIYSYLA.

[0086] CDR L The amino acid sequence of domain 2 is (SEQ ID NO:17): NTKTLPE.

[0087] CDR L The amino acid sequence of the 3-domain is (SEQ ID NO:18): QHHYGTPPWT.

[0088] CDR H The amino acid sequence of domain 1 is (SEQ ID NO:19): SYGMS.

[0089] CDR H The amino acid sequence of domain 2 is (SEQ ID NO:20): TINSGGSNTYYPDSLKG.

[0090] CDR H The amino acid sequence of the 3-domain is (SEQ ID NO:21): HDGGAMDY. C. Alternative humanized anti-B7-H3 antibody hmAb-A

[0091] Alternative humanized variable domains are generated to optimize binding activity and / or remove antigenic epitopes and / or remove potentially unstable amino acid residues. The amino acid sequence of the VL domain of hmAb-A (SEQ ID NO: 12) and the amino acid sequence of the light chain of hmAb-A containing the VL domain and CLκ domain of hmAb-A (SEQ ID NO: 13) are shown above.

[0092] The amino acid sequence of the alternative VH domain of hmAb-A (SEQ ID NO:22) is shown below (CDR). H(Residues are shown underlined). EVQLVESGGGLVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HX GGAMDY WGQG TTVTVSS

[0093] In some respects, alternative CDRs H The amino acid sequence of the 3-domain structure is (SEQ ID NO:23): H X GGAMDY. In some respects, alternative CDRs. H The 3-domain residue "X" can be any amino acid. In some respects, alternative CDRs are possible. H The 3-domain residue "X" is aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q). For example, the amino acid sequence of an exemplary alternative VH domain of hmAb-A (SEQ ID NO:24) is shown below (CDR). H Residues are shown underlined), where residue "X" is glutamic acid (E). EVQLVESGGGLVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HE GGAMDY WGQG TTVTVSS

[0094] An exemplary alternative heavy chain amino acid sequence (SEQ ID NO:25) containing the VH domain of hmAb-A and the CH1-H-CH2-CH3 domain of IgG1 is shown below: EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLEWVAT INSGGSNTYYPDSLKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARH EGGAMDYWGQG TTVTVSSAST KGPSVFPLAPSSKSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQY N STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKGQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X Where X is lysine (K) or is absent.

[0095] In SEQ ID NO:25, amino acids 1-117 correspond to the VH domain of hmAb-A (SEQ ID NO:24), amino acid residues 118-215 correspond to the IgG1 CH1 domain (SEQ ID NO:3), amino acid residues 216-230 correspond to the IgG1 hinge domain (SEQ ID NO:5), and amino acid residues 231-447 correspond to the IgG1 CH2-CH3 domain (SEQ ID NO:8). An N-linked glycosylation site is present at Kabat position 296 (underlined). The C-terminal residue "X" is lysine (K) or absent.

[0096] Exemplary alternative CDRs H The amino acid sequence of the 3-domain is (SEQ ID NO:26): HEGGAMDY. III. Modification of the Fc structural domain

[0097] The Fc domain of a molecule containing an Fc domain (e.g., antibodies and biantibodies) can be a complete Fc domain (e.g., the complete IgG Fc domain) or just a fragment of an Fc domain. Optionally, the Fc domain of a molecule containing an Fc domain lacks a C-terminal lysine amino acid residue.

[0098] In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system leads to a wide range of responses, from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signaling, such as regulation of lymphocyte proliferation and antibody secretion. All these interactions are initiated by the binding of the Fc domain of an antibody or immune complex to specialized cell surface receptors (individually referred to as “Fcγ receptors”, “FcγR”, and collectively as “FcγR”) found on the surfaces of various types of immune system cells, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. The diversity of cellular responses triggered by antibodies and immune complexes stems from the structural heterogeneity of three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A), and FcγRIII (CD16) are activating (i.e., immune system-enhancing) receptors; FcγRIIB (CD32B) is an inhibitory (i.e., immune system-suppressing) receptor. Furthermore, interaction with the neonatal Fc receptor (FcRn) mediates the recirculation of IgG molecules from the endostomy to the cell surface and their release into the bloodstream. The amino acid sequences of representative wild-type IgG1 (SEQ ID NO:8) and representative wild-type IgG4 (SEQ ID NO:9) are presented above.

[0099] Modification of the Fc domain can lead to altered phenotypes, such as altered serum half-life, altered stability, altered sensitivity to cellular enzymes, or altered effector function. Therefore, in some respects, the Fc domain of a molecule containing an Fc domain can be an engineered variant of the Fc domain. While the Fc domain of a molecule containing an Fc domain may have the ability to bind to one or more Fc receptors (e.g., FcγR), in particular, such variant Fc domains will alter the binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc domain), for example, enhancing the binding to activating receptors and / or having a significantly reduced ability to bind to inhibitory receptors or no ability to bind to inhibitory receptors. Therefore, the Fc domain of a molecule containing an Fc domain may include part or all of the CH2 domains and / or part or all of the CH3 domains of the intact Fc domain, or may include variant CH2 and / or variant CH3 sequences (which may include, for example, one or more insertions and / or one or more deletions of the CH2 or CH3 domains relative to the intact Fc domain). Such Fc domains may include non-Fc polypeptide portions, or may include portions of non-natural intact Fc domains, or may include CH2 and / or CH3 domains with non-natural orientations (such as, for example, two CH2 domains or two CH3 domains, or a CH3 domain attached to a CH2 domain in the N-terminal to C-terminal orientation, etc.).

[0100] In some respects, the Fc domain of the binding molecule exhibits reduced (or essentially no) binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type IgG1 Fc domain (SEQ ID NO:8). In some respects, the binding molecule includes an IgG Fc domain exhibiting reduced ADCC effector function. In such respects, the CH2-CH3 domain of the binding molecule includes any one, two, three, or four of the following substitutions: L234A, L235A, D265A, N297Q, and N297G. In another respect, the CH2-CH3 domain contains N297Q substitution, N297G substitution, L234A and L235A substitution, or D265A substitution, because these mutations eliminate FcR binding. Alternatively, the CH2-CH3 domain of a naturally occurring Fc domain may be used, which inherently exhibits reduced (or substantially no) binding to FcγRIIIA (CD16a) and / or diminished effector function (relative to the binding and effector function exhibited by the wild-type IgG1 Fc domain (SEQ ID NO:8)). In a particular aspect, the binding molecule includes the IgG4 Fc domain (SEQ ID NO:9). When using the IgG4 Fc domain, this disclosure also covers the introduction of stabilizing mutations, such as the hinge domain S228P substitution described herein (see, for example, SEQ ID NO:7).

[0101] The serum half-life of proteins containing an Fc domain can be increased by increasing the binding affinity of the Fc domain to FcRn. As used herein, the term "half-life" refers to the pharmacokinetic property of a molecule, which is a measure of the mean survival time of the molecule after administration. Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known number of molecules from the body of a subject (e.g., a human patient or other mammal) or from a specific compartment thereof, for example, as measured in serum (i.e., circulating half-life), or in other tissues. Generally, an increase in half-life results in an increase in the mean residence time (MRT) of the administered molecule in circulation. Modifications capable of increasing the half-life of molecules containing an Fc domain are known in the art and include, for example, M252Y, S254T, T256E, and combinations thereof. For example, see the modifications described in U.S. Patent Nos. 6,277,375, 7,083,784, 7,217,797 and 8,088,376; and U.S. Publications Nos. 2002 / 0147311, 2007 / 0148164 and 2011 / 0081347. IV.B7-H3-ADC

[0102] This disclosure relates to the anti-B7-H3 antibody hmAb-A, "B7-H3-ADC", conjugated with a cytotoxic drug as described above. Such B7-H3-ADCs enhance the cytotoxicity of anti-B7-H3 therapies, particularly in the treatment of cancer. As noted above, a B7-H3-ADC is represented by the following formula: Ab-(LM) m -(D) n , in: Ab is an antibody or its B7-H3-binding fragment that contains a humanized variable heavy chain (VH) domain and a humanized variable light chain (VL) domain, and; D represents the cytotoxic drug component; LM is a linker molecule that covalently connects Ab and D; m is an integer between 1 and n and represents the number of bonded or linker molecules in B7-H3-ADC; and n is an integer between 1 and 10 and represents the number of cytotoxic camptothecin moieties covalently linked to B7-H3-ADC.

[0103] In some respects, B7-H3-ADC contains the naturally occurring Fc domain of the IgG1 isotype. This type of Fc domain lacks the C-terminal lysine residue of the CH3 domain. In specific respects, B7-H3-ADC binds to tumor cells expressing B7-H3 and is then internalized into these cells via receptor-mediated endocytosis. Once inside the lysosome, B7-H3-ADC can be degraded, resulting in the release of the intracellular cytotoxic camptothecin moiety, leading to cell death. As will be understood, the mechanism of cell death can vary depending on the class of cytotoxic drug used. When dying cells release free drugs into the tumor environment during a process known as the bystander effect, neighboring cancer cells may also be killed (Panowski, S et al., (2014) "Site-Specific Antibody Drug Conjugates For Cancer Therapy," mAbs 6(1):34-45; Kovtun, YV et al., (2006) "Antibody-Drug Conjugates Designed To Eradicate Tumors With Homogeneous And Heterogeneous Expression Of The Target Antigen," Cancer Res. 66:3214-3221).

[0104] In some respects, the Ab of B7-H3-ADC is the B7-H3 antibody as described above.

[0105] In some respects, the Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) contains the CDRL1 sequence RASESIYSYLA (SEQ ID NO:16), the CDRL2 sequence NTKTLPE (SEQ ID NO:17), and the CDRL3 sequence QHHYGTPPWT (SEQ ID NO:18) in its variable light chain (VL) domain, and (ii) contains the CDRH1 sequence SYGMS (SEQ ID NO:19), the CDRH2 sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20), and the CDRH3 sequence HDGGAMDY (SEQ ID NO:21) or HEGGAMDY (SEQ ID NO:26) in its variable heavy chain (VH) domain.

[0106] In some respects, Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) in its variable light chain (VL) domain, comprises the CDRL1 sequence RASESIYSYLA (SEQ ID NO:16), the CDRL2 sequence NTKTLPE (SEQ ID NO:17), and the CDRL3 sequence QHHYGTPPWT (SEQ ID NO:18), and (ii) in its variable heavy chain (VH) domain, comprises the CDRH1 sequence SYGMS (SEQ ID NO:19), the CDRH2 sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20), and the CDRH3 sequence HDGGAMDY (SEQ ID NO:21).

[0107] In some respects, Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) comprises the CDRL1 sequence RASESIYSYLA (SEQ ID NO:16), the CDRL2 sequence NTKTLPE (SEQ ID NO:17), and the CDRL3 sequence QHHYGTPPWT (SEQ ID NO:18) in its variable light chain (VL) domain, and (ii) comprises the CDRH1 sequence SYGMS (SEQ ID NO:19), the CDRH2 sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20), and the CDRH3 sequence HEGGAMDY (SEQ ID NO:26) in its variable heavy chain (VH) domain.

[0108] In some respects, Ab includes: (i) a humanized VL domain comprising the amino acid sequence SEQ ID NO:12, and (ii) a humanized VH domain comprising the amino acid sequence SEQ ID NO:14 or SEQ ID NO:24.

[0109] In some respects, Ab includes: (i) a humanized VL domain comprising the amino acid sequence SEQ ID NO:12, and (ii) a humanized VH domain comprising the amino acid sequence SEQ ID NO:14.

[0110] In some respects, Ab includes: (i) a humanized VL domain comprising the amino acid sequence SEQ ID NO:12, and (ii) a humanized VH domain comprising the amino acid sequence SEQ ID NO:24.

[0111] In some respects, this disclosure provides antibody Abs that have undergone glycan remodeling. Examples of glycan remodeling are further described below. In some respects, the Abs have undergone glycan remodeling and include azidoses. In some respects, the azidoses are GalNAz (N-azidoacetylgalactosamine), 6-azido-Gal, or 6-azido-GalNAc. A. Connector molecule

[0112] This disclosure particularly considers B7-H3-ADCs having one or more linker molecules LM (i.e., m is an integer from 2 to n, where n is an integer from 2 to 10), each linker molecule LM covalently attaching the camptothecin D moiety to the Ab of such B7-H3-ADC.

[0113] This disclosure further provides a B7-H3-ADC, wherein the Ab is linked to more than one linker molecule LM, wherein all such linker molecules are identical. The camptothecin moiety D covalently linked to the Ab of such a B7-H3-ADC may be all identical, or may include 2, 3, 4 or more independent and distinct camptothecin moieties D.

[0114] This disclosure further provides such B7-H3-ADCs, wherein the Ab is linked to more than one linker molecule LM, wherein all such linker molecules are different and can be independently distinct. The camptothecin moiety D of the Ab linked to such B7-H3-ADCs can be all identical or can include 2, 3, 4 or more independently distinct camptothecin moieties D.

[0115] The above provides humanized VH and VL domains of antibodies binding to human B7-H3, as well as constant domains of human antibodies that can be included in B7-H3-ADCs. As described above, B7-H3-ADCs further include at least one cytotoxic drug moiety, which is covalently linked via amino acid residues of such VH or VL domains and / or constant domains through a linker molecule attached to the side chain and the drug moiety. The linker molecule can be a non-peptide molecule, a molecule containing both a non-peptide and a peptide moiety, or a molecule consisting only of amino acid residues. The amino acid residues of any such linker molecule can contain naturally occurring or non-naturally occurring amino acid residues, including D versions of naturally occurring amino acid residues, acetylphenylalanine, selenocysteine, etc. Optionally or additionally, specific residues having desired side chains (e.g., -CH2-SH side chain, -CH2-OH side chain, -CH(CH2)-SH side chain, -CH2-CH2-S-CH3 side chain; -CH2-C(O)-NH2 side chain, -CH2-CH2-C(O)-NH2 side chain, -CH2-C(O)OH- side chain, CH2-CH2-C(O)OH- side chain, -CH2-CH2-CH2-CH2-NH2 side chain, -CH2-CH2-CH2-NH-C(NH2)2 side chain, imidazole side chain, benzyl side chain, phenol side chain, indole side chain, etc.) can be engineered into B7-H3-ADC.

[0116] In some respects, the cytotoxic drug portion may be conjugated to the Ab of the B7-H3-ADC of this disclosure by methods known in the art (see, for example, WO2016053107; Yao, H. et al., (2016) "Methods to Design and Synthesize Antibody-Drug Conjugates (ADC)," Intl. J. Molec. Sci. 17(194):1-16); Behrens, CR 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). The thiol group of cysteine, the amino side group of lysine, glutamine, or arginine, or the carboxyl group of glutamic acid or aspartic acid can be employed to conjugate the linker molecule-cytotoxic drug moiety (LM-D) to the Ab of the B7-H3-ADC disclosed herein. Natural antibodies contain a large number of lysine conjugation sites and are therefore capable of linking multiple conjugation molecules per antibody. In fact, peptide mapping has identified conjugation occurring on both the heavy and light chains at approximately 20 different lysine residues (40 lysines per mAb). Therefore, more than one million different ADC species can be generated. Cysteine ​​conjugation occurs after the reduction of one to four interchain disulfide bonds and is therefore limited to eight exposed thiol groups in the native VL and VH domains. However, additional reactive residues (e.g., lysine, cysteine, selenocysteine, etc.) can be engineered into the antibody (e.g., within the VL and / or VH and / or constant domains) if desired. For example, one or more natural amino acid residues can be replaced by cysteine ​​residues.The amber-stop codon inhibitor tRNA / aaRS pair can be used to genetically integrate non-natural amino acids (e.g., acetylphenylalanine) into the antibody (see, for example, Behrens CR 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, an enzyme (e.g., glycosyltransferase) can be used to conjugate the adaptor molecule-cytotoxic drug moiety (LM-D) to the Ab of the B7-H3-ADC of this disclosure. A glycosyltransferase platform attaches the sugar moiety to a glycosylation site on an antibody (e.g., N297 of the Fc domain of a human IgG antibody), which can then serve as the adaptor molecule (LM) of this disclosure and couple the cytotoxic drug moiety (D) to the Ab of the B7-H3-ADC of this disclosure. Alternatively, transglutaminase can be used to catalyze the formation of a covalent bond between a free amino group and a glutamine side chain.

[0117] In some respects, the linker molecule LM attaches to the glycan moiety, which in turn attaches to the side chain of Ab. In some respects, the glycan moiety on the side chain is a naturally occurring glycan moiety. In some respects, the glycan moiety undergoes glycan remodeling before attaching to the linker molecule.

[0118] In some respects, glycan remodeling occurs by contacting the glycan moiety with an endoglucosidase and a glycosyltransferase. In some respects, glycan remodeling involves adding an azide sugar to the end of the glycan moiety. In some respects, the azide sugar is GalNAz (N-azidoacetylgalactosamine), 6-azido-Gal, or 6-azido-GalNAc. In some respects, a linker molecule LM attaches to the azide sugar on the glycan moiety. In some respects, the linker molecule LM attaches to the azide sugar on the glycan moiety by reacting the azido group on the azide sugar with a reactive group on the LM.

[0119] In some respects, glycan remodeling is performed by contacting the glycan moiety with an endoglycosidase and an N-acetylgalactosamine (GalNAc) transferase. In some respects, glycan remodeling adds an N-acetylgalactosamine (GalNAc) sugar to the end of the glycan moiety. In some respects, the GalNAc sugar includes an azide group. In some respects, a linker molecule LM attaches to the GalNAc sugar on the glycan moiety. In some respects, the linker molecule LM attaches to the GalNAc sugar on the glycan moiety through a reaction between the azide group on GalNAc and a reactive group on LM.

[0120] In some respects, the glycan reconstruction is performed using the GlycoConnect™ process (Synaffix). In other respects, the glycan reconstruction is performed as described in U.S. Patents US9,504,758, US10,745,488, US9,988,661, US10,858,641, US9,222,940, US10,239,807, and US11,358,921 (each of which is incorporated herein by reference).

[0121] In some respects, the linker molecule LM can be non-cleavable under physiological conditions, for example, by including hydrolyzable portions such as thioether linkers or hindered disulfide linkers. Hydrolyzable linkers are substantially stable in water and do not react with water at a useful pH, including but not limited to maintaining their position for an extended period under physiological conditions. Hydrolyzable or degradable linkers, on the other hand, are degradable in water or aqueous solutions (including, for example, blood).

[0122] In some alternative aspects, the linker molecule LM can be cleavable or may contain a cleavable moiety. Examples of such cleavable moieties include acid-labile linkers (e.g., 4-(4'-acetylphenoxy)butyric acid linkers that form hydrazine bonds), cleavable disulfide linkers (which are cleaved in a reducing intracellular environment), and protease-cleavable linkers. Acid-labile linkers are designed to be stable at the pH levels encountered in the blood but become unstable and degrade when encountering the low pH environment in lysosomes. Protease-cleavable linkers are also designed to be stable in blood / plasma but rapidly release free drugs into lysosomes in cancer cells after being 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 LM 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-alanine dipeptide-aminobenzyl alcohol linker (cAC10-mc-va-PABA), a valine-alanine dipeptide-aminobenzyl alcohol ester (Val-Ala-PABC or VA-PABC), a valine-citrulline dipeptide-aminobenzyl alcohol linker (cAC10-mc-vc-PABA), or a valine-citrulline dipeptide-aminobenzyl alcohol ester (Val-Cit-PABC or VC-PABC), which is selectively cleaved by lysosomal enzymes).Suitable cleavable linkers are known in the art; see, for example, de Groot, Franciscus MH, 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, Lysosomally-Cleavable Dipeptide Linkages." Bioorganic & Medicinal Chemistry Letters 12:1529-1532; U.S. Patent Nos. 5,547,667, 6,214,345, 7,585,491, 7,754,681, 8,080,250, 8,461,117 and WO 02 / 083180.

[0123] In some aspects, enzyme-instable or degradable linkers may be employed. Such linkers are degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linker molecules in the polymer backbone or in the linking groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linker molecules include, but are not limited to, ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolyzable linker molecules include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of amines and aldehydes; phosphate ester linkages formed by the reaction of alcohols with phosphate groups; hydrazone linkages that are products of the reaction of hydrazides and aldehydes; acetal linkages that are products of the reaction of aldehydes and alcohols; orthoester linkages that are products of the reaction of formate esters and alcohols; peptide linkages formed by amine groups and carboxyl groups of peptides (including, but not limited to, at the ends of polymers such as PEG); and oligonucleotide linkages formed by phosphoramidite groups and 5' hydroxyl groups of oligonucleotides (including, but not limited to, at the ends of polymers).

[0124] In some respects, the linker molecule LM includes a peptide linker. In some respects, the peptide linker is a valine-alanine dipeptide linker. In some respects, the linker molecule includes a cleavable linker. In some respects, the linker molecule includes a valine-alanine (Val-Ala) amino acid linker. In some respects, the Val-Ala linker is a Val-Ala-PABC linker.

[0125] In some respects, peptide linkers are valine-citrulline dipeptide linkers. In some respects, linker molecules include cleavable linkers. In some respects, linker molecules include valine-citrulline (Val-Cit) amino acid linkers. In some respects, Val-Cit linkers are Val-Cit-PABC linkers.

[0126] In one aspect, the linker molecule LM can be or may include a cleavable linker molecule comprising formula (4a) or (4b), or a salt thereof: in: a is either 0 or 1 independently; b is either 0 or 1 independently; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1 to 150; g is 0 or 1; i is 0 or 1; D represents the cytotoxic drug component; Q 1 It is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclic triazole group, or a cycloalkenyl group; Q 1 Functional groups attached to antibodies; Sp 1 Sp 2 Sp 3 and Sp 4 Independently select from the following groups: straight chain or branched chain C1-C 200 alkylene groups, C2-C 200 imide groups, C2-C 200 acetylenic group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkenyl groups, C8-C 200 Cycloynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imidene groups and C9-C 200The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynynyl group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally substituted by one or more heteroatoms and optionally spaced by one or more heteroatoms, wherein the one or more heteroatoms are selected from O, S, and NR. 3 The group, where R 3 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted; Z 1 It is Q 1 or SP 3 Connect to SP 2 O or C(O) or N(R) 1 () linking group; Z 2 Is it D or SP? 4 Connect to SP 1 、N(R 1 ), O or C(O) linking groups; Z 1 and Z 2 Choose independently from the following groups: -O-, -S-, -NR 2 -、-N=N-、-C(O)-、-C(O)NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 -NR 2 -C(O)-、-NR 2 -C(O)-O-、-NR 2 -C(O)-NR 2 -, -SC(O)-, -SC(O)-O-, -SC(O)-NR 2 -, -S(O)-, -S(O)2-, -OS(O)2-, -OS(O)2-O-, -OS(O)2-NR 2 -, -OS(O)-, -OS(O)-O-, -OS(O)-NR 2 -、-O-NR 2 -C(O)-、-O-NR 2 -C(O)-O-、-O-NR 2 -C(O)-NR 2 -、-NR2 -OC(O)-、-NR 2 -OC(O)-O-、-NR 2 -OC(O)-NR 2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-、-O-NR 2 -C(S)-NR 2 -、-NR 2 -OC(S)-、-NR 2 -OC(S)-O-、-NR 2 -OC(S)-NR 2 -, -OC(S)-, -OC(S)-O-, -OC(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-、-NR 2 -C(S)-NR 2 -, -SS(O)2-, -SS(O)2-O-, -SS(O)2-NR 2 -、-NR 2 -OS(O)-、-NR 2 -OS(O)-O-、-NR 2 -OS(O)-NR 2 -、-NR 2 -OS(O)2-、-NR 2 -OS(O)2-O-、-NR 2 -OS(O)2-NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O)2-O-、-O-NR 2 -S(O)2-NR 2 -、-O-NR 2 -S(O)2-、-OP(O)(R 2 )2-、-SP(O)(R 2 )2-、-NR 2 -P(O)(R 2 )2- and combinations of two or more of them, where R 2 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, ynyl groups, and cycloalkyl groups are optionally substituted; and R 1 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, the C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 The (hetero)arylalkyl group is optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, said one or more heteroatoms being selected from O, S, and NR. 3 , where R 3 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 It is D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 Sp 2 Sp 3 Sp 4 Z 1 Z 2 D, Q 1 b, c, d, e, g, and i are as defined above.

[0127] In some respects, Q1 is a triazole derivative of cyclooctyne.

[0128] In some respects, the connector molecule LM further includes Sp 1 Sp 2 Sp 3 and Sp 4If it exists, it is independently selected as either a straight chain or a branched chain C1-C. 20 The group consisting of alkylene groups, wherein the alkylene groups are optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, wherein the one or more heteroatoms are selected from O, S, and NR. 3 The group consisting of R 3 The group consisting of hydrogen and C1-C4 alkyl groups is selected independently.

[0129] In some respects, a detachable connector is a connector as disclosed in U.S. Patent No. 9,636,421, the disclosure of which is incorporated herein by reference.

[0130] In some respects, the linker molecule LM comprises one or more polar spacers. In some respects, the polar spacers are carbamoyl sulfonamides. Examples of usable spacers can be found in U.S. Patent Nos. US9,636,421 and US10,792,369 (which are incorporated herein by reference).

[0131] In some aspects, the B7-H3-ADC comprises two, three, four, five, six, seven, eight, nine, or ten cytotoxic drug moieties, which may be identical or independently identical or different from another cytotoxic drug moiety of the B7-H3-ADC. In one aspect, each such cytotoxic drug moiety is coupled to the Ab of the B7-H3-ADC via a separate adapter molecule. Alternatively, more than one cytotoxic drug moieties may be attached to the Ab of the B7-H3-ADC via the same adapter molecule.

[0132] In some respects, this disclosure contemplates a B7-H3-ADC comprising one or more linker molecules, specifically the camptothecin moiety D, wherein LM and D together comprise: B. Exemplary Cytotoxic Drugs Section

[0133] In some respects, the cytotoxic drug portion of B7-H3-ADC includes cytotoxins, radioisotopes, immunomodulators, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factors, hormones, hormone antagonists, enzymes, oligonucleotides, DNA molecules, RNA molecules, siRNA molecules, RNAi molecules, microRNA molecules, photoactive therapeutic agents, anti-angiogenic agents, pro-apoptotic agents, peptides, lipids, carbohydrates, chelating agents, or combinations thereof. 1. Topoisomerase inhibitors

[0134] In some respects, B7-H3-ADC may include a topoisomerase inhibitor cytotoxic drug portion. As used herein, the term "topoisomerase inhibitor" or "DNA topoisomerase inhibitor" refers to a compound that interferes with topoisomerase activity.

[0135] Topoisomerases play a crucial role in cell proliferation and replication, altering the supercoil of double-stranded DNA by catalyzing the breakage and rejoining of the phosphodiester backbone of the DNA strand during the normal cell cycle. DNA strand dissociation is essential for transcription and replication of the genome, which is performed by RNA and DNA polymerases (Pommier et al., “DNA topooisomerases and their poisoning by anticancer and antibacterial drugs,” Chem. & Biol. Review 17 (2010) 421-433). Due to the double helix structure of DNA, replication produces tandem progeny, which must be unwound by topoisomerases before cytokinesis. The two enzymes that play a role in this unwinding and rewinding process are topoisomerase I and topoisomerase II. They also play an important role in repairing DNA damage that occurs as a result of exposure to DNA damaging agents, such as radiation exposure or chemotherapy.

[0136] Topoisomerases are classified into type I and type II. Type I enzymes cleave one DNA strand at a time, while type II enzymes cleave two strands at a time to perform their catalytic function. All topoisomerases cleave the DNA phosphodiester backbone by catalyzing the nucleophilic attack of tyrosine residues linked to the phosphate terminus (PY) of the DNA break. These reactions are highly reversible, and the DNA sequence remains unchanged after topoisomerization (Pommier et al., “DNA topoisomerases and their poisoning by anticancer and antibacterial drugs,” Chem. & Biol. Review 17(2010) 421-433).

[0137] Numerous topoisomerase type I (Topo 1) inhibitors have been evaluated as anticancer therapeutic agents. Camptothecin was initially identified from Camptotheca acuminate (Wall et al., “The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminate,” J.Am. Chem. Soc. (1966) 88: 3888-3890). Numerous camptothecin derivatives, including, for example, topotecan, irinotecan, belotetan, gemmatostatin, letopotecan, diflunotecan, S39625, and eczetidine, have been further investigated as anticancer agents (Pommier et al., “DNA topoisomerases and their poisoning by anticancer and antibacterial drugs,” Chem. & Biol. Review 17 (2010) 421-433).

[0138] In addition to camptothecin derivatives, several non-camptothecin topoisomerase inhibitors have also been studied as anticancer agents, including indolocarbazole edotecarin, indomethacin NSC 706744 (MJ-III-65), NSC 725776 (LMP-776) and NSC 724998 (LMP-400), and dibenzonaphthyridiones such as topovale (ARC-111) and aromathinecinrosettacin (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433).

[0139] In one embodiment, the topoisomerase inhibitor is selected from topoisomerase I inhibitors. Known topoisomerase I inhibitors used in this invention include, but are not limited to: (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7] indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride (topotecan), (S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7] indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione (camptothecin), and (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-1 OH, 13H-benzopyrano(3′,4′:6,7) indene(1,2-b)quinoline-10,13-dione (exatecan), (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin (lurtotecan)) or (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14 -dioxo-1H-pyrano[3′,4′:6,7]-indo[1,2-b]quinoline-9-yl-[1,4′-bipiperidine]-1′-carboxylic acid ester (irinotecan), (R)-5-ethyl-9,10-difluoro-5-hydroxy-4,5-dihydrooxepino[3′,4′:6,7]indo[1,2-b]quinoline-3,15(1H, 13H)-dione (diflomotecan), (4S)-11-((E)-((1,1-dimethylethoxy)imino)methyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano(3′,4′:6,7) indo(1,2-b)quinoline-3,14(4H)-dione (gimatecan), (S)-8-ethyl-8-hydroxy-15-((4-methylpiperazin-1-yl) (Methyl)-11,14-dihydro-2H-[1,4]dioxane[2,3-g]pyrano[3′,4′:6,7] indo[1,2-b]quinoline-9,12(3H,8H)-dione (lurtotecan), (4S)-4-ethyl-4-hydroxy-11-[2-[(1-methylethyl)amino]ethyl]-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-3,14(4H, 12H)-dione (belotecan), 6-((1,3-dihydroxypropyl-2-yl)amino)-2,10-Dihydroxy-12-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (edotecarin), 8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxy-5H-dibenzo(c,h)(1,6)naphthidium-6-one (topovale), benzo[6,7]indo[1,2-b]quinoline-11(13H)-one (rosettacin), (S)-4-ethyl- 4-Hydroxy-11-(2-(trimethylsilyl)ethyl)-1H-pyrano[3′,4′:6,7] indano[1,2-b]quinoline-3,14(4H,12H)-dione (cositecan), tetra{(4S)-9-[([1,4′-bipiperidinyl]-1′-carbonyl)oxy]-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indano[1,2-b]quinoline-4-yl}N,N′,N″,N″′-{methanetetra[methylene poly(oxyethylene)oxy(1-oxyethylene)]}tetraglycine tetrahydrochloride (etirinotecan) pegol), 10-hydroxy-camptothecin (HOCPT), 9-nitrocamptothecin (rubitecan), SN38 (7-ethyl-10-hydroxycamptothecin) and 10-hydroxy-9-nitrocamptothecin (CPT109), (R)-9-chloro-5-ethyl-5-hydroxy-10-methyl-12-((4-methylpiperidin-1-yl)methyl)-4,5-dihydrooxazo[3′,4′:6,7] indo[1,2-b]quinoline-3,15(1H,13H)-dione (elmotecan).

[0140] In addition to Topo 1 inhibitors, a large number of anticancer agents targeting topoisomerase type II (Topo 2) enzymes have been investigated, including etoposide, teniposide, and DNA intercalation agents doxorubicin, daunorubicin, azarubicin, acridine, dexrazoxane, TAS-103, quinolones CP-115,963, ellipticine (including ellipticinium), azatoxin, genistein, VP-16, VM-26, mitoxantrone, amonafidem, and saintopin.

[0141] In one embodiment, the topoisomerase inhibitor is selected from topoisomerase II inhibitors. Known topoisomerase II inhibitors used in this invention include, but are not limited to: amonafide and its derivatives and analogs, etoposide, teniposide, doxorubicin, daunorubicin, azorubicin, acridine, dexrazoxane, TAS-103, quinolones CP-115,963, rose alkaloids including ellipticinium, azathioprine, genistein, VP-16, VM-26, mitoxantrone, amonafidem, and santoprine. 2. Camptothecin as a Cytotoxic Drug

[0142] In some respects, B7-H3-ADC may contain the camptothecin cytotoxic drug portion:

[0143] As used herein, the term "camptothecin" refers to a class of compounds that are considered camptothecin, camptothecin analogues, camptothecin derivatives, or camptothecin conjugates. These compounds are based on the pentacyclic skeleton characteristic of camptothecin.

[0144] Camptothecin (CPT) is a plant alkaloid discovered in the late 1950s to possess anticancer activity. Both substituted and unsubstituted camptothecin are thought to interfere with the mechanism of action of the ribozyme topoisomerase I (topo I), causing cell arrest in the S phase. Freely confined by theory, CPT is believed to achieve this by stabilizing the covalently linked DNA-topo I complex (called the cleavable complex), thereby preventing the progression of the replication fork. Collisions between the replication fork and the cleavable complex are thought to trigger the apoptosis pathway. (Z. Darzynkiewicz et al., The Cell Cycle Effects of Camptothecin, 803 Annals of the New York Academy of Sciences 93 (1996)). DNA strand breaks are also involved in the cytotoxic effects of CPT. (F. Traganos et al., Induction of Apoptosis by Camptothecin and Topotecan, 803 Annals of the New York Academy of Sciences 101 (1996)).

[0145] Examples of camptothecins include SN-38 (S-10-hydroxycamptothecin), irinotecan (CAMPTOSAR; 7-ethyl-10-[4-(1-piperidinyl)-1-piperidinyl]-carbonyloxycamptothecin), topotecan (HYCAMPTIN; (S)-9-N,N-dimethylaminoethyl-10-hydroxycamptothecin), 9-aminocamptothecin (9-amino-20(S)-camptothecin), 9-nitrocamptothecin (also known as rubitecan), letopotecan (7-(4-methylpiperazinemyl)-10,11-ethylenedioxy-20(S)-camptothecin), eczetine, calenitine, and holocrothecin. Structural and clinical information for some camptothecin compounds can be found in Garcia-Carbonero et al. Clin. Cancer Res. (March 2002) 8: 641-661. Examples of camptothecin compounds can also be found in U.S. Patent Nos. 4,604,463, 6,403,569, and 5,004,758, and WO 2004 / 012661, WO 2003 / 101998, WO2003 / 101996, WO 2003 / 101406, WO 2003 / 093274, WO 2003 / 086471, WO 01 / 76597, WO 01 / 64194, WO 00 / 70275, WO 00 / 53607, WO 99 / 17805, WO 99 / 17804, WO 99 / 05103, WO 98 / 35969, WO 97 / 28164, WO 97 / 25332, and WO 97 / 16454. (The contents of all these references are found by reference in this article.)

[0146] SN-38 (S-10-hydroxycamptothecin), topotecan, irinotecan, belotetan, and deruxtecan are approved CPT analogs and are currently used in cancer chemotherapy. SN-38 Drotecon

[0147] In some respects, camptothecin is partly a cytotoxic drug, specifically eczema. 3. Pyrrolobenzodiazepine dimer

[0148] In some respects, B7-H3-ADC may include the cytotoxic drug portion of a pyrrolobenzodiazepine (PBD) dimer. PBD dimers irreversibly bind to two guanines from opposing DNA strands in the minor groove of DNA without twisting the double helix. Because PBD dimers do not twist the double helix, they are less likely to be removed by DNA repair mechanisms. In some respects, PBD dimers are dimer PBD compounds, such as those provided in U.S. Patent Nos. 6,562,806 and 11,135,303 (each of which is incorporated herein by reference in its entirety). 4. Oristatin

[0149] In some aspects, B7-H3-ADC may include the cytotoxic drug portion of olarestatin. In some aspects, olarestatin is monomethylolarestatin (MMAE). In some aspects, olarestatin is monomethylolarestatin F (MMAF). In some aspects, olarestatin is olarestatin E (AE). In some aspects, olarestatin is the olarestatin disclosed in U.S. Patent Nos. 5,208,020; 5,416,064; 6,333,410; 6,340,701; 6,372,738; 6,436,931; 6,441,163; 6,596,757; 7,276,497; 7,585,857; or 7,851,432 (each of which is incorporated herein by reference in its entirety). C. MGC026

[0150] In some respects, B7-H3-ADC is MGC026. MGC026 comprises the light and heavy chains of anti-B7-H3hmAb-A conjugated with eczetidine loading. The amino acid sequences of Ab, the cytotoxic eczetidine moiety D, and the adaptor molecule LM in MGC026 are shown below: Ab includes: (i) A light chain containing the amino acid sequence SEQ ID NO:13; and (ii) A heavy chain containing the amino acid sequence SEQ ID NO:15; D includes eczema; and LM includes the connector molecules described above.

[0151] In some respects, the linker molecule co-coupled to Ab and eczetidine have the following structures: . V. Generation Method

[0152] The disclosed anti-B7-H3 antibody hmAb-A can be recombinantly prepared and expressed using any method known in the art to produce a recombinant protein. For example, a nucleic acid encoding a polypeptide chain of such a binding molecule can be constructed, introduced into an expression vector, and expressed in a suitable host cell. The binding molecule can be recombinantly generated in bacterial cells (e.g., *E. coli* cells) or eukaryotic cells (e.g., CHO, 293E, COS, NSO cells). Furthermore, the binding molecule can be expressed in yeast cells, such as *Pichia pastoris* or *Saccharomyces cerevisiae*.

[0153] To generate the anti-B7-H3 antibody hmAb-A, one or more polynucleotides encoding the molecule can be constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover the molecule (see, for example, the techniques described in Green, MR et al., (2012), “Molecular Cloning, A Laboratory Manual”, 4th edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., eds., 1998, “Current Protocols in Molecular Biology”, John Wiley & Sons, NY). The expression vector should have features that allow the vector to replicate in the host cell. The vector should also have promoter and signaling sequences necessary for expression in the host cell. Such sequences are well known in the art. In addition to the nucleic acid sequence encoding such a binding molecule, the recombinant expression vector may carry other sequences, such as sequences regulating vector replication in the host cell (e.g., origin of replication) and selectable marker genes. Another approach is to express the gene sequence in plants (e.g., tobacco) or transgenic animals. Suitable methods for recombinantly expressing such binding molecules 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; U.S. Patent No. 5,849,992; and Pollock et al., (1999) "Transgenic Milk As A Method For The Production of Recombinant Antibodies," J. Immunol Methods 231:147-157).

[0154] Once the anti-B7-H3 antibody hmAb-A has been recombinantly expressed, it can be purified from inside or outside the host cell (e.g., from a culture medium) using any method known in the art for the purification of peptides or polyproteins. Separation and purification methods commonly used for antibody purification (e.g., antigen-selectivity-based antibody purification protocols) can be used to separate and purify such molecules and are not limited to any particular method. For example, methods include column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, ion exchange chromatography, affinity chromatography (especially affinity for a specific antigen), sizing column chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography (Marshak et al., (1996) “Strategies for Protein Purification and Characterization: A Laboratory Course Manual.” (ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY). VI. Pharmaceutical Composition

[0155] Pharmaceutical compositions for formulating the B7-H3 ADC described herein include raw material pharmaceutical compositions that can be used to prepare pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions that can be used to prepare dosage forms (i.e., compositions suitable for administration to subjects or patients). Such compositions include preventative or therapeutically effective amounts of B7-H3-ADC as described herein, or combinations of such agents and pharmaceutically acceptable carriers. Preferably, the pharmaceutical composition includes preventative or therapeutically effective amounts of B7-H3-ADC and a pharmaceutically acceptable carrier. Further consideration is given to such pharmaceutical compositions comprising a secondary therapeutic antibody (e.g., a tumor-specific monoclonal antibody) specific to a particular cancer antigen and a pharmaceutically acceptable carrier.

[0156] As used herein, the term "pharmaceutically acceptable carrier" refers to a diluent, solvent, dispersion medium, antibacterial and antifungal agent, excipient, or vehicle that is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia as suitable for administration to animals, and more particularly to humans. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin. Salt solutions and solutions of dextran and glycerol can also be used as liquid carriers, particularly for injection solutions. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0157] Typically, the components of the composition may be provided individually or mixed together in dosage form (e.g., as a lyophilized powder or anhydrous concentrate, or as an aqueous solution in a sealed container (such as a vial, ampoule, or sachet) indicating the amount of active agent). When the composition is administered by infusion, it may be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules containing sterile water, saline, or other diluents may be provided for injection to allow mixing of the components prior to administration. VII. Drug Reagent Kit

[0158] This disclosure also provides pharmaceutical kits or pharmaceutical reagent kits comprising one or more containers containing one or more pharmaceutical compositions and instructional materials (e.g., precautions, packaging inserts, instructions, etc.). Furthermore, the pharmaceutical reagent kit may also include one or more other preventative or therapeutic agents for treating diseases. The containers of such pharmaceutical reagent kits may, for example, include one or more sealed vials, ampoules, pouches, etc., indicating the amount of active agent contained therein. When the composition is administered by infusion, the container may be an infusion bottle, bag, etc., containing a sterile pharmaceutical-grade solution (e.g., water, saline, buffer, etc.). When the composition is administered by injection, the pharmaceutical reagent kit may include ampoules for injection of sterile water, saline, or other diluents to facilitate mixing of the components of the pharmaceutical reagent kit for administration to a subject (e.g., a human patient or other mammal). In some aspects, the pharmaceutical kit or reagent kit includes a B7-H3-ADC pharmaceutical composition and instructional materials.

[0159] In one aspect, the B7-H3-ADC in such kits is provided in a sealed container as a dry, sterile lyophilized powder or anhydrous concentrate and can be reconstituted, for example, with water, saline, or other diluents, to a suitable concentration for administration to a subject. In another aspect, the B7-H3-ADC in such kits is provided in a sealed container as an aqueous solution and can be diluted, for example, with water, saline, or other diluents, to a suitable concentration for administration to a subject. The kit may further contain one or more other preventative and / or therapeutic agents that can be used to treat cancer in one or more containers; and / or the kit may further contain one or more cytotoxic antibodies that bind to one or more cancer antigens associated with cancer. In some aspects, the other preventative or therapeutic agents are chemotherapeutic agents. In other aspects, the preventative or therapeutic agents are biological agents or hormonal therapeutic agents.

[0160] The instruction materials included in a pharmaceutical kit may, for example, have the content and format prescribed by a government agency regulating the preparation, use, or sale of a drug or biological product, and may indicate the preparation, sale, or use of pharmaceutical compositions approved by that agency for human administration and / or human treatment. The instruction materials may, for example, provide information regarding the dosage of the pharmaceutical composition, how it can be prepared (e.g., reconstituted), and how it can be administered. Such instructions may further provide information regarding the dosage and administration of one or more pharmaceutical compositions not provided in the kit. Applications of VIII.B7-H3-ADC

[0161] The B7-H3-ADC described herein can be used to treat or prevent a variety of conditions, including cancer, including, for example, cancers in which B7-H3 is expressed. Therefore, this disclosure provides methods for treating cancer, such methods comprising administering B7-H3-ADC to a subject in need. In some aspects, this disclosure provides methods for treating cancer, such methods comprising administering MGC026 to a subject in need. As used herein, the term “subject” refers to a human (i.e., a human patient) or other mammal. This document provides non-limiting dosing regimens for administering such therapies to subjects in need.

[0162] In some respects, the cancers that B7-H3-ADC can treat, as described in this article, include cancers selected from the group consisting of: adrenal tumors, AIDS-related cancers, alveolar soft tissue sarcomas, astrocytic tumors, adrenal cancer, bladder cancer, bone cancer, brain and spinal cord cancers, metastatic brain tumors, B-cell cancers, breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, fibroproliferative small round cell tumors, ependymoma, Ewing's tumor, extraosseous myxoid chondrosarcoma, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, glioblastoma, hematologic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, renal cancer, leukemia (e.g., acute myeloid leukemia), and liposarcoma. Malignant lipomatous tumors, liver cancer, lymphoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), medulloblastoma, melanoma, meningioma, mesothelioma, laryngeal cancer, multiple endocrine tumors, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumors, childhood cancers, peripheral nerve sheath tumors, pheochromocytoma, pituitary adenoma, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumors in children (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck (SCCHN), gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (e.g., metastatic thyroid cancer), and uterine cancer.

[0163] In some areas, B7-H3-ADC can be used to treat adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, gastroesophageal junction cancer, glioblastoma, renal cancer, lung cancer, non-small cell lung cancer (NSCLC), acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, mesothelioma, pharyngeal cancer, non-Hodgkin lymphoma, small lymphocytic lymphoma, multiple myeloma, melanoma, ovarian cancer, and platinum-resistant ovarian cancer. Ovarian cancer (PROC), pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), sarcoma, skin cancer, renal cell carcinoma, small cell lung cancer (SCLC), extensive-stage small cell lung cancer (ES-SCLC), childhood small round blue cell tumors (including neuroblastoma and rhabdomyosarcoma), squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN), esophageal squamous cell carcinoma), testicular cancer, thyroid cancer (e.g., metastatic thyroid cancer), endometrial cancer, cervical cancer, clear cell renal cell carcinoma, hepatocellular carcinoma, small cell ovarian cancer, small cell colorectal cancer, and uterine cancer.

[0164] In some respects, B7-H3-ADC can be used to treat colorectal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck (SCCHN)).

[0165] In some respects, B7-H3-ADC can be used to treat melanoma.

[0166] In some respects, B7-H3-ADC can be used to treat ovarian cancer.

[0167] In some respects, B7-H3-ADC can be used to treat platinum-resistant ovarian cancer (PROC).

[0168] In some respects, B7-H3-ADC can be used to treat pancreatic cancer.

[0169] In some respects, B7-H3-ADC can be used to treat prostate cancer.

[0170] In some respects, B7-H3-ADC can be used to treat metastatic castration-resistant prostate cancer (mCRPC).

[0171] In some respects, B7-H3-ADC can be used to treat squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck (SCCHN)).

[0172] In some respects, B7-H3-ADC can be used to treat non-small cell lung cancer (NSCLC).

[0173] In some respects, B7-H3-ADC can be used to treat small cell lung cancer (SCLC).

[0174] In some respects, B7-H3-ADC can be used to treat extensive-stage small cell lung cancer (ES-SCLC). IX. Method of Application

[0175] The B7-H3-ADC disclosed herein can be administered to subjects, such as those in need, including human patients, via a variety of methods. For many applications, the administration route is one of the following: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection. Intra-articular delivery is also possible. Other parenteral administration modalities may also be used. Examples of such modalities include: intra-arterial, intrasheath, intra-bursal, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, intra-articular, sub-bursal, subarachnoid, intraspinal, epidural, and intrasternal injection.

[0176] The B7-H3-ADC disclosed herein can be administered as a weight-based dose or as a fixed dose. The dose can also be selected to reduce or avoid the production of antibodies against the administered molecule. Dosage regimens can be adjusted to provide a desired response, such as a therapeutic response or combined therapeutic effect. Typically, a dose of B7-H3-ADC (and optionally further agents) can be used to provide a bioavailable amount to the subject. As used herein, the term "dose" refers to a specified amount of the drug taken at one time. The term "dosage" refers to a dose administered in a specific amount, quantity, and frequency over a specific time period; therefore, the term dosage includes time characteristics such as duration and periodicity.

[0177] As used herein, the term "weight-based dose" refers to a discrete amount of molecule to be administered per unit of patient body weight, such as milligrams of drug per kilogram of subject body weight (mg / kg body weight, abbreviated as "mg / kg" herein). The calculated dose will be administered based on the subject's weight at baseline. As used herein, the term "fixed dose" refers to a dose independent of patient weight and comprises physically discrete units of molecules suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of drug. Typically, a significant (≥ 10%) change in weight from baseline or an established plateau weight usually prompts a recalculation of the dose. Single or multiple doses may be administered. Compositions comprising B7-H3-ADC may be administered to subjects in need via infusion.

[0178] As used herein, the term "fractionated dose" refers to two or more separate administrations of a molecule to be administered to achieve a specific desired dose. Fractionated dose provides that the desired dose can be divided into two or more separate administrations. The dose can be distributed in equal and / or unequal amounts between these two or more administrations. In some respects, a fractionated dose may be administered two or more separate times within a single cycle (e.g., a 3-week cycle or a 4-week cycle). Example

[0179] The foregoing aspects have now been generally described, and will be more readily understood by referring to the following examples. The following examples illustrate various methods of using the compositions in the diagnostic or therapeutic methods of this disclosure. These examples are intended to be illustrative, but in no way limit the scope of the appended claims. Example 1 In vitro cytotoxicity of MGC026

[0180] The ability of MGC026 to mediate cytotoxicity against A375.S2 human melanoma cells expressing B7-H3 in vitro was investigated. The MGC026 studied was the same as the ADC described above. The negative control ADC (Ctrl-SYNtecan E) was a humanized control ADC that did not bind to B7-H3 or any other human or mouse protein. Human tumor cells were cultured in DMEM / F-12 + 10% FBS. Cells were washed with PBS and lifted using 0.05% trypsin-EDTA. The antibody and ADC were diluted using a 9-point dose-response curve (plus antibody-free control wells), where the final maximum concentration was 10 μg / mL (67 nM antibody concentration), and the dilutions were 1:3 or 1:10, depending on the cell line sensitivity. The ADC was prepared at a 5X final concentration, and 20 μL was added to a 96-well tissue culture plate. Suspension cells were seeded at 5,000 cells / well and 80 μL / well of ADC was added to the ADC wells (total volume 100 μL / well).

[0181] Incubate the plates at 37°C for 7 days. Measure cell viability using alamarBlue (Trek Diagnostics #00-100; add 10 μL per well) and allow for expansion. Read the plates on a Gemini microplate reader (Molecular Devices) according to the alamarBlue instructions.

[0182] The data was analyzed using Graph Pad Prism (4-parameter curve fitting analysis) to determine the IC. 50 value.

[0183] The cytotoxicity curves of this study are presented in Figure 1In vitro, MGC026 mediated dose-dependent cytotoxicity against the A375.S2 human melanoma line, with an IC50 value of [missing information]. 50 = 31pM. The negative control ADC (Ctrl-SYNtecan E) had approximately 1000-fold lower activity than MGC026, confirming the specificity of MGC026's cytotoxic activity. Example 2 ADCC measurement

[0184] The ability of unconjugated MGA017 (an unconjugated B7-H3 antibody equivalent to the antibody used in the conjugate), conjugated MGC018 (a B7-H3 ADC with the same antibody and domikacin cytotoxic drug fraction; see WO2017 / 180813A1), and conjugated MGC026 (as described above) to mediate antibody-dependent cell cytotoxicity (ADCC) was evaluated. The ADCC assay utilized primary peripheral blood mononuclear cells (PBMCs) as a source of natural killer (NK) cells. The ADCC assay tested the antibody-bridged interaction between antigen-positive target cells and NK effector cells, and their subsequent ability to kill target cells via NK cells. Adhesive target tumor cells grown in F-12 / DMEM containing 10% fetal bovine serum (FBS) were isolated using 0.25% trypsin-EDTA solution and collected by centrifugation at 1000 rpm for 5 minutes. The collected tumor cells were washed once with PBS, then resuspended in assay medium (RPMI without phenol red + 5% FBS) and seeded at 20,000 cells / well into 96-well U-bottom cell culture plates. The assay antibody was serially diluted and seeded triplicate onto the cells. Then, 600,000 fresh PBMCs were added to the wells (30:1 effector:target ratio; E:T), and the plates were incubated at 37°C. 0 Incubate overnight at 5% CO2.

[0185] After incubation, 15 μL of 10X lysis solution (Promega # G182A) was added to the maximum release control wells for 10 minutes to completely lyse the target cells, and the plate was then centrifuged at 1200 rpm for 5 minutes. 50 μL of supernatant was transferred from each well to a clear flat-bottom ELISA plate, and 50 μL of lactate dehydrogenase (LDH) substrate solution (Promega # G1780) was added to each well. The plates were incubated in the dark at room temperature for 5–10 minutes, followed by the addition of 50 μL of stop solution. The optical density was measured at 490 nm over 1 hour using an Emax microplate reader (Molecular Devices). The percentage of cytotoxicity was calculated as described below and further analyzed using GraphPad Prism5 software.

[0186] The following formula is used to calculate specific cell lysis based on optical density (OD) data, incorporating maximum release (MR), antibody-independent cytotoxicity (AICC), and spontaneous cell release (SR): Cytotoxicity (%) = 100 × (OD of sample - OD of AICC) / (OD of MR - OD of SR)

[0187] ADCC measurement results are presented in Figures 2A-2E In this study, MGA017 and MGC018 were tested for ADCC mediated by five tumor cell lines expressing B7-H3. MGC026 did not mediate ADCC in any of the five tumor cell lines. Example 3 Surface Plasmon Resonance Analysis

[0188] Surface plasmon resonance (SPR) was used to analyze the binding with B7-H3. The anti-penta-His-tagged mAb was immobilized on the SPR CM5 sensor chip according to the manufacturer's recommended procedure. Briefly, the carboxyl groups on the sensor chip surface were activated by injecting a solution containing 0.2 M N-ethyl-N-(3-diethylamino-propyl)carbodiimide and 0.05 M N-hydroxy-succinimide. The mAb (5 µg / mL) was injected at a flow rate of 5 μL / min into the activated CM5 surface in 10 mM sodium acetate (pH 5.0), followed by the injection of 1 M ethanolamine to deactivate the remaining amine-reactive groups.

[0189] His-tagged human or cynomolgus monkey B7-H3(4Ig) extracellular domain protein was injected at a flow rate of 20 μL / min for 10 seconds to achieve a capture ligand of approximately 30 resonance units (RU) suitable for kinetic studies.

[0190] MGC026 or MGA017 (both as shown above) was injected into HBS-EP buffer at a flow rate of 30 µL / min (in duplicate) for 120 seconds at concentrations of 0, 12.5, 25, 50, 100, and 200 nM. The immobilized anti-5-His mAb surface was regenerated by pulse injection of 10 mM glycine (pH 1.5).

[0191] Reference curves were obtained by injecting each dilution of MGC026 or MGA017 onto a treated surface without protein fixation. The binding curve at zero concentration was subtracted to serve as a blank. The association / dissociation (k...) was then analyzed. a / k d The interaction curves and the divalent analyte interaction model (BIA evaluation software v4.1) are used for overall analysis to evaluate the kinetic constant k of the single-arm affinity interaction. a and k dDissociation equilibrium constant (K) D ) Calculated as K D = k d / k a .

[0192] Antibodies MGC026 and MGA017 both exhibited single-arm affinity binding similar to that of captured human and cynomolgus monkey B7-H3 (4Ig). Figure 3A Table 1 shows the kinetic constants calculated for the single-arm interaction using the divalent analyte model. K is determined for the interaction with captured human B7-H3(4Ig). D The values ​​were similar for all interactions and varied between 16 and 18 nM or 6 and 8 nM for human or cynomolgus monkey B7-H3(4Ig), respectively. The differences in the overall binding response magnitude are likely due to differences in the available binding sites for the two antigens captured on the sensor chip surface and do not reflect kinetic parameters. *The average data from two independent experiments (shown in parentheses)

[0193] His-tagged human CD16A (FcγRIIIIA) extracellular domain protein was injected at a flow rate of 20 μL / min for 10 seconds to achieve a capture ligand of approximately 120 resonance units (RU). MGC026 or MGA017 was injected (in duplicate) at a flow rate of 30 μL / min into HBS EP buffer for 120 seconds at concentrations of 0, 62.5, 125, 250, 500, and 1000 nM. Regeneration of the immobilized anti-His mAb surface was performed by pulsed injection of 10 mM glycine (pH 1.5). The equilibrium binding response of the antibody's Fc moiety to the captured human CD16A allele was fitted to a steady-state affinity model to obtain the KB value. D value.

[0194] Unconjugated antibody MGA017 binds to the captured human CD16 allele. As expected, MGA017 binds with a higher affinity for the CD16A 158V allele compared to the lower affinity CD16A 158F allele. MGC026 (conjugated antibody) does not bind to either CD16A allele. Figure 3BTherefore, it is expected that MGC026 will not mediate effector function via the Fcγ receptor CD16A, which is consistent with the fact that MGC026 cannot mediate ADCC in vitro (see Example 2). The loss of effector function is potentially advantageous for MGC026 because the binding of ADCs to effector cells can reduce tumor localization, inhibit internalization, and lead to off-target toxicity (McDonagh, Mol Cancer Ther 2008; Perez, DrugDiscovery Today 2013).

[0195] Surface plasmon resonance was also used to analyze the binding of recombinant B7-H3 to MGA017 and MGA017 (D96E) (MGA017 with a D96E mutation in the heavy chain), which contains amino acids L29 to G245 of human B7-H3 fused to HSV and a 10xHis tag at the C-terminus. Binding studies were performed in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant). MGA017 or MGA017 (D96E) was captured by a polyclonal goat anti-human Fc antibody immobilized on a CM4 chip. Binding of the recombinant B7-H3 protein was analyzed at concentrations of 12.5, 50, and 200 nM. The equilibrium dissociation constant (K0) was determined. D ), association rate (k a ) and dissociation rate (k d The values ​​were determined by the overall fit of the binding curve to the Langmuir 1:1 binding model (BIA evaluation software, version 4.1), as shown in Table 2. MGA017 (D96E) Figure 3D It exhibits similar binding affinity to recombinant B7-H3 as MGA017. Figure 3C ). Example 4 MGC026 exhibits potent in vivo activity.

[0196] To further demonstrate the antitumor activity of MGC026, the in vivo toxicity of the aforementioned MGC026 molecule was evaluated in a CD-1 nude mouse model using different tumor cell lines. Briefly, approximately 5 x 10⁻⁶ cells suspended in 1:1 serum-free medium and Matrigel basement membrane matrix were used. 6 One surviving tumor cell was subcutaneously inoculated into the lateral portion of CD-1 nude mice (Charles River Laboratories). The tumors reached an average volume of approximately 100-140 mm. 3In the study, mice were randomly assigned to groups and administered MGC026 or a vector control intravenously. Selected studies included a non-targeted control ADC or MGC018 (as described above). In these studies, a single dose of MGC026, MGC018, a non-targeted control ADC, or a vector control was administered once weekly (QW). Tumors were measured twice weekly using an orthogonal measurement method with electronic calipers, where tumor volume was calculated as (length x width x height) / 2. Animals were considered to have partial regression (“PR”) when the tumor volume decreased by 50% or more compared to the tumor volume on the day of the first dose administration. During the study period, treated animals achieved a tumor volume reduction to < 5 mm. 3 The finding was considered to indicate a complete response (“CR”). Tumor volume (relative to the vector control) was determined (“T / C”). Antitumor activity was assessed according to the National Cancer Institute (NCI) criteria; a T / C ≤ 42% was the minimum level of antitumor activity, while a T / C value > 42% was inactive. A T / C < 10% was considered highly active. In vivo activity against Calu-6 non-small cell lung cancer tumor cells

[0197] The results of this study regarding subcutaneous inoculation of Calu-6 lung adenocarcinoma cells are presented in Table 3 and... Figure 4 The study also demonstrated responsiveness to Calu-6 tumor cells. a%T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. When the vector tumor volume reached a mean of 1147 mm on day 49... 3 It is calculated in time. b. Antitumor activity: Assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0198] Female CD-1 nude mice (homozygous) (n=6 mice / group) were subcutaneously implanted with Calu-6 (lung adenocarcinoma) tumor cells (5×10⁻⁶ cells) suspended in serum-free culture medium / Matrigel (1:1). 6(cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 22. 3 (104 ± 25 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 22 (arrow) with either a vector control (1X PBS) or a targeted ADC (MGC026 or MGC018) at the indicated dose level. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM bar visible above. Antitumor activity was observed at all three dose levels treated with MGC026 and at two dose levels treated with MGC018.

[0199] MGC026 reduced tumor volume by 99% at all tested dose levels on day 49 and was highly active at every dose (1% T / C) according to NCI criteria. MGC018 was highly active at 10 mg / kg (1% T / C) and active at 3 mg / kg (22% T / C). MGC026 induced partial regression in 6 / 6 animals at doses of 10 mg / kg, 6 mg / kg, and 3 mg / kg, and complete regression in 5 / 6, 6 / 6, and 4 / 6 animals, respectively, at doses of 10 mg / kg, 6 mg / kg, and 3 mg / kg. MGC018 induced partial regression in 6 / 6 animals at 10 mg / kg and in 2 / 6 animals at 3 mg / kg, with complete regression in 4 / 6 animals at a dose of 10 mg / kg. MGC026 maintained its antitumor activity and tumor regression at all tested dose levels until the end of the study, while MGC018 maintained tumor regression at a dose of 10 mg / kg until the end of the study. The tumor H score was determined to be 95 by immunohistochemistry (IHC). In vivo activity against Calu-6 lung adenocarcinoma tumor cells at the minimum effective dose

[0200] The results of this study regarding subcutaneous inoculation of Calu-6 lung adenocarcinoma cells are presented in Table 4 and... Figure 5 The study also demonstrated responsiveness to Calu-6 tumor cells. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. When the vector tumor volume reached a mean of 1168 mm on day 41... 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0201] Female CD-1 nude mice (homozygous) (n=5 mice / group) were subcutaneously implanted with Calu-6 (lung adenocarcinoma) tumor cells (5×10⁻⁶ cells) suspended in serum-free culture medium / Matrigel (1:1). 6 (cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 16. 3 (100 ± 22 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 16 (arrow) with either a vector control (1X PBS) or a targeted ADC (MGC026 or MGC018) at the indicated dose level. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed after treatment with MGC026 and MGC018.

[0202] MGC026 reduced tumor volume by 87% by day 41 and was active at the tested dose level (3 mg / kg) based on NCI criteria (13% T / C). MGC018 was active at 3 mg / kg (34% T / C). MGC026 induced partial regression in 2 / 5 of the animals, but not complete regression. MGC018 did not induce partial or complete regression in 0 / 5 of the animals at 3 mg / kg. The tumor H score was determined to be 210 by IHC. In vivo activity against A375.S2 melanoma tumor cells

[0203] The results of this study regarding subcutaneous inoculation of A375.S2 melanoma tumor cells are presented in Table 5 and 6. Figure 6 The study also demonstrated responsiveness to A375.S2 tumor cells. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The mean tumor volume in the vector group reached 1133 mm on day 57. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0204] Female CD-1 nude mice (homozygous) (n=6 / group) were subcutaneously implanted with A375.S2 (melanoma) tumor cells (5×10⁻¹⁰) suspended in serum-free culture medium / Matrigel (1:1). 6 (cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 24. 3 (102 ± 33 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 25 (arrow) with either a vector control (1X PBS) or a targeted ADC (MGC026 or MGC018) at the indicated dose level. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at all three dose levels of MGC026 and two dose levels of MGC018.

[0205] Tumor volume reduction was observed after treatment with 10 mg / kg MGC026, with a 99% reduction by day 57. Treatment with 6 mg / kg and 3 mg / kg MGC026 resulted in a 100% reduction in tumor volume. Based on NCI criteria, MGC026 was highly active at 10 mg / kg (1% T / C), 6 mg / kg (0% T / C), and 3 mg / kg (0% T / C). MGC018 was highly active at both 10 mg / kg and 3 mg / kg (0% T / C). MGC026 induced partial and complete regression in 6 / 6 animals at doses of 10 mg / kg, 6 mg / kg, and 3 mg / kg. MGC018 induced partial and complete regression in 6 / 6 animals at doses of 10 mg / kg and 3 mg / kg. For all tested dose levels, the antitumor activity and complete tumor regression of MGC026 and MGC018 persisted until the end of the study. The tumor H score was determined to be 180 by IHC. In vivo activity against A375.S2 melanoma tumor cells at minimum effective dose

[0206] The results of this study regarding subcutaneous inoculation of A375.S2 melanoma tumor cells are presented in Table 6 and... Figures 7A-7C The study also demonstrated responsiveness to A375.S2 tumor cells. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The vector tumor volume reached a mean of 1090 mm on day 80. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0207] Female CD-1 nude mice (homozygous) (n=5 mice / group) were subcutaneously implanted with A375.S2 (melanoma) tumor cells (5×10⁻⁶ cells) suspended in serum-free culture medium / Matrigel (1:1). 6(cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 26. 3 (87 ± 21 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 26 (arrow) with either a vector control (1X PBS) or a targeted ADC (MGC026 or MGC018) at the indicated dose levels (A, B, C) for a total of one dose. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed after treatment with the three tested doses of MGC026 and MGC018.

[0208] MGC026 reduced tumor volume by 100% by day 80 and was highly active at 3 mg / kg (0% T / C) and 1 mg / kg (7% T / C) according to NCI criteria, and was active at 0.3 mg / kg (16% T / C). Treatment with MGC018 was highly active at 3 mg / kg (0% T / C), active at 1 mg / kg (41% T / C), and inactive at 0.3 mg / kg (54% T / C). Although the non-targeted control ADC showed antitumor activity at 3 mg / kg and 0.3 mg / kg, the activity was limited and significantly lower than that observed with targeted MGC026, and was considered inactive at 3 mg / kg and 0.3 mg / kg (74%, 50% T / C) according to NCI criteria. MGC026 induced partial regression in 5 / 5 of animals at dose levels of 3 mg / kg and 1 mg / kg, and in 4 / 5 at a dose level of 0.3 mg / kg. Complete regression was observed in 5 / 5 and 3 / 5 at 3 mg / kg and 1 mg / kg, respectively. MGC018 induced partial regression in 5 / 5 and 3 / 5 at 3 mg / kg and 1 mg / kg, respectively, and complete regression was observed in 4 / 5 of animals at 3 mg / kg. Antitumor control was maintained with treatment with MGC026 and MGC018 at a dose level of 3 mg / kg. The tumor H score was determined to be 210 by IHC. In vivo activity against FaDu pharyngeal and head and neck squamous cell carcinoma tumor cells

[0209] The results of this study regarding subcutaneously inoculated FaDu pharyngeal and head and neck squamous cell carcinoma tumor cells are presented in Table 7 and... Figure 8 The study also demonstrated responsiveness to FaDu tumor cells. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The vector tumor volume reached a mean of 1243 mm on day 35. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0210] Female CD-1 nude mice (homozygous) (n=7 mice / group) were subcutaneously implanted with FaDu (pharyngeal and head and neck squamous cell carcinoma) tumor cells (5×10⁻¹⁰) suspended in serum-free culture medium / Matrigel (1:1). 6 (cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 15. 3 (125 ± 25 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 15 (arrow) with a single dose of either the carrier control (1X PBS), the targeted ADC (MGC026), or the non-targeted control ADC at the indicated dose level. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Dose-responsive antitumor activity was observed at all three dose levels of MGC026.

[0211] Tumor volume reduction was observed after treatment with 10 mg / kg MGC026, with an 88% reduction in tumor volume by day 35 compared to the vector control. Treatment with 6 mg / kg and 3 mg / kg MGC026 resulted in tumor volume reductions of 93% and 86%, respectively. Based on NCI criteria, MGC026 was highly active at 6 mg / kg (7% T / C), and active at 10 mg / kg (12% T / C) and 3 mg / kg (14% T / C). Although the non-targeted control ADC showed antitumor activity, the activity was limited and significantly lower than that observed with MGC026, and was considered inactive at 10 mg / kg, 6 mg / kg, and 3 mg / kg (60%, 74%, and 87% T / C), respectively, based on NCI criteria. MGC026 induced partial regression in 6 / 7, 4 / 7, and 3 / 7 animals, respectively, at doses of 10 mg / kg, 6 mg / kg, and 3 mg / kg, and complete regression in 1 / 7 animals at all three tested doses. In contrast, the non-targeted control ADC showed 1 / 7 partial regression at doses of 6 mg / kg and 3 mg / kg, and 1 / 7 complete regression at dose of 6 mg / kg. The tumor H score was determined to be 200 by IHC. In vivo activity against Hs700T pancreatic adenocarcinoma tumor cells

[0212] The results of this study regarding subcutaneously inoculated Hs700T pancreatic adenocarcinoma cells are presented in Table 8 and... Figure 9 The study also demonstrated responsiveness to Hs700T tumor cells. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. When the vector tumor volume reached a mean of 1286 mm on day 96... 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0213] Female CD-1 nude mice (homozygous) (n=7 mice / group) were subcutaneously implanted with Hs700T (pancreatic adenocarcinoma) tumor cells (5×10⁻¹⁰) suspended in serum-free culture medium / Matrigel (1:1). 6 (cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 46. 3 (114 ± 27 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 46 (arrow) with a single dose of either the carrier control (1X PBS), the targeted ADC (MGC026), or the non-targeted control ADC at the indicated dose level. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. The MGC026 dose level differed from the non-targeted control ADC dose level because the test certificate for MGC026 was corrected after the start of the study. Antitumor activity was observed at all three dose levels of MGC026 compared to the carrier control.

[0214] Dose-response was observed at three dose levels of MGC026. Tumor volume reduction was observed after treatment with 7.4 mg / kg MGC026, with a 96% reduction in tumor volume at day 96 compared to the vector control. Treatment with 4.5 mg / kg and 2.2 mg / kg MGC026 resulted in 93% and 85% reductions in tumor volume at day 96, respectively. Based on NCI criteria, MGC026 was highly active at 7.4 mg / kg (4% T / C) and 4.5 mg / kg (7% T / C), and active at 2.2 mg / kg (15% T / C), while the non-targeted control ADC was inactive at 10 mg / kg, 6 mg / kg, and 3 mg / kg (70%, 69%, and 77% T / C). MGC026 induced partial regression in 5 / 7, 4 / 7, and 2 / 7 animals, respectively, at doses of 7.4 mg / kg, 4.5 mg / kg, and 2.2 mg / kg, and complete regression in 1 / 7 and 1 / 7 animals, respectively, at doses of 7.4 mg / kg and 2.2 mg / kg. In contrast, the non-targeted control ADC group showed no partial or complete regression at any of the three dose levels. The tumor H score was determined to be 295 by IHC. In vivo activity against 22Rv1 prostate cancer tumor cells

[0215] The results of this study regarding subcutaneous inoculation of 22Rv1 prostate cancer cells are presented in Table 9 and... Figure 10 The study also demonstrated responsiveness to 22Rv1 tumor cells. a %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The mean tumor volume in the vector group reached 1161 mm on day 72. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0216] Female CD-1 nude mice (homozygous) (n=7 mice / group) were subcutaneously implanted with 5 × 10⁻⁶ 22Rv1 (prostate cancer) tumor cells suspended in serum-free culture medium / Matrigel (1:1). 6 (cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 31. 3 (126 ± 26 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 32 (arrow) with a single dose of either the carrier control (1X PBS), the targeted ADC (MGC026), or the non-targeted control ADC at the indicated dose level. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. The MGC026 dose level differed from the non-targeted control ADC dose level because the test certificate for MGC026 was corrected after the start of the study. Antitumor activity was observed with MGC026 at all three dose levels.

[0217] Dose-response was observed at three dose levels of MGC026. Tumor volume reduction was observed after treatment with 7.4 mg / kg MGC026, with a 78% reduction in tumor volume at day 72 compared to the vector control. Treatment with 4.5 mg / kg and 2.2 mg / kg MGC026 resulted in tumor volume reductions of 77% and 65%, respectively. Based on NCI criteria, MGC026 was active at 7.4 mg / kg (22% T / C), 4.4 mg / kg (23% T / C), and 2.2 mg / kg (35% T / C), while the non-targeted control ADC was inactive at 10 mg / kg, 6 mg / kg, and 3 mg / kg (81%, 72%, and 78% T / C), respectively. MGC026 induced partial regression in 5 / 7, 3 / 7, and 2 / 7 animals, respectively, but not complete regression. In contrast, the non-targeted control ADC group showed no partial or complete regression at any three dose levels. The tumor H score, determined by IHC, was 155.

[0218] These in vivo studies indicate that the tested MGC026 exhibited dose-dependent antitumor activity against B7-H3-positive tumors in xenograft models of mouse lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, and melanoma. Example 5 MGC026 exhibits enhanced potency in vivo.

[0219] To further analyze the antitumor activity of MGC026, the antitumor activity of MGC026 or DS-mAb-Dxd (an ADC with an M30-H1-L4 antibody conjugated to drutecan (Dxd) that binds to B7-H3) was evaluated using two different tumor cell lines; see WO2012147713A1. In vivo activity against Calu-6 lung adenocarcinoma tumor cells

[0220] The results of this study regarding subcutaneous inoculation of Calu-6 lung adenocarcinoma cells are presented in Table 10 and... Figure 11A-11C The study also demonstrated responsiveness to Calu-6 tumor cells. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The mean tumor volume in the vector control group reached 1168 mm on day 41. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0221] Female CD-1 nude mice (homozygous) (n=5 mice / group) were subcutaneously implanted with Calu-6 (lung adenocarcinoma) tumor cells (5×10⁻⁶ cells) suspended in serum-free culture medium / Matrigel (1:1). 6 The dose was 0.1 mL per mouse (cells). When the tumor reached approximately 100 mm on day 16... 3 (100 ± 22 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 16 (arrow) with either a vector control (1X PBS) or a targeted ADC (MGC026 or DS-mAb-Dxd) at the indicated dose levels (A, B, C) for a total of one dose. Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at a dose level of 3 mg / kg after treatment with MGC026, while no antitumor activity was observed at a dose level of 3 mg / kg after treatment with DS-mAb-Dxd.

[0222] MGC026 reduced tumor volume by 87% at 3 mg / kg on day 41 and was active at this dose based on NCI criteria (13% T / C). DS-mAb-Dxd was inactive at 3 mg / kg. MGC026 induced partial regression in 2 / 5 of the animals at 3 mg / kg, but not complete regression, while DS-mAb-Dxd did not induce either partial or complete regression at 3 mg / kg. The tumor H score was determined to be 210 by immunohistochemistry (IHC). In vivo activity against A375.S2 melanoma tumor cells

[0223] The antitumor activity of conjugated MGC026 or DS-mAb-Dxd was evaluated using A375.S2 melanoma tumor cells. Results from this study regarding subcutaneous inoculation of A375.S2 melanoma tumor cells are presented in Table 11 and... Figures 12A-12CThe study also demonstrated responsiveness to A375.S2 tumor cells. a. %T / C = The percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The vector tumor volume reached an average of 1090 mm on day 80. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0224] Female CD-1 nude mice (homozygous) (n=5 mice / group) were subcutaneously implanted with A375.S2 (melanoma) tumor cells (5×10⁻⁶ cells) suspended in serum-free culture medium / Matrigel (1:1). 6 (cells), 0.1 mL volume per mouse. When the tumor reached approximately 100 mm on day 26. 3 (87 ± 21 mm 3 Mice were randomly assigned to groups at mean tumor volume ± standard deviation and treated intravenously on day 26 (arrow) with either a vector control (1X PBS) or a targeted ADC (MGC026 or DS-mAb-Dxd) at the indicated dose levels (A, B, C) for a total of one dose. Tumor volume is shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed after treatment with three test doses of MGC026 and 3 mg / kg of DS-mAb-Dxd.

[0225] MGC026 reduced tumor volume by 100% on day 80 and was highly active at 3 mg / kg (0% T / C) and 1 mg / kg (7% TC) according to NCI criteria, and active at 0.3 mg / kg (16% T / C). Treatment with DS-mAb-Dxd was active at 3 mg / kg (13% T / C) and inactive at 1 mg / kg (84% T / C) and 0.3 mg / kg (100% T / C). MGC026 induced partial regression in 5 / 5 of the animals at dose levels of 3 mg / kg and 1 mg / kg, and in 4 / 5 of the animals at dose level of 0.3 mg / kg. Complete regression was observed in 5 / 5 and 3 / 5 of the animals at 3 mg / kg and 1 mg / kg, respectively. D-mAb-Dxd induced partial regression in 5 / 5 of the animals and complete regression in 1 / 5 of the animals at 3 mg / kg. Treatment with MGC026 maintained antitumor control at a dose level of 3 mg / kg. The tumor H score was determined to be 210 by IHC. Example 6 Tolerance study of MGC026 in cynomolgus monkeys

[0226] To determine the tolerability of MGC026 (as described above), preclinical animal studies were conducted using cynomolgus monkeys. These studies included both non-GLP and GLP studies.

[0227] In the non-GLP study, MGC026 was administered to each of two animals via intravenous (IV) infusion over 4 minutes at doses of 7.4, 22.2, and 44.4 mg / kg, at two-week (Q2W) intervals, for a total of two doses, in a total of six cynomolgus macaques (Table 12).

[0228] MGC026 was well tolerated when administered at 7.4, 22.2, or 44.4 mg / kg / dose Q2W (days 1 and 15 of the study). Study evaluations included: toxicokinetics, body weight, food consumption, clinical observation, clinicopathology (hematology, clinical chemistry), and anatomical pathology. Results were limited to anatomical pathology, and cytogenic (lymphocytic) thymus was observed in one of the two animals administered 7.4 mg / kg (minimum) and in the two animals administered 44.4 mg / kg (minimum and mild).

[0229] In the GLP study, MGC026 was administered intravenously (IV) at doses of 10, 30, and 50 mg / kg to 6, 10, and 10 animals respectively over 15 minutes, at three-week (Q3W) intervals, for a total of 26 cynomolgus monkeys (Table 13).

[0230] Q3W (days 1, 22, and 43 of the study) administration of 10, 30, or 50 mg / kg / dose of MGC026 was well tolerated. Study assessments included: toxicokinetic assessment, body weight, food consumption, clinical observation, clinicopathology (hematology, clinical chemistry, coagulation, or urinalysis), ECGs, blood pressure, respiratory rate, heart rate, or neurological assessment (response to environmental stimuli, body temperature, eye function, motor reflexes, and proprioceptive function) and anatomical pathology. max Both the AUC and MGC026 parameters increased with increasing MGC026 dose levels and were close to dose-proportional. Results were limited to anatomical pathology. At the final autopsy (day 49 of the study), a decrease in thymus weight associated with thymic (lymphocyte) reduction was observed in three of ten animals administered 30 mg / kg (mild to moderate) and in six of ten animals administered 50 mg / kg (mild to moderate). This finding was not considered an adverse outcome because animals with moderate severity had significant thymic tissue remnants, lacked evidence of opportunistic infections, and other components of the immune system (spleen, mesenteric lymph nodes, mandibular lymph nodes, and gut-associated immune system) were normal. Recovery autopsy (day 91 of the study) revealed no findings. Example 7 Tolerance study of MGC018 in cynomolgus monkeys

[0231] To determine the tolerability of MGC018 (as described above), preclinical animal studies were conducted using cynomolgus monkeys. These studies included both non-GLP and GLP studies.

[0232] Non-GLP studies included single-dose non-GLP PK studies and repeated-dose non-GLP toxicology studies. In the single-dose non-GLP PK studies, 14 animals were given MGC018 at doses of 1, 3, 10, 20, or 27 mg / kg. In the repeated-dose non-GLP toxicology studies, 6 animals (3 per group) were given MGC018 at doses of 6 or 20 mg / kg every 2 weeks (Q2W) (Table 14).

[0233] Data collected from two non-GLP MGC018 studies indicated that a single dose of 27 mg / kg was well tolerated based on body weight, food consumption, clinical observation, clinicopathology (hematology, clinical chemistry), and anatomical pathology. However, repeated dosing at 20 mg / kg Q2W was not tolerated, and animals required veterinary management for fever (possible infection), skin changes (including dryness, erythema, and / or weeping abrasion / erosion), and watery stools. Veterinary management did not affect the study data or interpretation because the effects of the treatment are well-known and / or short-lived. One animal receiving a 20 mg / kg dose on days 1 and 15 of the study developed erythema in the left groin, which progressed to dry, erythematous skin in the groin and axilla, and erosion of the skin in the left groin. From day 20, the animal exhibited decreased activity, kyphosis, and loss of appetite. By day 22, skin lesions were present in the groin and axilla, as well as on the left hind limb. Skin lesions were cleaned with a topical antibacterial agent (chlorhexidine) and treated with a topical soothing agent (Douxo) and antibiotics (Neo-predef) as needed from days 14 to 22. A nonsteroidal anti-inflammatory drug (NSAID) (Metacam) was administered on days 21 and 22 to address pain and decreased mobility. Cleaning of the skin lesions on day 22 resulted in skin peeling, and in extreme cases, the animals were euthanized depending on the size of the lesions. The remaining two animals, given a dose of 20 mg / kg / day, were also euthanized on day 22 to assess the extent of skin erosion, dryness, and hyperpigmentation.

[0234] Following repeated administration of 6 and 20 mg / kg doses Q2W, other MGC018-related clinical symptoms included sporadic watery stools, increased occurrence of dry skin (starting from day 8 or later), and skin discoloration (red, black, and brown). Other observations with MGC018 at a dose of 20 mg / kg included green stools, decreased activity, loss of appetite, kyphosis, increased occurrence of chafing (primarily in the groin area), and yellow discharge (from chafing) within 3 days of administration, which were most pronounced on day 18 or later.

[0235] The smallest mean weight loss was observed in animals after two doses of 20 mg / kg MGC018 (a decrease of -6.5% between day -1 and day 21), which may be due to the loss of appetite observed in all animals at the 20 mg / kg dose level.

[0236] Beginning on day 15, a moderate decrease in erythrocyte mass was more pronounced at a dose of 20 mg / kg / day and was associated with a decrease in absolute reticulocyte count. These were associated with MGC018, and in two of the three animals at a dose of 20 mg / kg / day, the decrease or inhibition of hematopoiesis was associated with a reduction in hematopoietic cells in the sternal bone marrow. From day 15, a decrease in lymphocytes (mild), neutrophils (moderate), and platelets (mild) was also observed at a dose of 20 mg / kg / day. At a later time point at a dose of 6 mg / kg / day, similar levels of decrease in lymphocyte and platelet counts were observed, while no decrease in neutrophils was observed. These decreases were associated with MGC018 and were associated with thymic lymphoid depletion in two of the three animals at a dose of 20 mg / kg / day.

[0237] The simultaneous occurrence of increased fibrinogen (moderate), CRP (mild to moderate), globulin (mild), and decreased albumin (mild) concentrations indicated an inflammatory response, and these occurred after animals received a second dose of 20 mg / kg / day (on day 22). These were thought to be associated with MGC018 and most likely with skin lesions on the forelimbs, hindlimbs, and ventral abdomen of these animals. Findings in animals receiving a 6 mg / kg / dose were limited to a mild increase in CRP.

[0238] Animals receiving doses of MGC018 of 6 or 20 mg / kg / day were observed to have a range of associated skin findings at multiple sites, including the face, forelimbs, hindlimbs, and ventral abdomen. These findings varied in severity between sites within an individual animal and across animals, but the characteristics of these findings were generally similar across dose groups, present in all animals at these doses, and showed a clear dose-response in terms of the severity and extent of the findings.

[0239] At a dose of 20 mg / kg, abrasions / scabs were observed on the forelimbs, hindlimbs, and ventral abdomen, and were microscopically associated with ulcers. Minimal to mild lymphocytic inflammation was present at the interface between the epidermis and dermis and in the surrounding superficial dermal vessels, with edema frequently observed within the superficial dermis in these areas. Minimal to mild single-cell necrosis was present in the overlying epidermis, ranging from vacuolar formation and degeneration in the epidermal basal layer to single epithelial cell necrosis. Occasionally, minimal to mild epidermal hyperplasia with increased keratinization was observed. Minimal to moderate epidermal detachment occurred in areas with inflammation, consisting of separation of the epidermis from the underlying basement membrane and dermis. The detachment occurred below the basal cell layer and formed multiple small to larger conjoined vesicles. As the detachment became more extensive, the epidermis ceased to exist, leaving ulcers of varying sizes, visible to the naked eye, ranging in severity from minimal to severe, and associated with macroscopically visible abrasions / scabs. Minimal erosions / ulcers were present in the perianal skin and inside the anus in one of the three animals.

[0240] At a dose of 6 mg / kg, skin darkening was observed on the face, forelimbs, hindlimbs, and / or ventral abdomen, with a minimal increase in pigmentation microscopically associated with the presence of intraepidermal pigmentation, occasionally extending into the superficial dermis. Minimal to mild lymphocytic inflammation was observed at the interface between the epidermis and dermis and in the surrounding superficial dermal vessels, with varying amounts of edema present in the superficial dermis of these areas. Minimal single-cell necrosis was observed in the overlying epidermis and occasionally in hair follicles, ranging from vacuolation and degeneration in the epidermal basal layer to necrosis of individual epithelial cells. Minimal epidermal hyperplasia and increased keratinization were also occasionally present.

[0241] The GLP study was an 8-week repeated-dose GLP toxicology study in which 40 animals (10 per group) received MGC018 every 3 weeks (Q3W, 3 doses for a total of 1, 3, 6 or 10 mg / kg (Table 15).

[0242] The assessment in the MGC018 GLP toxicology study included: toxicokinetic assessment, body weight, food consumption, clinical observation, clinicopathology (hematology, clinical chemistry, coagulation or urine analysis), ECG, blood pressure, respiratory rate, heart rate or neurological assessment (response to environmental stimuli, body temperature, eye, motor reflexes and proprioceptive function) and anatomical pathology.

[0243] In cynomolgus monkeys, MGC018 PK was linear between 6 and 10 mg / kg / dose, but showed nonlinearity at doses ≤ 6 mg / kg, which may be due to target-dependent clearance at low doses and / or saturation of target-specific clearance mechanisms at high doses.

[0244] Clinical symptoms associated with MGC018 are primarily dose-dependent and include findings in the skin such as hyperpigmentation (males at ≥ 1 mg / kg / dose and females at ≥ 3 mg / kg / dose) and dry skin ± erythema at ≥ 3 mg / kg / dose, increased occurrence of soft / watery stools primarily at 3 and 6 mg / kg / dose, occasional loss of appetite, soft / watery stools, emaciation (in single females at > 6 mg / kg / dose), and sparse hair, particularly found in the subgroup of animals at ≥ 6 mg / kg / dose.

[0245] Changes in clinicopathological parameters associated with MGC018 include indicators of acute inflammatory response (transient increases in CRP and fibrinogen), transient decreases in neutrophils and lymphocytes, decreased or suppressed erythropoiesis (decreased red blood cell mass and reticulocyte count), and transient increases in AST and / or ALT. All of these are dose-responsive in nature, reversible before or during the recovery period without microscopic correlation, and are not considered adverse because the changes often remain within or very close to the historical control range of these parameters in cynomolgus monkeys.

[0246] Microscopically, a range of findings were observed in the skin of routine sections (lateral ventral), infusion sites, and other locations (including forelimbs, hindlimbs, and head). These findings varied in severity and / or presence across individual sites and between animals, but were generally similar in character across dose groups and exhibited a marked dose-response in severity and extent, typically more pronounced at doses ≥6 mg / kg / dose, and included increased pigmentation, lymphocytic inflammation, epidermal hyperplasia, and single-cell necrosis. Inflammation and single-cell necrosis reversed by the end of the recovery period, while pigmentation changes and hyperplasia resolved persistently by the end of the recovery period. Example 8 Comparison of toxicological studies of MGC026 and MGC018 in cynomolgus monkeys

[0247] Table 16 provides a comparison of the toxicological studies of MGC026 and MGC018 conducted in cynomolgus monkeys as described in Examples 6 and 7. This comparison shows that MGC026 has a more distinct profile compared to MGC018, which caused hyperpigmentation, lymphocytic inflammation, epidermal hyperplasia, and single-cell necrosis in the animals' skin. These findings were more severe as the dose interval in Q2W decreased, leading to euthanasia in animals receiving 20 mg / kg. Example 9 MGC026 exhibited in vivo activity in patient-derived xenografts.

[0248] To further demonstrate the antitumor activity of MGC026, tumor fragments from different patients were used in AthymicNude-Foxn1. nuThe in vivo toxicity of the MGC026 molecule described above was evaluated in a mouse model (Envigo, Charles River Laboratories). In short, the in vivo toxicity of the molecule was assessed by implanting a low-passage tumor fragment into Athymic Nude-Foxn1. nu In stock mice. When enough stock mice have tumors reaching 1000-1500 mm. 3 At that time, tumors were harvested, and tumor fragments were unilaterally reimplanted into the left side of pre-research mice. When the tumor reached 150-300 mm... 3 Before the study, mice were randomly assigned to groups based on tumor volume and treated with either MGC026 or a carrier control (formulation buffer (FB)). Mice were assigned to each group and administered the drug intravenously via tail vein injection (approximately 100 µL). In these studies, either MGC026 or the carrier control was administered every two weeks for a total of two doses (Q2W×2). Tumors were measured twice weekly using an orthogonal measurement method with electronic calipers, where tumor volume was calculated as: (width×2)×length×0.52. Animals were considered to have partial regression (“PR”) when their tumor volume decreased by 50% or more compared to the tumor volume on the day of the first dose administration. During the study period, treated animals achieved a tumor volume reduction to ≤ 5 mm. 3 The finding was considered to indicate complete remission (“CR”). Tumor volume (relative to vector control) was determined (“T / C”). Antitumor activity was assessed according to the National Cancer Institute (NCI) criteria; a T / C ≤ 42% was the minimum level of antitumor activity, while a T / C value > 42% was inactive. A T / C < 10% was considered highly active. In vivo activity against tumors derived from small cell lung cancer patients

[0249] The results of this study regarding tumor fragments derived from subcutaneously inoculated small cell lung cancer patients in Model 1 are presented in Table 17 and... Figure 13 The study also demonstrated responsiveness to tumor fragments derived from small cell lung cancer patients. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. When the vector tumor volume reached a mean of 1776 mm on day 10... 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0250] Female Athymic naked-Foxn1 nu Mice (n=3 mice / group) were subcutaneously implanted with tumor fragments derived from small cell lung cancer patients in stock mice. The tumors reached an average size of 150-300 mm. 3 (257 ± 46 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0251] MGC026 reduced tumor volume by 78% on day 10 at a dose level of 10 mg / kg Q2W×2 and was active according to NCI criteria (22% T / C). At this dose level, MGC026 induced 1 / 3 partial regression, but no complete regression. In vivo activity against tumors derived from small cell lung cancer patients

[0252] Results from this study regarding tumor fragments derived from subcutaneously inoculated small cell lung cancer patients in Model 2 are presented in Table 18 and... Figure 14 The study also demonstrated responsiveness to tumor fragments derived from small cell lung cancer patients. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The mean tumor volume in the vector group reached 1733 mm on day 52. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0253] Female Athymic naked-Foxn1 nu Mice (n=3 mice / group) were subcutaneously implanted with tumor fragments derived from small cell lung cancer patients from stock mice. The tumors reached an average size of 150-300 mm. 3 (268 ± 10 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0254] MGC026 reduced tumor volume by 99% on day 52 at a dose level of 10 mg / kg Q2W×2 and was highly active according to NCI criteria (1% T / C). At this dose level, MGC026 induced partial regression in 3 / 3 and complete regression in 1 / 3. Treatment with MGC026 at a dose level of 10 mg / kg Q2W×2 was maintained at an antitumor control level until the end of the study. In vivo activity against tumors derived from small cell lung cancer patients

[0255] The results of this study regarding tumor fragments derived from subcutaneously inoculated small cell lung cancer patients in Model 3 are presented in Table 19 and... Figure 15 The study also demonstrated responsiveness to tumor fragments derived from small cell lung cancer patients. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The vector tumor volume reached a mean of 2565 mm on day 23. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0256] Female Athymic naked-Foxn1 nu Mice (n=3 mice / group) were subcutaneously implanted with tumor fragments derived from small cell lung cancer patients from stock mice. The tumors reached an average size of 150-300 mm. 3 (236 ± 80 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0257] MGC026 reduced tumor volume by 100% on day 23 at a dose level of 10 mg / kg Q2W x 2 and was highly active according to NCI criteria (0% T / C). At this dose level, MGC026 induced partial regression in 3 / 3 and complete regression in 2 / 3. Treatment with MGC026 at a dose level of 10 mg / kg Q2W x 2 was maintained at an antitumor control level until the end of the study. In vivo activity against tumors derived from ovarian cancer patients

[0258] The results of this study regarding subcutaneous inoculation of tumor fragments derived from ovarian cancer patients are presented in Table 20 and... Figure 16 The study also demonstrated responsiveness to tumor fragments derived from ovarian cancer patients. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The vector tumor volume reached a mean of 2302 mm on day 48. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0259] Female Athymic naked-Foxn1 nu Mice (n=3 mice / group) were subcutaneously implanted with tumor fragments derived from ovarian cancer patients from stock mice. The tumors reached an average size of 150-300 mm. 3 (211 ± 82 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0260] MGC026 reduced tumor volume by 98% on day 48 at a dose level of 10 mg / kg Q2W×2 and was highly active based on NCI criteria (2% T / C). At this dose level, MGC026 induced 3 / 3 partial regression, but not complete regression. Treatment with MGC026 at a dose level of 10 mg / kg Q2W×2 maintained antitumor control until the end of the study. In vivo activity against tumors derived from melanoma patients

[0261] The results of this study regarding subcutaneous inoculation of tumor fragments derived from melanoma patients are presented in Table 21 and... Figure 17 The study also demonstrated responsiveness to tumor fragments derived from melanoma patients. a %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The mean tumor volume in the vector group reached 3003 mm on day 30. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0262] Female Athymic naked-Foxn1 nu Mice (n=3 / group) were subcutaneously implanted with tumor fragments derived from melanoma patients from stock mice. The tumors reached an average size of 150-300 mm. 3 (202 ± 78 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0263] MGC026 reduced tumor volume by 97% on day 30 at a dose level of 10 mg / kg Q2W×2 and was highly active (3% T / C) according to NCI criteria. At this dose level, MGC026 induced partial regression in 2 / 3 and complete regression in 1 / 3. Treatment with MGC026 at a dose level of 10 mg / kg Q2W×2 was maintained at an antitumor control level until the end of the study. In vivo activity against tumors derived from colorectal cancer patients

[0264] The results of this study regarding subcutaneous inoculation of tumor fragments derived from colorectal cancer patients are presented in Table 22 and... Figure 18 The study also demonstrated responsiveness to tumor fragments derived from colorectal cancer patients. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. The mean tumor volume in the vector group reached 1102 mm on day 29. 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0265] Female Athymic naked-Foxn1 nu Mice (n=3 / group) were subcutaneously implanted with tumor fragments derived from colorectal cancer patients from stock mice. The tumors reached an average size of 150-300 mm. 3 (248 ± 93 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0266] MGC026 reduced tumor volume by 76% on day 29 at a dose level of 10 mg / kg Q2W×2 and was active based on NCI criteria (24% T / C). At this dose level, MGC026 induced partial regression of 1 / 3, but not complete regression. In vivo activity against tumors derived from patients with squamous cell carcinoma of the head and neck (SCCHN)

[0267] The results of this study regarding subcutaneous inoculation of tumor fragments from SCCHN patients are presented in Table 23 and... Figure 19 The study also demonstrated responsiveness to tumor fragments derived from SCCHN patients. %T / C = the percentage of the mean tumor size (T) in the treatment group relative to the mean tumor size (C) in the vector control group. When the vector tumor volume reached a mean of 1292 mm on day 34... 3 It is calculated in time. b. Antitumor activity: assessed based on the criteria of the National Cancer Institute (NCI); T / C ≤ 42% is the minimum level of antitumor activity, while T / C > 42% is inactive. T / C < 10% is considered highly active. Abbreviations: Ab: antibody; mg: milligram; kg: kilogram; IV: intravenous; PR: partial regression (tumor volume decreases by 50% or more from the date of administration during the study); CR: complete regression (defined as no obvious tumor is detectable during the study process (0 mm)). 3 When the tumor volume is unmeasurable (≤ 5mm) 3 )hour).

[0268] Female Athymic naked-Foxn1 nu Mice (n=3 mice / group) were subcutaneously implanted with SCCHN patient-derived tumor fragments from stock mice. The tumors reached an average size of 150-300 mm. 3 (258 ± 50 mm 3 Mice were randomly assigned to groups based on tumor volume before the study, and treated with either MGC026 or a carrier control (formulation buffer) at the indicated dose levels on days 0 and 14 (arrows). Tumor volumes are shown as group mean ± standard error of the mean (SEM), with the SEM scale bar visible above. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0269] MGC026 reduced tumor volume by 92% on day 34 at a dose level of 10 mg / kg Q2W x 2 and was highly active (8% T / C) based on NCI criteria. At this dose level, MGC026 induced 3 / 3 partial regression, with no complete regression.

[0270] All publications and patents mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated herein by reference in its entirety. While specific aspects are described herein, it should be understood that further modifications are possible, and this application is intended to cover any changes, uses, or alterations to these aspects.

Claims

1. An anti-B7-H3 antibody-drug conjugate (B7-H3-ADC), comprising the formula: Ab-(LM) m -(D) n , in: Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) Contains CDRs within its variable light chain (VL) domain. L 1. Sequence RASESIYSYLA (SEQ ID NO:16), CDR L 2. Sequence NTKTLPE (SEQ ID NO:17) and CDR L 3. Sequence QHHYGTPPWT (SEQ ID NO:18), and (ii) Contains CDRs within its variable heavy chain (VH) domain. H 1. Sequence SYGMS (SEQ ID NO:19), CDR H 2. Sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20) and CDR H 3 sequences HDGGAMDY (SEQ ID NO:21) or HEGGAMDY (SEQ ID NO:26); D is the camptothecin portion; LM is a linker molecule that covalently connects Ab and D; m is an integer between 1 and n and represents the number of linker molecules in B7-H3-ADC; as well as n is an integer between 1 and 10 and represents the number of cytotoxic camptothecin moieties covalently linked to the B7-H3-ADC molecule.

2. The B7-H3-ADC according to claim 1, wherein Ab comprises: (i) Contains a humanized VL domain containing the amino acid sequence SEQ ID NO:12, and (ii) A humanized VH domain containing the amino acid sequence SEQ ID NO:14 or SEQ ID NO:

22.

3. The B7-H3-ADC according to claim 1 or 2, wherein Ab is an antibody.

4. The B7-H3-ADC according to any one of claims 1-3, wherein Ab is an antigen-binding fragment of the antibody.

5. The B7-H3-ADC according to any one of claims 1-4, wherein Ab comprises the Fc domain of human IgG.

6. The B7-H3-ADC according to claim 5, wherein the human IgG is human IgG1, IgG2, IgG3 or IgG4.

7. The B7-H3-ADC according to claim 5 or 6, wherein the Fc domain is a variant Fc domain comprising: (a) One or more amino acid modifications that reduce the affinity of the variant Fc domain for FcγR; and / or (b) One or more amino acid modifications that enhance the serum half-life of the variant Fc domain.

8. The B7-H3-ADC of claim 7, wherein the modification that reduces the affinity of the variant Fc domain for FcγR comprises the following substitutions: L234A; L235A; or L234A and L235A, wherein the number is the EU index number in Kabat.

9. The B7-H3-ADC according to claim 7 or 8, wherein the modification of the serum half-life of the enhancing variant Fc domain comprises the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, wherein the number is the EU index number in Kabat.

10. The B7-H3-ADC of claim 1, wherein the LM comprises a peptide linker.

11. The B7-H3-ADC of claim 1, wherein the LM includes a detachable connector.

12. The B7-H3-ADC according to claim 1, wherein the LM comprises formula (4a) or (4b) or a salt thereof: in: a is either 0 or 1 independently; b is either 0 or 1 independently; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1 to 150; g is 0 or 1; i is 0 or 1; D is the cytotoxic camptothecin moiety; Q 1 It is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclic triazole group, or a cycloalkenyl group; Q 1 Functional groups attached to antibodies; Sp 1 Sp 2 Sp 3 and Sp 4 Independently select from the following groups: C1-C of straight chains or branches. 200 alkylene groups, C2-C 200 imide groups, C2-C 200 Imynyl group, C3-C 200 Cycloalkyl groups, C5-C 200 Cycloalkenyl groups, C8-C 200 Cycloynyl group, C7-C 200 alkylarylene groups, C7-C 200 arylalkylene groups, C8-C 200 aryl imide groups and C9-C 200 The arylynyl group, wherein the alkylene group, alkenyl group, ynynyl group, cycloalkylene group, cycloalkenyl group, cycloynynyl group, alkylaryl group, arylalkylene group, arylalkenyl group, and arylynyl group are optionally selected from O, S, and NR. 3 One or more heteroatoms of the group are substituted and optionally selected from O, S and NR. 3 One or more heteroatom spacings in the group, where R 3 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted; Z 1 It is Q 1 or SP 3 Connect to SP 2 O or C(O) or N(R) 1 () linking group; Z 2 Is it D or SP? 4 Connect to SP 1 、N(R 1 ), O or C(O) linking groups; where Z 1 and Z 2 are independently selected from the group consisting of: -O-, -S-, -NR 2 -, -N=N-, -C(O)-, -C(O)NR 2 -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-NR 2 -, -NR 2 -C(O)-, -NR 2 -C(O)-O-, -NR 2 -C(O)-NR 2 -, -S-C(O)-, -S-C(O)-O-, -S-C(O)-NR 2 -, -S(O)-, -S(O)2-, -O-S(O)2-, -O-S(O)2-O-, -O-S(O)2-NR 2 -, -O-S(O)-, -O-S(O)-O-, -O-S(O)-NR 2 -, -O-NR 2 -C(O)-, -O-NR 2 -C(O)-O-, -O-NR 2 -C(O)-NR 2 -, -NR 2 -O-C(O)-, -NR 2 -O-C(O)-O-, -NR 2 -O-C(O)-NR 2 -, -O-NR 2 -C(S)-, -O-NR 2 -C(S)-O-, -O-NR 2 -C(S)-NR 2 -, -NR 2 -O-C(S)-, -NR 2 -O-C(S)-O-, -NR 2 -O-C(S)-NR 2 -, -O-C(S)-, -O-C(S)-O-, -O-C(S)-NR 2 -, -NR 2 -C(S)-, -NR 2 -C(S)-O-, -NR 2 -C(S)-NR 2 -, -S-S(O)2-, -S-S(O)2-O-, -S-S(O)2-NR 2 -, -NR 2 -O-S(O)-, -NR 2 -O-S(O)-O-, -NR 2 -OS(O)-NR 2 -、-NR 2 -OS(O)2-、-NR 2 -OS(O)2-O-、-NR 2 -OS(O)2-NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O)2-O-、-O-NR 2 -S(O)2-NR 2 -、-O-NR 2 -S(O)2-、-OP(O)(R 2 )2-、-SP(O)(R 2 )2-、-NR 2 -P(O)(R 2 )2- and combinations of two or more, wherein R 2 Independently select from the following groups: hydrogen, C1-C 24 Alkyl groups, C2-C 24 alkenyl groups, C2-C 24 alkynyl group and C3-C 24 Cycloalkyl groups, wherein the alkyl groups, alkenyl groups, ynyl groups, and cycloalkyl groups are optionally substituted; and R 1 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups, the C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 The (hetero)arylalkyl group is optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, said one or more heteroatoms being selected from O, S, and NR. 3 , where R 3 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 It is D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 Sp 2 Sp 3 Sp 4 Z 1 Z 2 D, Q 1 b, c, d, e, g, and i are defined as above.

13. The B7-H3-ADC according to claim 12, wherein Sp 1 Sp 2 Sp 3 and Sp 4 If present, independently selected from the group consisting of: straight or branched C1-C 20 An alkylene group, wherein the alkylene group is optionally substituted with one or more heteroatoms and optionally spaced by one or more heteroatoms, the one or more heteroatoms being selected from O, S, and NR. 3 The group consisting of R 3 The group consisting of hydrogen and C1-C4 alkyl groups is selected independently.

14. The B7-H3-ADC according to any one of claims 1-13, wherein the LM comprises a valine-alanine (Val-Ala) amino acid linker.

15. The B7-H3-ADC according to claim 14, wherein the Val-Ala connector is a Val-Ala-PABC connector.

16. The B7-H3-ADC according to any one of claims 1-15, wherein the camptothecin portion is selected from the group consisting of: SN-38 (S-10-hydroxycamptothecin), topotecan (HYCAMPTIN; (S)-9-N,N-dimethylaminoethyl-10-hydroxycamptothecin), 9-aminocamptothecin (9-amino-20(S)-camptothecin), 9-nitrocamptothecin (also known as rubitecan), letopotecan (7-(4-methylpiperazinemethylene)-10,11-ethylenedioxy-20(S)-camptothecin), eczetine, belotetine, calenitine, and holoconetin.

17. The B7-H3-ADC according to any one of claims 1-16, wherein the camptothecin portion is eczetidine.

18. The B7-H3-ADC according to any one of claims 1-17, wherein LM and D together comprise: 。 19. An anti-B7-H3 antibody-drug conjugate (B7-H3-ADC), comprising the formula: Ab-(LM) m -(D) n , in: Ab is a humanized B7-H3 antibody or its B7-H3 binding fragment that binds to B7-H3, and: (i) Contains CDRs within its variable light chain (VL) domain. L 1. Sequence RASESIYSYLA (SEQ ID NO:16), CDR L 2. Sequence NTKTLPE (SEQ ID NO:17) and CDR L The sequence QHHYGTPPWT (SEQ ID NO:18) and (ii) Contains CDRs within its variable heavy chain (VH) domain. H 1. Sequence SYGMS (SEQ ID NO:19), CDR H 2. Sequence TINSGGSNTYY PDSLKG (SEQ ID NO:20) and CDR H 3 sequences HDGGAMDY (SEQ ID NO:21) or HEGGAMDY (SEQ ID NO:26); D and LM together include: m is an integer between 0 and n and represents the number of linker molecules in the B7-H3-ADC; and n is an integer between 1 and 10 and represents the number of cytotoxic camptothecin moieties covalently linked to the B7-H3-ADC molecule.

20. The B7-H3-ADC of claim 19, wherein Ab comprises: (i) Contains a humanized VL domain comprising the amino acid sequence SEQ ID NO:12, and (ii) A humanized VH domain containing the amino acid sequence SEQ ID NO:14 or SEQ ID NO:

22.

21. A pharmaceutical composition comprising an effective amount of B7-H3-ADC according to any one of claims 1-20 and a pharmaceutically acceptable carrier, excipient, or diluent.

22. Use of the B7-H3-ADC according to any one of claims 1-20 or the pharmaceutical composition according to claim 21 in the treatment of diseases or conditions associated with or characterized by B7-H3 expression.

23. A method for treating a disease or condition associated with or characterized by B7-H3 expression, the method comprising administering to a subject a B7-H3-ADC according to any one of claims 1-20 or a pharmaceutical composition according to claim 21.

24. The use according to claim 22 or the method according to claim 23, wherein the disease or condition associated with or characterized by B7-H3 expression is cancer.

25. The use or method according to claim 24, wherein the cancer is selected from the group consisting of: adrenal tumors, AIDS-related cancers, alveolar soft tissue sarcomas, astrocytic tumors, adrenal carcinoma, bladder cancer, bone cancer, brain and spinal cord cancers, metastatic brain tumors, B-cell carcinoma, breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, fibroproliferative small round cell tumors, ependymoma, Ewing's tumor, extraosseous myxoid chondrosarcoma, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, glioblastoma, hematologic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, renal cancer, leukemia (e.g., acute myeloid leukemia), liposarcoma / malignant tumors. Lipomas, liver cancer, lymphoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), medulloblastoma, melanoma, meningioma, mesothelioma, laryngeal cancer, multiple endocrine tumors, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumors, childhood cancers, peripheral nerve sheath tumors, pheochromocytoma, pituitary adenoma, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumors in children (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck (SCCHN)), gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (e.g., metastatic thyroid cancer), and uterine cancer.

26. The use or method according to claim 25, wherein the cancer is selected from the group consisting of: adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, renal cancer, non-small cell lung cancer (NSCLC), acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, mesothelioma, laryngeal cancer, non-Hodgkin lymphoma, small lymphocytic lymphoma, Multiple myeloma, melanoma, ovarian cancer, platinum-resistant ovarian cancer (PROC), pancreatic cancer, prostate cancer, metastatic castration-resistant prostate cancer (mCRPC), skin cancer, renal cell carcinoma, small cell lung cancer (SCLC), extensive-stage small cell lung cancer (ES-SCLC), childhood small round blue cell tumors (including neuroblastoma and rhabdomyosarcoma), squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN), testicular cancer, thyroid cancer (e.g., metastatic thyroid cancer), and uterine cancer cells selected from the adrenal gland cells.

27. The use or method according to claim 24, wherein the cancer is melanoma.

28. The use or method according to claim 24, wherein the cancer is lung cancer.

29. The use or method according to claim 24, wherein the cancer is squamous cell carcinoma of the head and neck (SCCHN).

30. The use or method according to claim 24, wherein the cancer is pancreatic cancer.

31. The use or method according to claim 24, wherein the cancer is prostate cancer.

32. The use or method according to claim 24, wherein the cancer is small cell lung cancer (SCLC).

33. The use or method according to claim 24, wherein the cancer is small cell ovarian cancer.

34. The use or method according to claim 24, wherein the cancer is small cell colorectal cancer.

35. The use or method according to claim 24, wherein the cancer is esophageal squamous cell carcinoma.

36. The use or method according to claim 24, wherein the cancer is non-small cell lung cancer (NSCLC).

37. The use or method according to claim 24, wherein the cancer is bladder cancer.

38. The use or method according to claim 24, wherein the cancer is a sarcoma.

39. The use or method according to claim 24, wherein the cancer is endometrial cancer.

40. The use or method according to claim 24, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC).

41. The use or method according to claim 24, wherein the cancer is breast cancer.

42. The use or method according to claim 24, wherein the cancer is ovarian cancer.

43. The use or method according to claim 24, wherein the cancer is cervical cancer.

44. The use or method according to claim 24, wherein the cancer is colorectal cancer.

45. The use or method according to claim 24, wherein the cancer is gastric cancer or gastroesophageal junction cancer.

46. ​​The use or method according to claim 24, wherein the cancer is clear cell renal cell carcinoma.

47. The use or method according to claim 24, wherein the cancer is hepatocellular carcinoma.

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