B7-H3 antibody-drug conjugate

Abstract

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 camptothecin drug moiety. This disclosure also relates to pharmaceutical compositions containing the above B7-H3-ADCs, as well as methods involving the use of any of the above 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 drug portion. This disclosure also relates to pharmaceutical compositions containing the B7-H3-ADCs, and methods involving the use of any of the B7-H3-ADCs in the treatment of cancer and other diseases and conditions related to or characterized by B7-H3 expression. [Background technology]

[0002] I.B7 Super Family and B7-H3 B7-H3 is a member of the B7-CD28 superfamily, and B7-H3 expressed on antigen-presenting cells is unique in that the major human form contains two extracellular tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC) (Non-Patent Literature 1). The 4-immunoglobulin extracellular domain variant ("4Ig-B7-H3") was initially thought to contain only two Ig domains (IgV-IgC) (see, for example, NCBI sequence NP_079516), but it has been identified and discovered to be a more common human form of the above protein (Non-Patent Literature 2; see also, for example, NCBI sequence NP_001019907). B7-H3 mRNA expression has been found in the heart, kidney, testes, lungs, liver, pancreas, prostate, colon, and osteoblasts (Non-Patent Literature 1). At the protein level, B7-H3 is found in the human liver, lungs, bladder, testes, prostate, breast, placenta, and lymphatic organs (Non-Patent Literature 3).

[0003] B7-H3 is not expressed on resting B or T cells, monocytes, or dendritic cells, but is induced on dendritic cells by IFN-γ and on monocytes by GM-CSF (Non-Patent Literature 2). The mode of action of B7-H3 is complex, and this protein has been reported to mediate both T cell co-stimulation and co-inhibition (Non-Patent Literature 3, 4). B7-H3 mediates T cell co-inhibition by binding to one or more unidentified receptors. Furthermore, B7-H3 acts as an inhibitor on NK cells and osteoblasts through interaction with one or more unknown receptors (Non-Patent Literature 3).

[0004] II. B7-H3 expressing tumors B7-H3 is also expressed on a variety of cancer cells (e.g., neuroblastoma, gastric cancer, ovarian cancer, non-small cell lung cancer, etc., see Non-Patent Document 5, for example) and cultured tumor stem-like cells. Several independent studies have shown that human malignant tumor cells exhibit a significant increase in B7-H3 protein expression, and that this increase in expression is associated with increased disease severity (Non-Patent Documents 6, 7), suggesting that B7-H3 is utilized by tumors as an immune evasion pathway (Non-Patent Document 3).

[0005] The role of B7-H3 in inhibiting the immune system, and the increased expression of B7-H3 on human tumors, suggest that this molecule may function as a therapeutic target for cancer treatment. It has been proposed to treat tumors and / or upmodulate immune responses using anti-B7-H3 antibodies and other molecules that modulate B7-H3 expression (see Non-Patent Documents 8-10; also see Patent Documents 1-20). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 7,279,567 [Patent Document 2] U.S. Patent No. 7,527,969 [Patent Document 3] U.S. Patent No. 7,718,774 [Patent Document 4] U.S. Patent No. 8,779,098 [Patent Document 5] U.S. Patent No. 8,802,091 [Patent Document 6] U.S. Patent Application Publication No. 2002 / 0168762 [Patent Document 7] U.S. Patent Application Publication No. 2008 / 0081346 [Patent Document 8] U.S. Patent Application Publication No. 2008 / 0116219 [Patent Document 9] U.S. Patent Application Publication No. 2013 / 0078234 [Patent Document 10] U.S. Patent Application Publication No. 2015 / 0274838 [Patent Document 11] International Publication No. 2009 / 073533 [Patent Document 12] International Publication No. 2008 / 066691 [Patent Document 13] International Publication No. 2006 / 016276 [Patent Document 14] International Publication No. 2008 / 116219 [Patent Document 15] International Publication No. 2001 / 094413 [Patent Document 16] International Publication No. 2002 / 32375 [Patent Document 17] International Publication No. 2004 / 093894 [Patent Document 18] International Publication No. 2006 / 016276 [Patent Document 19] International Publication No. 2008 / 116219 [Patent Document 20] International Publication No. 2011 / 109400 [Non-patent literature]

[0007] [Non-Patent Document 1] Collins, M. et al. (2005) "The B7 Family Of Immune-Regulatory Ligands," Genome Biol. 6:223.1-223.7 [Non-Patent Document 2] Sharpe, AH et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2:116-126 [Non-Patent Document 3] Hofmeyer, K. et al. (2008) "The Contrasting Role Of B7-H3," Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278 [Non-Patent Document 4] Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation And Anti-Tumor Immunity," Semin. Cancer Biol. 17(4):288-298 [Non-Patent Document 5] Modak, S., et al. (2001) "Monoclonal antibody 8H9 targets a novel cell surface antigen expressed by a wide spectrum of human solid tumors," Cancer Res 61:4048-54 [Non-Patent Document 6] 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 [Non-Patent Document 7] Wang, L., et al. (2013) "B7-H3 Mediated Tumor Immunology: Friend Or Foe?," Int. J. Cancer 134(12):2764-2771 [Non-Patent Document 8] Loo, D. et al. (2012) "Development of an Fc-Enhanced Anti-B7-H3 Monoclonal Antibody with Potent Antitumor Activity," Clin Cancer Res; 18: 3834-3845 [Non-Patent Document 9] Ahmed, M. et al. (2015) "Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loop of B7-H3," J. Biol. Chem. 290: 30018-30029 [Non-Patent Document 10] 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 [Overview of the project] [Problems that the invention aims to solve]

[0008] 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 drug portion. This disclosure relates to pharmaceutical compositions containing the B7-H3-ADCs, and methods involving the use of any of the B7-H3-ADCs in the treatment of cancer and other diseases and conditions. [Means for solving the problem]

[0009] In multiple embodiments, this disclosure relates to the formula: Ab-(LM) m -(D) n We offer an anti-B7-H3 antibody drug conjugate (B7-H3-ADC) containing: Ab is bound to B7-H3, and: (i) Within its variable light chain (VL) domain, CDR L 1 Sequence RASESIYSYLA (SEQ ID NO: 16), CDR L 2 sequences NTKTLPE (sequence number 17), and CDR L 3-sequence QHHYGTPPWT (sequence number 18), (ii) Within its variable heavy chain (VH) domain, CDR H 1. Sequence SYGMS (SEQ ID NO: 19), CDR H 2 sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H 3-sequence HDGGAMDY (sequence number 21) or HEGGAMDY (sequence number 26) A humanized B7-H3 antibody or its B7-H3 binding fragment, D is the cytotoxic drug portion; LM is a linker molecule that covalently bonds Ab and D; m is an integer between 1 and n, representing the number of linker molecules in B7-H3-ADC; n is an integer between 1 and 10, representing the number of cytotoxic drug moieties covalently bonded to the B7-H3-ADC molecule.

[0010] In multiple embodiments, this disclosure relates to the formula: Ab-(LM) m -(D) n Provided is an anti-B7-H3 antibody drug conjugate (B7-H3-ADC) comprising: Ab binds to B7-H3 and: (i) within its variable light chain (VL) domain, CDR 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), (ii) within its variable heavy chain (VH) domain, CDR H 1 sequence SYGMS (SEQ ID NO: 19), CDR H 2 sequence TINSGGSNTYY PDSLKG (SEQ ID NO: 20), and CDR H 3 sequence HDGGAMDY (SEQ ID NO: 21) or HEGGAMDY (SEQ ID NO: 26) is a humanized B7-H3 antibody or a B7-H3 binding fragment thereof, D is a cytotoxic camptothecin moiety; LM is a linker molecule that covalently attaches Ab and D; m is an integer from 1 to n, representing the number of linker molecules of B7-H3-ADC; n is an integer from 1 to 10, representing the number of cytotoxic camptothecin moieties covalently attached to the B7-H3-ADC molecule.

[0011] In multiple embodiments, the disclosure further provides the above B7-H3-ADC, wherein Ab is: a) a humanized VL domain comprising the amino acid sequence of SEQ ID NO: 12, and b) a humanized VH domain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 22 In multiple embodiments, the disclosure further provides the above B7-H3-ADC, wherein Ab is an antibody. In multiple embodiments, the disclosure further provides the above B7-H3-ADC, wherein Ab is an antigen-binding fragment of an antibody. [[ID=​​

[0013] In several embodiments, the Disclosure further provides the B7-H3-ADC wherein Ab comprises the Fc domain of human IgG. In several embodiments, the Disclosure further provides the B7-H3-ADC wherein human IgG is human IgG1, IgG2, IgG3, or IgG4.

[0014] In several embodiments, the disclosure further states that an Fc domain is a variant Fc domain: (a) One or more amino acid modifications that reduce the affinity of the variant Fc domain to FcγR: and / or (b) One or more amino acid modifications that increase the serum half-life of the variant Fc domain The above B7-H3-ADC is provided, which is a variant Fc domain containing the above.

[0015] In multiple embodiments, the Disclosure further provides the B7-H3-ADC, wherein the modification for reducing the affinity of the variant Fc domain to FcγR includes substitutions of L234A;L235A; or L234A and L235A, and the numbering is an EU index numbering similar to that in Kabat. In multiple embodiments, the Disclosure further provides the B7-H3-ADC, wherein the modification for increasing the serum half-life of the variant Fc domain includes substitutions of M252Y;M252Y and S254T;M252Y and T256E;M252Y, S254T and T256E; or K288D and H435K, and the numbering is an EU index numbering similar to that in Kabat.

[0016] In several embodiments, the disclosure further provides the B7-H3-ADC in which the LM comprises a peptide linker. In several embodiments, the disclosure further provides the B7-H3-ADC in which the LM comprises a cleavable linker.

[0017] In several embodiments, the Disclosure further relates that LM is formula (4a) or (4b), or a salt thereof: [ka] The above B7-H3-ADC is provided, including: a is independently either 0 or 1; b is independently either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is an integer in the range of 1 to 150; g is either 0 or 1; i is either 0 or 1; D is the cytotoxic drug portion; Q 1 is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclotriazole group, or a cycloalkenyl group; Q 1 It is bound to the functional group of the antibody; Sp 1 , Sp 2 , Sp 3 , and Sp 4 These are independently linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkylene group, C7-C 200 Alkylalylene group, C7-C 200 Arylalkylene group, C8-C 200 Arylalkenylene group, and C9-C 200 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylalylene group, arylalkylene group, arylalkenylene group, and arylalkylene group may be optionally substituted, and optionally O, S, and NR 3 It is interrupted by one or more heteroatoms selected from the group, where R 3 Independently, hydrogen, C1-C 24Alkyl alkyl group, C2-C 24 Alkenyl group, C2-C 24 Alkynyl group, and C3-C 24 Selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group may be optionally substituted; Z 1 Q 1 or Sp 3 Sp 2 , O or C(O) or N(R 1 It is a connecting group that connects to ); Z 2 is D or Sp 4 Sp 1 , N(R 1 A connecting element that connects to O, or C(O); Z 1 and Z 2 These are independently -O-, -S-, and -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 -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​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ R 1 is hydrogen, C1-C 24 Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3-C 24 Alkyl (hetero)aryl group, and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the above C1-C 24 Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3-C 24 Alkyl (hetero)aryl group, and C3-C 24 The (hetero)arylalkyl group may be optionally substituted, and may also be O, S, and NR. 3 It is interrupted by one or more heteroatoms selected from, where R 3 These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D], or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ] and here 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.

[0018] In several embodiments, the Disclosure further relates that LM is formula (4a) or (4b), or a salt thereof: [ka] The above B7-H3-ADC is provided, including: a is independently either 0 or 1; b is independently either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is an integer in the range of 1 to 150; g is either 0 or 1; i is either 0 or 1; D is the cytotoxic camptothecin moiety; Q 1 is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclotriazole group, or a cycloalkenyl group; Q 1 It is bound to the functional group of the antibody; Sp 1 , Sp 2 , Sp 3 , and Sp 4 These are independently linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkylene group, C7-C 200 Alkylalylene group, C7-C 200 Arylalkylene group, C8-C 200 Arylalkenylene group, and C9-C 200 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylalylene group, arylalkylene group, arylalkenylene group, and arylalkylene group may be optionally substituted, and optionally O, S, and NR 3 It is interrupted by one or more heteroatoms selected from the group, where R 3 Independently, hydrogen, C1-C 24 Alkyl alkyl group, C2-C 24 Alkenyl group, C2-C24 Alkynyl group, and C3-C 24 Selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group may be optionally substituted; Z 1 Q 1 or Sp 3 Sp 2 , O or C(O) or N(R 1 It is a connector that connects to ); Z 2 is D or Sp 4 Sp 1 , N(R 1 A connecting element that connects to O, or C(O); Z 1 and Z 2 These are independently -O-, -S-, and -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 -, -NR2 -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 selected from the group consisting of two or more combinations of these, where R 2 Independently, hydrogen, C1-C 24 Alkyl alkyl group, C2-C 24 Alkenyl group, C2-C 24 Alkynyl group, and C3-C 24 Selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group may be optionally substituted; R 1 is hydrogen, C1-C 24Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3-C 24 Alkyl (hetero)aryl group, and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the above C1-C 24 Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3-C 24 Alkyl (hetero)aryl group, and C3-C 24 The (hetero)arylalkyl group may be optionally substituted, and may also be O, S, and NR. 3 It is interrupted by one or more heteroatoms selected from, where R 3 These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D], or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ] and here 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.

[0019] In several embodiments, this disclosure further includes Sp 1 , Sp 2 , Sp 3 , and Sp 4 If they exist, they can be linear or branched C1-C independently. 20 Selected from the group consisting of alkylene groups, the alkylene groups may be optionally substituted, and may also be O, S, and NR.3 It is interrupted by one or more heteroatoms selected from the group consisting of R, where R 3 This independently provides the above B7-H3-ADC, which is selected from the group consisting of hydrogen and C1-C4 alkyl groups.

[0020] In several embodiments, the Disclosure further provides the B7-H3-ADC in which LM comprises a valine-alanine (Val-Ala) amino acid linker. In several embodiments, the Disclosure further provides the B7-H3-ADC in which the Val-Ala linker is a Val-Ala-PABC linker.

[0021] In several embodiments, the present disclosure further provides the B7-H3-ADC in which the camptothecin moiety 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), lulutotecan (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin), exatecan, calenitecin, and homocamptothecin.

[0022] In several embodiments, the disclosure further provides the B7-H3-ADC wherein the camptothecin portion is exatecan.

[0023] In several embodiments, this disclosure further states that LM and D together: [ka] The above-mentioned B7-H3-ADC is provided, which constitutes the above.

[0024] In several embodiments, this disclosure further relates to the formula: Ab-(LM) m -(D) n We offer an anti-B7-H3 antibody drug conjugate (B7-H3-ADC) containing: Ab is bound to B7-H3, and: (i) Within its variable light chain (VL) domain, CDR L 1 Sequence RASESIYSYLA (SEQ ID NO: 16), CDR L 2 sequences NTKTLPE (sequence number 17), and CDR L 3-sequence QHHYGTPPWT (sequence number 18), (ii) Within its variable heavy chain (VH) domain, CDR H 1. Sequence SYGMS (SEQ ID NO: 19), CDR H 2 sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H 3-sequence HDGGAMDY (sequence number 21) or HEGGAMDY (sequence number 26) A humanized B7-H3 antibody or its B7-H3 binding fragment, D and LM are both: [ka] Constitute, m is an integer between 0 and n, representing the number of linker molecules in B7-H3-ADC; n is an integer between 1 and 10, representing the number of cytotoxic camptothecin moieties covalently bonded to the B7-H3-ADC molecule.

[0025] In several embodiments, the Disclosure further provides the B7-H3-ADC molecule wherein Ab comprises a humanized VL domain comprising the amino acid sequence of SEQ ID NO: 12 and a humanized VH domain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 22.

[0026] In several embodiments, the Disclosure further provides pharmaceutical compositions comprising an effective amount of the above-mentioned B7-H3-ADC and a pharmaceutically acceptable carrier, excipient, or diluent.

[0027] In several embodiments, the Disclosure further provides the use of the B7-H3-ADC or the pharmaceutical composition in the treatment of diseases or conditions related to or characterized by the expression of B7-H3.

[0028] In several embodiments, the Disclosure further provides a method for treating a disease or condition related to or characterized by B7-H3 expression, comprising the step of administering the B7-H3-ADC or the pharmaceutical composition to a subject.

[0029] In several embodiments, the Disclosure further provides the above-mentioned uses or methods in which the disease or condition associated with or characterized by the expression of B7-H3 is cancer.

[0030] In multiple aspects, the Disclosure further provides the above uses or methods for cancers selected from the group consisting of: adrenal tumors, AIDS-related cancers, alveolar soft part sarcomas, astrocytic tumors, adrenal carcinomas, bladder cancers, bone cancers, cancers of the brain and spinal cord, metastatic brain tumors, B-cell carcinomas, breast cancers, carotid body tumors, cervical cancers, chondrosarcomas, chordomas, chromophobe renal cell carcinomas, clear cell carcinomas, colon cancers, colorectal cancers, benign fibrous histiocytomas, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancers, gastric cancers. Cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, glioblastoma, hematopoietic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (e.g., acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), medulloblastoma, melanoma, meningioma, mesothelioma / pharyngeal cancer (mesothelioma pharyngeal cancer) Cancer), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastasis, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumors in childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN)), stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (e.g., metastatic thyroid cancer); and uterine cancer.

[0031] In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is selected from the group consisting of: adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute 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. 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), advanced small cell lung cancer (ES-SCLC), small round blue cell tumors in childhood (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.

[0032] In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is melanoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is lung cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is head and neck squamous cell carcinoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is pancreatic cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is prostate cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is small cell lung cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is ovarian small cell carcinoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is colorectal small cell carcinoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is esophageal squamous cell carcinoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is non-small cell lung cancer (NSCLC). In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is bladder cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is sarcoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is endometrial cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC). In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is breast cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is ovarian cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is cervical cancer. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is colorectal cancer. In several embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer, wherein the cancer is gastric cancer or gastroesophageal junction cancer.In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer in which the cancer is clear cell renal cell carcinoma. In multiple embodiments, the Disclosure further provides the above-mentioned use or method for the treatment of cancer in which the cancer is hepatocellular carcinoma. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 is a plot of percentage cell viability against ADC concentration in an in vitro cytotoxicity study of B7-H3 expressing A375.S2 human melanoma cells, as described in Example 1. [Figure 2A-2E] Figures 2A-2E show plots of percentage cytotoxicity against 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. [Figure 3A-3D] Figures 3A-3B show plots of resonance units (RUs) over time in surface plasmon resonance (SPR) assays of MGC026 and MGA017 using His-tagged human or cynomolgus monkey B7H3(4Ig) extracellular domain protein (3A) and His-tagged human CD16A extracellular domain protein (3B), as described in Example 3. Figures 3C-3D show plots of resonance units (RUs) over time in surface plasmon resonance (SPR) assays of MGA017(3C) and MGA017(D96E)(3D) using recombinant B7-H3, as described in Example 3. [Figure 4] Figure 4 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 and MGC018 against Calu-6 lung adenocarcinoma tumor cells, as described in Example 4. [Figure 5]Figure 5 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 and MGC018 against Calu-6 lung adenocarcinoma tumor cells at the minimum effective dose, as described in Example 4. [Figure 6] Figure 6 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 and MGC018 against A375.S2 melanoma tumor cells, as described in Example 4. [Figure 7A-7C] Figures 7A-7C show plots of mean tumor volume over time, illustrating 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. [Figure 8] Figure 8 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against FaDu pharyngeal, head and neck squamous cell carcinoma ("HNSCC") tumor cells, as described in Example 4. [Figure 9] Figure 9 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against Hs700T pancreatic adenocarcinoma tumor cells, as described in Example 4. [Figure 10] Figure 10 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against 22Rv1 prostate cancer cells, as described in Example 4. [Figure 11A-11C] Figures 11A-11C show plots of mean tumor volume over time, illustrating the antitumor activity of MGC026 and DS-mAb-Dxd (an ADC using a B7-H3-binding M30-H1-L4 antibody conjugated with deruxtecan (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. [Figure 12A-12C]Figures 12A-12C show plots of mean tumor volume over time, illustrating 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. [Figure 13] Figure 13 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from small cell lung cancer (SCLC) patients from Model 1, as described in Example 9. [Figure 14] Figure 14 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from small cell lung cancer (SCLC) patients from Model 2, as described in Example 9. [Figure 15] Figure 15 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from small cell lung cancer (SCLC) patients from Model 3, as described in Example 9. [Figure 16] Figure 16 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from ovarian cancer patients, as described in Example 9. [Figure 17] Figure 17 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from melanoma patients, as described in Example 9. [Figure 18] Figure 18 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from colorectal cancer patients, as described in Example 9. [Figure 19] Figure 19 shows a plot of mean tumor volume over time, illustrating the antitumor activity of MGC026 against tumor fragments derived from patients with head and neck squamous cell carcinoma (SCCHN), as described in Example 9. [Modes for carrying out the invention]

[0034] 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 drug portion. This disclosure also relates to pharmaceutical compositions containing the above B7-H3-ADCs, and methods involving the use of any of the above B7-H3-ADCs in the treatment of cancer and other diseases and conditions. Specific B7-H3-ADCs, and their use in the treatment of cancer, are described, for example, in International Publication No. 2017 / 180813, which is expressly incorporated herein by reference.

[0035] I. Antibodies and their binding domains The antibodies of this disclosure are immunoglobulin molecules that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, and polypeptides by at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. Accordingly, the B7-H3-ADC of this disclosure includes an antibody that binds to B7-H3. As used herein, the terms “antibody” and “antibodies” include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fv(scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bonded bispecific Fv(sdFv), intracellular antibodies, and any of the above epitope-binding fragments. 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), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. Antibodies can "immunospecifically bind" (or bind to these molecules in an "immunospecific manner") to polypeptides, proteins, or non-protein molecules because a specific domain, part, or structure ("epitope") exists on such molecules. Epitope-containing molecules can have immunological activity, thereby inducing an antibody-producing response in animals. Such molecules are called "antigens."

[0036] As used herein, an antibody, diabody, or other epitope-binding molecule is said to “immunospecifically” bind to a region (i.e., an epitope) of another molecule if it reacts or binds to that epitope more frequently, more rapidly, for a longer period, and / or with higher affinity than an alternative epitope. For example, an antibody that immunospecifically binds to a certain viral epitope is an antibody that binds to that viral epitope with higher affinity, higher binding activity, more readily, and / or for a longer period than immunospecific binding to other viral epitopes or non-viral epitopes. Reading this definition, it is also understood that an antibody (or part or epitope) that immunospecifically binds to a first target may or may not bind specifically or preferentially to a second target. Thus, “immunospecific binding” does not necessarily require (but may include) exclusive binding. Overall, references to binding mean “immunospecific” binding, but not necessarily so. Two molecules are said to be able to bind to each other in a "physiospecific" manner if their binding to each other exhibits the same specificity as the binding of receptors to the ligands of each of the two molecules.

[0037] The term "monoclonal antibody" refers to a homogeneous group of antibodies, where monoclonal antibodies are composed of amino acids (naturally occurring or not naturally occurring) that are involved in the selective binding of antigens. Monoclonal antibodies are highly specific and direct towards a single epitope (or antigen site). The term "monoclonal antibody" encompasses not only complete and full-length monoclonal antibodies, but also their fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) binding molecules, their variants, fusion proteins containing the antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules containing antigen recognition sites that have the necessary specificity and binding ability to a given antigen. No limitation is intended with respect to the source of the antigen or the method of producing the antigen (e.g., by hybridoma, phage selection, recombinant expression, genetically modified animals, etc.). This term includes whole immunoglobulins and the fragments mentioned above in the definition of "antibody." Methods for producing monoclonal antibodies are known in the art. One possible method is the method or a modification thereof described in Kohler, G. et al. (1975) “Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity,” Nature 256:495-497. Typically, monoclonal antibodies are produced in mice, rats, or rabbits. The antibodies are produced by immunizing animals with an immunogenous amount of cells, cell extracts, or protein preparations containing the desired epitope. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissues. Alternatively, existing monoclonal antibodies and other equivalent antibodies with immunospecificity for a desired pathogenic epitope can be recombinantly sequenced and produced by any means known in the art. In one embodiment, such antibodies are sequenced, and the polynucleotide sequence is subsequently cloned into a vector for expression or proliferation.The sequence encoding the antibody of interest can be retained in a vector within a host cell, which can then be grown and frozen for future use. The polynucleotide sequences of such antibodies can be used for genetic engineering to improve the affinity or other characteristics of the antibody by generating monospecific or multispecific (e.g., bispecific, triplicate, and quadruplicate) molecules, as well as molecules with optimized affinity, chimeric antibodies, humanized antibodies, and / or canine antibodies. The general principle of humanizing antibodies is to replace the remaining non-human portion of the antibody with a human antibody sequence while retaining the base sequence of the antigen-binding portion.

[0038] Natural antibodies (such as IgG antibodies) are composed of two "heavy chains" and two "light chains" complexed together. Each light chain contains one variable domain ("VL") and one constant domain ("CL"). Each heavy chain contains one variable domain ("VH"), three constant domains ("CH1", "CH2", and "CH3"), and a "hinge" region ("H") located between the CH1 and CH2 domains. Therefore, the basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is a trimer with one light chain and two heavy chains, usually expressed as a glycoprotein of approximately 150,000 Da. The amino-terminus ("N-terminus") of each chain contains a variable domain of approximately 100-110 amino acids or more, which plays a major role in antigen recognition. The carboxyl terminus ("C-terminus") of each chain defines a constant region; the light chain has a single constant domain, and the heavy chain typically has three constant domains and one hinge domain. Therefore, the structure of the light chain of an IgG molecule is n-VL-CL-c, and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c (where n and c represent the N-terminus and C-terminus of the polypeptide, respectively).

[0039] A. Characterization of antibody variable domains The variable domain of the IgG molecule consists of multiple complementarity-determining regions ("CDRs") containing residues that contact the epitope, and a non-CDR segment called a framework segment ("FR"). The framework segment generally maintains the structure of the CDR loop to enable the aforementioned contact and determines its position (although certain framework 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 CDRs determines whether the antibody can bind to a particular 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.

[0040] The amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are specified by their position within the chain. Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991)) describes numerous amino acid sequences for antibodies, identifies amino acid consensus sequences for each subgroup, assigns residue numbers to each amino acid, and identifies CDRs and FRs according to Kabat's definition (CDR as defined by Chothia, C. & Lesk, AM ((1987) “Canonical structures for the hypervariable regions of immunoglobulins,”. J. Mol. Biol. 196:901-917)). H(It should be understood that 1 starts 5 residues earlier). Kabat's numbering scheme can be extended to antibodies not included in the Kabat list by referencing conserved amino acids and aligning the antibody in question with one of the consensus sequences in Kabat. Such methods for assigning residue numbers have become standard in the art and allow for easy identification of amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of a human antibody light chain occupies the equivalent position of the amino acid at position 50 of a mouse antibody light chain. Thus, the positions within the VL and VH domains where CDRs begin and end are clearly defined and can be confirmed 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 Dubel, S. (eds.), Springer-Verlag Berlin Heidelberg, Chapter 3 (pages 33-51))).

[0041] The polypeptides that are the first, second, and third CDRs of the antibody light chain (or can function as the first, second, and third CDRs) are referred to herein as CDRs, respectively. L 1 domain, CDR L 2 domains, and CDR L These are called 3 domains. Similarly, polypeptides that are the first, second, and third CDRs of the antibody heavy chain (or can function as the first, second, and third CDRs) are referred to as CDRs in this specification, respectively. H 1 domain, CDR H 2 domains, and CDR H It is called the 3 domains. Therefore, CDR L 1 domain, CDR L 2 domains, CDR L 3 domains, CDR H 1 domain, CDR H2 domains, and CDR H The term "3-domain" refers to polypeptides that, when incorporated into a protein, enable that protein to bind to a specific epitope, regardless of whether the protein is an antibody having light and heavy chains, a diabody or 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 immunospecifically binding to a particular epitope. An epitope-binding fragment may contain one, two, three, four, or five CDR domains of an antibody, or all six CDR domains of an antibody, and may exhibit immunospecificity, affinity, or selectivity for such an epitope that differs from that of the antibody, although it may bind immunospecifically to such an epitope. However, preferably, the epitope-binding fragment contains all six of the CDR domains of such an antibody. The epitope-binding fragment of an antibody may be a single polypeptide chain (e.g., scFv) or may comprise two or more polypeptide chains, each having an amino-terminus and a carboxyl-terminus (e.g., a diabody, a Fab fragment, a Fab'2 fragment, etc.). Unless otherwise specified, the domain order of the protein molecules described herein is "N-terminal to C-Terminal".

[0042] This disclosure particularly encompasses single-stranded variable domain fragments ("scFv") containing humanized anti-B7-H3-VL and / or VH domains. The single-stranded variable domain fragments include VL and VH domains linked together using a short "linker" peptide. Such linkers can be modified to provide additional functions, such as enabling drug attachment or attachment to a solid support. Single-stranded variants can be produced by recombination or synthesis. Automated synthesizers can be used for the synthetic production of scFv. For the recombinant production of scFv, a suitable plasmid containing the polynucleotide encoding scFv can be introduced into a suitable host cell, such as a eukaryotic cell like yeast, plant, insect, or mammalian cell, or a prokaryotic cell like Escherichia coli. The polynucleotide encoding the scFv of interest can be prepared by conventional operations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0043] This disclosure particularly encompasses binding molecules (including antibodies and diabodies) containing the VL and / or VH domains of humanized antibodies. The term “humanized” antibody refers to a chimeric molecule, generally prepared using recombinant technology, having an epitope-binding site of an immunoglobulin derived from a non-human species and an immunoglobulin structure of the remaining molecule based on the structure and / or sequence of a human immunoglobulin. The polynucleotide sequence of the variable domain of such an antibody can be used for genetic engineering to produce derivatives of the antibody, thereby improving the affinity or other characteristics of the antibody. Both heavy and light chain variable domains are known to contain three complementarity-determining regions (CDRs) that change in response to the antigen in question to determine binding ability, flanked by four framework regions (FRs) that are relatively conserved in some species and are presumed to provide a scaffold for the CDRs. When preparing a non-human antibody against a particular antigen, the variable domain can be “reshaped” or “humanized.” The general principle of humanizing antibodies is to replace the remaining non-human portion of the antibody with a human antibody sequence while preserving the base sequence of the epitope-binding region. There are four general steps to humanizing a monoclonal antibody. These steps are as follows: (1) determining the nucleotides and predicted amino acid sequences of the light chain and heavy chain variable domains of the initiating antibody; (2) designing the humanized or canine antibody, i.e., determining the antibody framework region to be used during the humanization or canine process; (3) the actual humanization or canine method / technique; and (4) transfection and expression of the humanized antibody. See, for example, U.S. Patent No. 4,816,567; U.S. Patent No. 5,807,715; U.S. Patent No. 5,866,692; and U.S. Patent No. 6,331,415.

[0044] Numerous humanized antibody molecules containing epitope-binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies having a rodent or modified rodent variable domain fused to a human constant domain with a related complementarity-determining region (CDR) (see, for example, Lobuglio et al. (1989) “Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989)). Other references describe rodent CDRs grafted onto human supporting framework regions (FRs) before fusing with appropriate human antibody constant domains (see, e.g., 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 recombinantly veneered rodent framework regions; see, e.g., European Publication No. 519,596. These “humanized” molecules are designed to minimize undesirable immunological responses to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic application of these parts in human recipients.Other methods that can be utilized to humanize an antibody are disclosed by Daugherty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Mouse Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins,” Nucl. Acids Res. 19:2471-2476 as well as U.S. Patent No. 6,180,377; U.S. Patent No. 6,054,297; U.S. Patent No. 5,997,867; and U.S. Patent No. 5,866,692. In multiple embodiments, a humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other multiple embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, or six) that differ in sequence from the original antibody.

[0045] B. Characterization of Antibody Constant Domains 1. Constant Domain of the Light Chain As described above, each light chain of an antibody contains a variable domain (“VL”) and a constant domain (“CL”).

[0046] A 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 NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC is.

[0047] 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 GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS is.

[0048] 2. Constant domain of the heavy chain As described above, the heavy chain of an antibody may include CH1, hinge domain, CH2, and CH3 constant domains. The CH1 domains of the two heavy chains of an antibody complex with the CL constant domain of the light chain of the antibody and attach to the heavy chain CH2 domain via an intervening hinge domain.

[0049] A 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 HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV is.

[0050] 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 HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV is.

[0051] A 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.

[0052] 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 may contain stabilizing mutations such as S228P substitution. The amino acid sequence of the S228P-stabilized human IgG4 hinge domain is (SEQ ID NO: 7): ESKYGPPCPPCP.

[0053] The CH2 and CH3 domains of the two heavy chains of an antibody interact to form an "Fc domain." This is a domain recognized by cellular Fc receptors, including but not limited to the Fcγ receptor (FcγR). As used herein, the term "Fc domain" is used to define the C-terminal region of an IgG heavy chain. An Fc domain is said to belong to a particular IgG isotype, class, or subclass if its amino acid sequence most closely matches that of a particular IgG isotype compared to other IgG isotypes. Antibodies have been shown to be useful as therapeutic agents, in addition to their known uses in diagnostics.

[0054] The amino acid sequence of the CH2-CH3 domain of a typical human IgG1 is (SEQ ID NO: 8): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X This is numbered by an EU index, similar to Kabat, where X is either lysine (K) or absent.

[0055] The amino acid sequence of the CH2-CH3 domain of a typical human IgG4 is (SEQ ID NO: 9): 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X This is numbered by an EU index, similar to Kabat, where X is either lysine (K) or absent.

[0056] Throughout this specification, the numbering of residues in the constant region of the IgG heavy chain is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5 thThis is the EU index numbering according to Ed. Public Health Service, NH1, MD (1991) (which is expressly incorporated herein by reference). The term "EU index as in Kabat" refers to the numbering of the constant domains of human IgG1 EU antibodies.

[0057] Polymorphisms have been observed at numerous different locations within the antibody constant region (including, but not limited to, Fc positions 270, 272, 312, 315, 356, and 358, as numbered by the EU index as described in Kabat), and therefore slight differences may exist between the sequences presented here and those of prior art. Polymorphic forms of human immunoglobulins have been well-characterized. Currently, the following 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 incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype, or haplotype of the sequences presented herein. Furthermore, depending on the expression system, the C-terminal amino acid residue of the CH3 domain (shown in bold above) can be removed post-translation. Therefore, the C-terminal residue of the CH3 domain is an optional amino acid residue. Specifically included by this disclosure is B7-H3-ADC lacking the C-terminal residue of the CH3 domain. Also specifically included by this disclosure are such constructs containing the C-terminal lysine residue of the CH3 domain.

[0058] The present disclosure particularly encompasses B7-H3-ADCs comprising anti-B7-H3 variable domains (i.e., VL and / or VH domains) that immunospecifically bind to epitopes of human B7-H3 polypeptide. Such B7-H3-ADCs can immunospecifically bind to human B7-H3. As used herein, the B7-H3 variable domains are referred to as "anti-B7-H3-VL" and "anti-B7-H3-VH", respectively.

[0059] II. Anti-B7-H3 antibody mAb-A A representative anti-B7-H3 antibody designated "mAb-A" was isolated from hybridoma cells generated by immunization with cells expressing human B7-H3, its B7-H3 polypeptide or peptide epitope. Antibody mAb-A was humanized.

[0060] Antibody mAb-A was found to have cross-reactivity against cynomolgus B7-H3. The amino acid sequences of the VL and VH domains of mAb-A are provided below. The B7-H3-ADC comprises all three CDRs of the VH domain of humanized monoclonal antibody mAb-A ("hmAb-A") H , all three CDRs of the VL domain L , and optionally, the entire VH and VL domains of humanized monoclonal antibody mAb-A ("hmAb-A").

[0061] A. Mouse anti-B7-H3 antibody mAb-A 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 underlined): DIQMTQSPAS LSVSVGETVT ITC RASESIY SYLA WYQQKQ GKSPQLLVY N TKTLPE GVPS RFSGSGSGTQ FSLKINSLQP EDFGRYYC QH HYGTPPWT FG GGTNLEIK

[0062] The amino acid sequence of the VH domain of anti-B7-H3 mAb-A (SEQ ID NO: 11) is shown below (CDR H The residues are indicated by underlines): EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMS WVRQT PDKRLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNTLY LQMRSLKSED TAMYYCAR HD GGAMDY WGQG TSVTVSS

[0063] B. Humanized anti-B7-H3 antibody hmAb-A The variable domain of the anti-B7-H3 antibody mAb-A was humanized to generate humanized mAb-A ("hmAb-A"). In some cases, alternative humanized variable domains were generated to optimize binding activity and / or to remove antigenic epitopes and / or to remove potentially unstable amino acid residues.

[0064] The amino acid sequence of the VL domain of hmAb-A (SEQ ID NO: 12) is shown below (CDR L The residues are indicated by underlines): DIQMTQSPSS LSASVGDRVT ITC RASESIY SYLA WYQQKP GKAPKLLVY N TKTLPE GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QHHYGTPPWT FG QGTRLEIK

[0065] The amino acid sequence of the light chain of hmAb-A, including the VL domain and CLκ domain, is shown below (SEQ ID NO: 13): DIQMTQSPSS LSASVGDRVT ITCRASESIY SYLAWYQQKP GKAPKLLVYN TKTLPEGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH HYGTPPWTFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

[0066] In 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 light chain κ constant region (SEQ ID NO: 1).

[0067] The amino acid sequence of the VH domain of hmAb-A (SEQ ID NO: 14) is shown below (CDR H The residues are indicated by underlines): EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HD GGAMDY WGQG TTVTVSS

[0068] The amino acid sequence of the heavy chain containing the VH domain and the IgG1 CH1-H-CH2-CH3 domain of hmAb-A (SEQ ID NO: 15) is shown below: EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLEWVAT INSGGSNTYY PDSLKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHD GGAMDYWGQG TTVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQY N STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X Here, X is either ricin (K) or absent.

[0069] 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 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). The glycosylation site linked to N is located at Kabat position 296 (indicated by an underline). The C-terminal residue "X" is either lysine (K) or absent.

[0070] CDR L The amino acid sequence of one domain (SEQ ID NO: 16) is:RASESIYSYLA.

[0071] CDR L The amino acid sequence of the two domains (SEQ ID NO: 17) is NTKTLPE.

[0072] CDR L The amino acid sequence of the 3 domains (SEQ ID NO: 18) is:QHHYGTPPWT.

[0073] CDR H The amino acid sequence of one domain (SEQ ID NO: 19) is SYGMS.

[0074] CDR H The amino acid sequence of the two domains (SEQ ID NO: 20) is:TINSGGSNTYYPDSLKG.

[0075] CDR H The amino acid sequence of the 3 domains (SEQ ID NO: 21) is:HDGGAMDY.

[0076] C. Alternative humanized anti-B7-H3 antibody hmAb-A Alternative humanized variable domains were generated to optimize binding activity, and / or to remove antigenic epitopes, and / or to 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, including the VL domain and CLκ domain (SEQ ID NO: 13) are shown above.

[0077] The amino acid sequence of the VH domain of the alternative hmAb-A (SEQ ID NO: 22) is shown below (CDR H The residues are indicated by underlines): EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HX GGAMDY WGQG TTVTVSS

[0078] In multiple ways, alternative CDRs H The amino acid sequence of the 3 domains (SEQ ID NO: 23) is:H X GGAMDY. Alternative CDR in multiple ways HThe residue "X" in the 3 domain is any amino acid. In several embodiments, alternative CDRs H The residue "X" in the 3-domain is aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q).

[0079] For example, the amino acid sequence of the exemplary alternative VH domain of hmAb-A (SEQ ID NO: 24) is shown below (CDR H (The residues are indicated by underlining), where residue "X" is glutamine (E): EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HE GGAMDY WGQG TTVTVSS

[0080] The following is an exemplary alternative heavy chain amino acid sequence (SEQ ID NO: 25) containing the VH domain of hmAb-A and the IgG1 CH1-H-CH2-CH3 domain: EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLEWVAT INSGGSNTYY PDSLKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARH E GGAMDYWGQG TTVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQY N STY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X Here, X is either ricin (K) or absent.

[0081] 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). The glycosylation site linked to N is located at Kabat position 296 (indicated by an underline). The C-terminal residue "X" is either lysine (K) or absent.

[0082] Exemplary alternative CDR H The amino acid sequence of the 3 domains is (SEQ ID NO: 26): HEGGAMDY.

[0083] III. Modification of the Fc domain The Fc domain of an Fc domain-containing molecule (e.g., an antibody and a diabody) may be a complete Fc domain (e.g., a complete IgG Fc domain) or only a fragment of an Fc domain. Optionally, the Fc domain of an Fc domain-containing molecule may not contain a C-terminal lysine amino acid residue.

[0084] In conventional immune function, the interaction between antibody-antigen complexes and immune system cells results in a wide range of responses, from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis, to immunomodulatory signals that regulate lymphocyte proliferation and antibody secretion. All of these interactions are initiated by binding to receptors (individually called "Fc gamma receptors (FcγRs)," "FcγRs," and collectively "FcγRs") found on the surface of specific cell surfaces on hematopoietic cells, particularly on the surfaces of several types of immune system cells (e.g., B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells). All of these interactions are initiated by the binding of the Fc domain of an antibody or immune complex to specific cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes is due to the structural heterogeneity of the 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 attenuation) receptor. Furthermore, interaction with the neonatal Fc receptor (FcRn) mediates the recirculation of IgG molecules from endosomes 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) have already been presented.

[0085] Modification of the Fc domain can lead to phenotypic changes, such as changes in serum half-life, stability, sensitivity to cellular enzymes, or effector function. Therefore, in certain embodiments, the Fc domain of an Fc domain-containing molecule may be an engineered variant Fc domain. The Fc domain of an Fc domain-containing molecule may have the ability to bind to one or more Fc receptors (e.g., one or more FcγR), but in particular, the variant Fc domain exhibits altered binding to FcγRIA(CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), FcγRIIIA(CD16a), or FcγRIIIB(CD16b) (compared to the binding exhibited by the wild-type Fc domain), for example, enhanced binding to activating receptors and / or reduced or no ability to bind to one or more inhibitory receptors. Therefore, the Fc domain of an Fc domain-containing molecule may include part or all of the CH2 domain and / or part or all of the CH3 domain of a complete Fc domain, or may include variant CH2 and / or variant CH3 sequences (for example, including one or more insertions and / or one or more deletions to the CH2 or CH3 domain of a complete Fc domain). Such an Fc domain may include a non-Fc polypeptide moiety, or a portion of a complete Fc domain that does not exist naturally, or an orientation of the CH2 and / or CH3 domains that does not exist naturally (for example, two CH2 domains or two CH3 domains, or a CH3 domain and a CH2 domain linked thereto in the direction from the N-terminus to the C-terminus, etc.).

[0086] In certain embodiments, the Fc domain of the binding molecule exhibits reduced binding (or near-absence of binding) to FcγRIA(CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), FcγRIIIA(CD16a), or FcγRIIIB(CD16b) (compared to the binding exhibited by the wild-type IgG1 Fc domain (SEQ ID NO: 8)). In certain embodiments, the binding molecule contains an IgG Fc domain with reduced ADCC effector function. In such embodiments, the CH2-CH3 domain of the binding molecule contains any one, two, three, or four of the following substitutions: L234A, L235A, D265A, N297Q, and N297G. In another embodiment, the CH2-CH3 domain contains N297Q substitution, N297G substitution, L234A and L235A substitution, or D265A substitution, because these mutations cause the loss of FcR binding. Alternatively, the CH2-CH3 domain of a naturally occurring Fc domain is utilized, which has inherently low (or almost no) binding to FcγRIIIA (CD16a) and / or inherently low effector function (compared to the binding and effector function exhibited by the wild-type IgG1 Fc domain (SEQ ID NO: 8)). In a specific embodiment, the binding molecule includes the IgG4 Fc domain (SEQ ID NO: 9). When utilizing the IgG4 Fc domain, the present invention also includes the introduction of stabilizing mutations such as the hinge domain S228P substitution described herein (see, for example, SEQ ID NO: 7).

[0087] The serum half-life of proteins containing an Fc domain can be extended 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 for 50 percent (50%) of the known amount of the molecule to be eliminated from the subject’s body (e.g., a human patient or another mammal) or its particular body cavity, when measured in serum (i.e., circulating half-life) or other tissues. Generally, an extension of half-life leads to an extension of the mean residence time (MRT) of the administered molecule in circulation. Modifications that can extend the half-life of Fc domain-containing molecules are known in the art, including, for example, M252Y, S254T, T256E, and combinations thereof. See, for example, U.S. Patent No. 6,277,375; U.S. Patent No. 7,083,784; U.S. Patent No. 7,217,797; and U.S. Patent No. 8,088,376; U.S. Patent Publication No. 2002 / 0147311; U.S. Patent Publication No. 2007 / 0148164; and U.S. Patent Publication No. 2011 / 0081347 for the modifications described therein.

[0088] IV.B7-H3-ADC This disclosure relates to the above-mentioned anti-B7-H3 antibody hmAb-A, i.e., "B7-H3-ADC," conjugated with a cytotoxic agent. Such B7-H3-ADC enhances the cytotoxicity of anti-B7-H3 therapy, particularly in the treatment of cancer. As described above, B7-H3-ADC is formulated with the formula: Ab-(LM) m -(D) n Represented by: Ab is an antibody that binds to B7-H3 containing a humanized variable heavy chain (VH) domain and a humanized variable light chain (VL) domain, or a B7-H3 binding fragment thereof; D is the cytotoxic drug portion; LM is a linker molecule that covalently bonds Ab and D; m is an integer between 1 and n, representing the number of B7-H3-ADC bonds or linker molecules; n is an integer between 1 and 10, representing the number of cytotoxic camptothecin moieties covalently bound to B7-H3-ADC.

[0089] In certain embodiments, B7-H3-ADC contains a naturally occurring Fc domain of the IgG1 isotype. Such an Fc domain does not contain the C-terminal lysine residue of the CH3 domain. In specific embodiments, B7-H3-ADC binds to tumor cells expressing B7-H3 and is internalized into the cells by receptor-mediated endocytosis. Upon entering the lysosome, B7-H3-ADC can be degraded, triggering the release of a cytotoxic camptothecin moiety within the cell, which leads to cell death. As understood, the mechanism of action of this cell death may vary depending on the class of cytotoxic drug used. In a process known as the bystander effect, when a free drug is released into the tumor environment by a dead cell, adjacent 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).

[0090] In several embodiments, the Ab of B7-H3-ADC is the B7-H3 antibody described above.

[0091] In several embodiments, Ab is a humanized B7-H3 antibody or B7-H3 binding fragment that binds to B7-H3: (i) its variable light chain (VL) domain contains CDR L1 Sequence RASESIYSYLA (SEQ ID NO: 16), CDR L 2 sequences NTKTLPE (sequence number 17), and CDR L (ii) the sequence QHHYGTPPWT (sequence number 18), and (ii) its variable heavy chain domain (VH), CDR H 1. Sequence SYGMS (SEQ ID NO: 19), CDR H 2 sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H It contains 3 sequences HDGGAMDY (sequence number 21) or HEGGAMDY (sequence number 26).

[0092] In several embodiments, Ab is a humanized B7-H3 antibody or B7-H3 binding fragment that binds to B7-H3: (i) its variable light chain (VL) domain contains CDR L 1 Sequence RASESIYSYLA (SEQ ID NO: 16), CDR L 2 sequences NTKTLPE (sequence number 17), and CDR L (ii) the sequence QHHYGTPPWT (sequence number 18), and (ii) its variable heavy chain domain (VH), CDR H 1. Sequence SYGMS (SEQ ID NO: 19), CDR H 2 sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H It contains the 3-sequence HDGGAMDY (sequence number 21).

[0093] In several embodiments, Ab is a humanized B7-H3 antibody or B7-H3 binding fragment that binds to B7-H3: (i) its variable light chain (VL) domain contains CDR L 1 Sequence RASESIYSYLA (SEQ ID NO: 16), CDR L 2 sequences NTKTLPE (sequence number 17), and CDR L (ii) the sequence QHHYGTPPWT (sequence number 18), and (ii) its variable heavy chain domain (VH), CDR H 1. Sequence SYGMS (SEQ ID NO: 19), CDR H 2 sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H Includes sequence HEGGAMDY (sequence number 26).

[0094] In several embodiments, Ab comprises: (i) a humanized VL domain containing the amino acid sequence of SEQ ID NO: 12, and (ii) a humanized VH domain containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 24.

[0095] In several embodiments, Ab comprises: (i) a humanized VL domain containing the amino acid sequence of SEQ ID NO: 12, and (ii) a humanized VH domain containing the amino acid sequence of SEQ ID NO: 14.

[0096] In several embodiments, Ab comprises: (i) a humanized VL domain containing the amino acid sequence of SEQ ID NO: 12, and (ii) a humanized VH domain containing the amino acid sequence of SEQ ID NO: 24.

[0097] In several embodiments, the present disclosure provides an antibody Ab that has undergone glycan remodeling. Examples of glycan remodeling are described below. In several embodiments, Ab has undergone glycan remodeling and contains an azido sugar. In several embodiments, the azido sugar is GalNAz (N-azidoacetylgalactosamine), 6-azido-Gal, or 6-azido-GalNAc.

[0098] A. Linker molecule Therefore, this disclosure particularly envisions a B7-H3-ADC having one or more linker molecules LM (i.e., m is an integer from 2 to n, and n is an integer from 2 to 10), wherein each linker molecule LM covalently bonds the cytotoxic camptothecin moiety D to Ab of the B7-H3-ADC.

[0099] The disclosure further provides a B7-H3-ADC in which Ab is bound to two or more linker molecules LM, all of which are identical. The cytotoxic camptothecin moieties D covalently bound to Ab of the B7-H3-ADC may all be identical, or may comprise two, three, four, or five or more individually distinct cytotoxic camptothecin moieties D.

[0100] The disclosure further provides a B7-H3-ADC in which Ab is bound to two or more linker molecules LM, and the linker molecules are not identical but are individually different. The cytotoxic camptothecin moieties D bound to Ab of the B7-H3-ADC may all be identical, or may comprise two, three, four, or five or more individually different cytotoxic camptothecin moieties D.

[0101] Humanized VH and VL domains of antibodies that bind to human B7-H3, as well as human antibody constant domains that may be included in B7-H3-ADC, have already been presented. As described above, B7-H3-ADC further includes at least one cytotoxic drug moiety, which is covalently bound to the amino acid residues of the VH domain or VL domain and / or constant domain via a linker molecule attached to the side chain and drug moiety. This linker molecule may be a non-peptide molecule, a molecule containing both a non-peptide and a peptide moiety, or a molecule composed solely of amino acid residues. The amino acid residues of any such linker molecule may include naturally occurring or non-naturally occurring amino acid residues, including D versions of naturally occurring amino acid residues, p-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.) may be incorporated into B7-H3-ADC.

[0102] In several embodiments, the cytotoxic drug portion may be conjugated to Ab of the B7-H3-ADC of this disclosure by means known in the art (see, for example, International Publication No. 2016053107; 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 linker molecule-cytotoxic drug moiety (LM-D) can be conjugated to the Ab of the B7-H3-ADC of this disclosure using the thiol group of cysteine, the amino side group of lysine, glutamine or arginine, or the carboxyl group of glutamic acid or aspartic acid. Since the native antibody contains numerous lysine conjugation sites, each antibody can be linked to multiple conjugated molecules. In practice, peptide mapping has been determined in which conjugation occurs in both the heavy and light chains with approximately 20 different lysine residues (40 lysines per mAb). Thus, more than one million different ADC species can be generated. Since the cysteine ​​conjugation occurs after the reduction of 1 to 4 interchain disulfide bonds, this conjugation is limited to eight exposed sulfhydryl groups in the native VL and VH domains. However, additional reactive residues (e.g., lysine, cysteine, selenocysteine, etc.) may be incorporated into the antibody (e.g., within the VL domain and / or VH domain and / or constant domain) as desired. For example, one or more native amino acid residues may be substituted with cysteine ​​residues.Using Amber stop codon repressor tRNA / aaRS pairs, non-natural amino acids (e.g., p-acetylphenylalanine) may be genetically incorporated into antibodies (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 International Publication No. 2008 / 070593). Alternatively, an enzyme (e.g., glycotransferase) may be used to conjugate the linker molecule-cytotoxic drug moiety (LM-D) to Ab of the B7-H3-ADC of the present disclosure. The glycotransferase platform can attach the sugar moiety to a glycosylation site on an antibody (e.g., the N297 position of the Fc domain of a human IgG antibody), which functions as the linker molecule (LM) of the present disclosure to conjugate the cytotoxic drug moiety (D) to Ab of the B7-H3-ADC of the present disclosure. Alternatively, a transglutaminase may be used to catalyze the formation of a covalent bond between the free amino group and the glutamine side chain.

[0103] In several embodiments, the linker molecule LM attaches to a glycan portion attached to the side chain of Ab. In several embodiments, the glycan portion on the side chain is a naturally occurring glycan portion. In several embodiments, the glycan portion undergoes glycan remodeling before attachment to the linker portion.

[0104] In several embodiments, glycan remodeling is carried out by contacting the glycan portion with endoglycosidase and glycosyltransferase. In several embodiments, glycan remodeling involves adding an azido sugar to the end of the glycan portion. In several embodiments, the azido sugar is GalNAz (N-azidoacetylgalactosamine), 6-azido-Gal, or 6-azido-GalNAc. In several embodiments, a linker molecule LM attaches to the azido sugar of the glycan portion. In several embodiments, the linker molecule LM attaches to the azido sugar of the glycan portion by reacting the azido group of the azido sugar with the reactant group of LM.

[0105] In several embodiments, glycan remodeling is carried out by contacting the glycan portion with endoglycosidase and N-acetylgalactosamine (GalNAc) transferase. In several embodiments, glycan remodeling involves adding N-acetylgalactosamine (GalNAc) sugar to the end of the glycan portion. In several embodiments, the GalNAc sugar contains an azide group. In several embodiments, the linker molecule LM attaches to the GalNAc sugar of the glycan portion. In several embodiments, the linker molecule LM attaches to the GalNAc sugar of the glycan portion by reacting the azide group of GalNAc with the reactant group of LM.

[0106] In several embodiments, the glycosylation remodeling performed is the GlycoConnect® process (Synaffix). In several embodiments, the glycosylation remodeling is performed as described in U.S. Patent Nos. 9,504,758, 10,745,488, 9,988,661, 10,858,641, 9,222,940, 10,239,807, and 11,358,921, which are incorporated herein by reference, respectively.

[0107] In several embodiments, the linker molecule LM may be incapable of cleavage under physiological conditions and may consist of a hydrolysis-stable moiety, for example, a thioether linker or a hindered disulfide linker. Hydrolysis-stable linkers are substantially stable in water and do not react with water at useful pH values, including but not limited to physiological conditions, over long periods. In contrast, hydrolysis-unstable or degradable linkers decompose in water or in aqueous solutions, such as blood.

[0108] Alternatively, the linker molecule LM may be cleavable or may contain cleavable moieties. Examples of such cleavable moieties include acid-unstable linkers (e.g., 4-(4'-acetylphenoxy)butanoic acid linkers that form hydrazine bonds), cleavable disulfide linkers (cleaved in a reducing intracellular environment), and protease-cleavable linkers. Acid-unstable linkers are designed to be stable at the pH levels encountered in the blood but unstable and degraded when encountered in the low pH environment of lysosomes. Protease-cleavable linkers are designed to be stable in blood / plasma but to rapidly release free drugs within lysosomes in cancer cells upon cleavage 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 enzymatically cleavable substrate, such as a cleavable peptide (e.g., a valine-alanine dipeptide para-aminobenzyl alcohol linker (cAC10-mc-va-PABA), a valine-alanine dipeptide para-aminocarbamate (Val-Ala-PABC or VA-PABC), a valine-citrulline dipeptide para-aminobenzyl alcohol linker (cAC10-mc-vc-PABA), or a valine-citrulline dipeptide para-aminocarbamate (Val-Cit-PABC or VC-PABC) that is selectively cleaved by a lysosomal enzyme), or may contain an enzymatically cleavable substrate.Suitable cleavable linkers are known in the art, 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 See 12:1529–1532; U.S. Patent No. 5,547,667; U.S. Patent No. 6,214,345; U.S. Patent No. 7,585,491; U.S. Patent No. 7,754,681; U.S. Patent No. 8,080,250; U.S. Patent No. 8,461,117; and International Publication No. 02 / 083180.

[0109] Enzyme-unstable or degradable linkers can be employed. Such linkers are degraded by one or more enzymes. As just one example, PEG and related polymers may contain one or more degradable linker molecules within the polymer backbone or within the linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such one or more 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, such ester groups generally hydrolyze under physiological conditions to release the bioactive agent. Other hydrolyzable linker molecules include, but are not limited to, carbonate bonds; imine bonds resulting from the reaction of amines and aldehydes; phosphate ester bonds formed by the reaction of alcohols and phosphate groups; hydrazone bonds, which are reaction products of hydrazines and aldehydes; acetal bonds, which are reaction products of aldehydes and alcohols; orthoester bonds, which are reaction products of formic acid and alcohols; peptide bonds formed by amine groups, including but not limited to those at the ends of polymers such as PEG, and carboxyl groups of peptides; and oligonucleotide bonds formed by phosphoramidite groups, including but not limited to those at the ends of polymers, and 5'-hydroxyl groups of oligonucleotides.

[0110] In multiple aspects, the linker molecule LM comprises a peptide linker. In multiple aspects, the peptide linker is a valine-alanine dipeptide linker. In multiple aspects, the linker molecule comprises a cleavable linker. In multiple aspects, the linker molecule comprises a valine-alanine (Val-Ala) amino acid linker. In multiple aspects, the Val-Ala linker is a Val-Ala-PABC linker.

[0111] In multiple embodiments, the peptide linker is a valine-citrulline dipeptide linker. In multiple embodiments, the linker molecule comprises a cleavable linker. In multiple embodiments, the linker molecule comprises a valine-citrulline (Val-Cit) amino acid linker. In multiple embodiments, the Val-Cit linker is a Val-Cit-PABC linker.

[0112] In one embodiment, the linker molecule LM is of formula (4a) or (4b), or a salt thereof: [ka] A cleavable linker molecule containing, or may contain, such cleavable linker molecule: a is independently either 0 or 1; b is independently either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is an integer in the range of 1 to 150; g is either 0 or 1; i is either 0 or 1; D is the cytotoxic drug portion; Q 1 is an alkenyl group, a (hetero)cycloalkenyl group, a bicyclotriazole group, or a cycloalkenyl group; Q 1 It is bound to the functional group of the antibody; Sp 1 , Sp 2 , Sp 3 , and Sp 4 These are independently linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkylene group, C7-C 200 Alkylalylene group, C7-C 200Arylalkylene group, C8-C 200 Arylalkenylene group, and C9-C 200 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylalylene group, arylalkylene group, arylalkenylene group, and arylalkylene group may be optionally substituted, and optionally O, S, and NR 3 It is interrupted by one or more heteroatoms selected from the group, where R 3 Independently, hydrogen, C1-C 24 Alkyl alkyl group, C2-C 24 Alkenyl group, C2-C 24 Alkynyl group, and C3-C 24 Selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group may be optionally substituted; Z 1 Q 1 or Sp 3 Sp 2 , O or C(O) or N(R 1 It is a connecting group that connects to ); Z 2 is D or Sp 4 Sp 1 , N(R 1 A connecting element that connects to O, or C(O); Z 1 and Z 2 These are independently -O-, -S-, and -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-NR2 -、-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-NR 2 -S(O)2-O-、-O-NR 2 -S(O)2-NR 2 -、-O-NR 2 -S(O)2-、-O-P(O)(R2 )2-,-SP(O)(R 2 )2-, -NR 2 -P(O)(R 2 )2-, and selected from the group consisting of two or more combinations of these, where R 2 Independently, hydrogen, C1-C 24 Alkyl alkyl group, C2-C 24 Alkenyl group, C2-C 24 Alkynyl group, and C3-C 24 Selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group may be optionally substituted; R 1 is hydrogen, C1-C 24 Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3-C 24 Alkyl (hetero)aryl group, and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the above C1-C 24 Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3-C 24 Alkyl (hetero)aryl group, and C3-C 24 The (hetero)arylalkyl group may be optionally substituted, and may also be O, S, and NR. 3 It is interrupted by one or more heteroatoms selected from, where R 3 These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 1 D, -[(Sp 1 ) b (Z 2 ) e -(Sp 4 ) i -D], or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ] and here Sp 1 , Sp2 , Sp 3 , Sp 4 , Z 1 , Z 2 , D, Q 1 b, c, d, e, g, and i are as defined above.

[0113] In some cases, Q 1 It is a triazole derivative of cyclooctin.

[0114] In some embodiments, the linker molecule LM is further Sp 1 , Sp 2 , Sp 3 , and Sp 4 And if these exist, independently, linear or branched C1-C 20 A group selected from the group consisting of alkylene groups, Sp 1 , Sp 2 , Sp 3 , and Sp 4 The alkylene group may be optionally substituted, and may also be optionally O, S, and NR 3 It is interrupted by one or more heteroatoms selected from the group consisting of R, where R 3 These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

[0115] In several embodiments, the severable linker is the linker disclosed in U.S. Patent No. 9,636,421, which is incorporated herein by reference.

[0116] In several embodiments, the linker molecule LM comprises one or more polar spacers. In several embodiments, the polar spacer is a carbamoyl sulfamide. Examples of usable spacers can be found in U.S. Patent No. 9,636,421 and U.S. Patent No. 10,792,369, which are incorporated herein by reference.

[0117] In several embodiments, B7-H3-ADC comprises two, three, four, five, six, seven, eight, nine, or ten cytotoxic drug moieties, which may independently be identical or different from other cytotoxic drug moieties of B7-H3-ADC. In some embodiments, each of these cytotoxic drug moieties is conjugated to the Ab of B7-H3-ADC via a separate linker molecule. Alternatively, two or more cytotoxic drug moieties may be attached to the Ab of B7-H3-ADC via the same linker molecule.

[0118] In several embodiments, the present disclosure comprises one or more linker molecules LM and camptothecin moieties D, wherein LM and D together: [ka] The B7-H3-ADC is intended to be the component of this.

[0119] B. Exemplary cytotoxic drug portion In multiple embodiments, the cytotoxic drug portion of B7-H3-ADC comprises 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, phototherapeutic agents, anti-angiogenic agents, apoptosis promoters, peptides, lipids, carbohydrates, chelating agents, or combinations thereof.

[0120] 1. Topoisomerase inhibitors In several embodiments, B7-H3-ADC may contain a topoisomerase inhibitor cytotoxic drug moiety. As used herein, the terms “topoisomerase inhibitor” or “DNA topoisomerase inhibitor” refer to compounds that inhibit the activity of topoisomerase.

[0121] Topoisomerase enzymes play a crucial role in cell proliferation and replication, altering the superhelical structure of double-stranded DNA by catalyzing the cleavage and recombination of the phosphodiester backbone of DNA strands during the normal cell cycle. Separation of DNA strands is essential for transcription and replication of the genome by the copying of each base by RNA polymerase and DNA polymerase (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433). Because DNA has a double helix structure, replication produces linked offspring, which need to be cleaved by topoisomerases before cytokinesis. Two enzymes that play a role in this helix relaxation and reformation process are topoisomerase I and topoisomerase II. They also play an important role in repairing DNA damage resulting from exposure to DNA damaging factors, such as radiation exposure or chemotherapy.

[0122] Topoisomerases are classified into type I and type II. Type I enzymes cleave one DNA strand at a time, while type II enzymes cleave both strands to exert catalytic function. All topoisomerases cleave the DNA phosphodiester backbone by nucleophilic attack from a catalytic tyrosine residue linked to the phosphate terminus (PY) of the DNA cleavage site. These reactions are highly reversible and do not alter the DNA sequence after topoisomerization (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433).

[0123] Numerous type I topoisomerase (Topo 1) inhibitors have been evaluated as anticancer agents. Camptothecin was initially identified from the Chinese tree 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 topotecan, irinotecan, berotecan, gimatecan, lulutotecan, diflomotecan, S39625, and exatecan, 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).

[0124] In addition to camptothecin derivatives, several non-camptothecin topoisomerase inhibitors have also been investigated as anticancer agents, including the indolocarbazole edtecarin, indenoisoquinolines NSC706744 (MJ-III-65), NSC725776 (LMP-776), and NSC724998 (LMP-400), dibenzonaphthilidione (ARC-111) such as topoval, and the aromasesin rosettacin (Pommier et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs, Chem. & Biol. Review 17 (2010) 421-433).

[0125] In one embodiment, the topoisomerase inhibitor is selected from topoisomerase I inhibitors. Known topoisomerase I inhibitors that can be used in the present invention include, but are not limited to, the following: (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride (topotecan), (S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14-(4H,12H)-dione (camptothecin), (1S,9S )-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo(de)pyrano(3',4':6,7)indolidino(1,2-b)quinoline-10,13-dione(exatecan), (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecan(lulutotecan), or (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3',4 ':6,7]-Indolidino[1,2-b]quinoline-9-yl-[1,4'-bipiperidine]-1'-carboxylate (irinotecan), (R)-5-ethyl-9,10-difluoro-5-hydroxy-4,5-dihydrooxepino[3',4':6,7]indolidino[1,2-b]quinoline-3,15(1H,13H)-dione (diflomothecan), (4S)-11-((E)-((1,1-dimethylethoxy)imino)methyl)-4-ethyl-4-hydroxy-1,12-dihydro-14H-pyrano(3',4':6,7) Indlidino(1,2-b)quinoline-3,14(4H)-dione(gimatecan), (S)-8-ethyl-8-hydroxy-15-((4-methylpiperazine-1-yl)methyl)-11,14-dihydro-2H-[1,4]dioxyno[2,3-g]pyrano[3',4':6,7]indlidino[1,2-b]quinoline-9,12(3H,8H)-dione(lulutotecan), (4S)-4-ethyl-4-hydroxy-11-[2-[(1-methylethyl)amino]ethyl]-1H-pyrano[3',4':6,7]indlidino[1,2-b]Quinoline-3,14(4H,12H)-dione (berotecan), 6-((1,3-dihydroxypropane-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-indoro[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)naphthyridine-6-one (Topoval), benzo[6,7]indolidino[1,2-b]quinoline-11(13H)-one (Rosettasin), (S)-4-ethyl-4-hydroxy-11-(2-(trimethylsilyl)ethyl)-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-3,14(4H,12H)-dione (cocitecane), tetrakis{( 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]indolidino[1,2-b]quinoline-4-yl}N,N',N'',N'''-{methanetetrayltetrakis[methylenepoly(oxyethylene)oxy(1-oxoethylene)]}tetraglycinate tetrahydrochloride (ethilinotecan pegol), 10-hydroxycan Putothecin (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-methylpiperidine-1-yl)methyl)-4,5-dihydrooxepino[3',4':6,7]indolidino[1,2-b]quinoline-3,15(1H,13H)-dione (eromothecan).

[0126] In addition to Topo 1 inhibitors, numerous anticancer drugs targeting the type II topoisomerase (Topo 2) enzyme have been investigated. These include etoposide, teniposide, and DNA intercalators such as doxorubicin, daunorubicin, acralubicin, amsacrin, dexrazoxane, TAS-103, quinolones CP-115,963, ellipticins including ellipticinium, azatoxins, genistein, VP-16, VM-26, mitoxantrone, amonafide, and saintopein.

[0127] In one embodiment, the topoisomerase inhibitor is selected from topoisomerase II inhibitors. Known topoisomerase II inhibitors that can be used in the present invention include, but are not limited to, amonafide and its derivatives, etoposide, teniposide, doxorubicin, daunorubicin, acralubicin, amsacrin, dexrazoxane, TAS-103, quinolone CP-115,963, erlipticins including erlipticinium, azatoxins, genistein, VP-16, VM-26, mitoxantrone, amonafide, and saintopine.

[0128] 2. Camptothecin cytotoxic drug portion In multiple embodiments, B7-H3-ADC is a camptothecin cytotoxic drug moiety: [ka] It can include...

[0129] As used herein, the term "camptothecin" means a class of compounds that are considered to be camptothecin, camptothecin analogs, camptothecin derivatives, or camptothecin conjugates. These compounds are based on the characteristic five-ring skeleton of camptothecin.

[0130] The plant alkaloid camptothecin (CPT) was discovered to possess anticancer activity in the late 1950s. Camptothecin, whether substituted or not, is thought to intervene in the mechanism of action of the nuclear enzyme topoisomerase I (topo I), causing cells to be arrested in the S phase. While not theoretically constrained, CPT is thought to achieve this by stabilizing the covalent complex DNA-topo I (called the cleavable complex), thereby halting the progression of the replication fork. Such a collision between the replication fork and the cleavable complex is thought to trigger the apoptotic pathway. (Z. Darzynkiewicz et al., The Cell Cycle Effects of Camptothecin, 803 Annals of the New York Academy of Sciences 93 (1996)). DNA strand cleavage is also thought to be 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).

[0131] Examples of camptothecin include SN-38(S-10-hydroxycamptothecin), irinotecan (CAMPTOSAR; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin), topotecan (HYCAMPTIN; (S)-9-N,N-dimethylaminoethyl-10-hydroxycamptothecin), 9-aminocamptothecin (9-amino-20(S)-camptothecin), 9-nitrocamptothecin (also called rubitecan), lulutotecan (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin), exatecan, calenitecin, and homocamptothecin. Structural and clinical information on several camptothecin compounds can be found in Garcia-Carbonero, et al., Clin. Cancer Res. (March 2002) 8: 641-661. Examples of camptothecin compounds are found in U.S. Patent Nos. 4,604,463, 6,403,569, and 5,004,758, as well as in International Publication Nos. 2004 / 012661, 2003 / 101998, 2003 / 101996, 2003 / 101406, 2003 / 093274, 2003 / 086471, and 01 / 76597. This can also be confirmed in the specification, International Publication No. 01 / 64194, International Publication No. 00 / 70275, International Publication No. 00 / 53607, International Publication No. 99 / 17805, International Publication No. 99 / 17804, International Publication No. 99 / 05103, International Publication No. 98 / 35969, International Publication No. 97 / 28164, International Publication No. 97 / 25332, and International Publication No. 97 / 16454, and the contents of all these documents are incorporated into this application by reference.

[0132] SN-38 (S-10-hydroxycamptothecin), topotecan, irinotecan, berotecan, and deruxtecan are CPT analogs that are currently approved and used in cancer chemotherapy: [ka] [ka] [ka] [ka] [ka]

[0133] In several embodiments, the cytotoxic drug portion of camptothecin is exatecan. [ka]

[0134] 3. Pyrrolobenzodiazepine dimer In several embodiments, B7-H3-ADC may contain a pyrrolobenzodiazepine (PBD) dimeric cytotoxic drug moiety. The PBD dimer irreversibly binds to two guanines derived from opposing DNA strands in the DNA subgroove without distorting the double helix. Because the PBD dimer does not distort the double helix, it is difficult for the DNA repair mechanism to remove it. In several embodiments, the PBD dimer is a dimeric PBD compound such as those provided in U.S. Patent No. 6,562,806 and U.S. Patent No. 11,135,303, both of which are incorporated herein by reference in their entirety.

[0135] 4. Auristatin In several embodiments, B7-H3-ADC may contain an auristatin cytotoxic drug moiety. In several embodiments, auristatin is monomethyl auristatin (MMAE). In several embodiments, auristatin is monomethyl auristatin F (MMAF). In several embodiments, auristatin is auristatin E (AE). In several embodiments, auristatin is the auristatin disclosed in U.S. Patent No. 5,208,020; U.S. Patent No. 5,416,064; U.S. Patent No. 6,333,410; U.S. Patent No. 6,340,701; U.S. Patent No. 6,372,738; U.S. Patent No. 6,436,931; U.S. Patent No. 6,441,163; U.S. Patent No. 6,596,757; U.S. Patent No. 7,276,497; U.S. Patent No. 7,585,857; or U.S. Patent No. 7,851,432, each of which is incorporated herein by reference in its entirety.

[0136] C.MGC026 In certain embodiments, B7-H3-ADC is MGC026. MGC026 contains the light and heavy chains of anti-B7-H3 hmAb-A conjugated to an exatecan payload. The amino acid sequences of Ab, the cytotoxic exatecan moiety D, and the linker molecule LM in MGC026 are shown below: Ab is: (i) Light chain containing the amino acid sequence of Sequence ID No. 13; and (ii) Heavy chain containing the sequence of Sequence ID No. 15 Includes; D contains exatecan; LM contains the linker molecule mentioned above.

[0137] In several embodiments, when a linker molecule and exatecan are combined and conjugated to Ab, the following structure is obtained: [ka] It holds.

[0138] V. Manufacturing method The anti-B7-H3 antibody of this disclosure can be synthesized and expressed by recombinant means of any method known in the art for the production of recombinant proteins. For example, a nucleic acid encoding the polypeptide chain of the binding molecule can be constructed, introduced into an expression vector, and expressed in a suitable host cell. The binding molecule can be recombinantly produced in bacterial cells (e.g., Escherichia coli cells) or eukaryotic cells (e.g., CHO, 293E, COS, NS0 cells). Furthermore, the binding molecule can be expressed in yeast cells such as those of the genera Pichia or Saccharomyces.

[0139] To produce the anti-B7-H3 antibody hmAb-A, one or more polynucleotides encoding this molecule can be constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biological techniques are used to prepare the recombinant expression vector, transfect it into host cells, select transformants, culture the host cells, and recover the molecule (see, for example, the techniques described in Green, MR et al., (2012), Molecular Cloning, A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY). One or more expression vectors must have features that allow for vector replication in host cells. The vectors must also have promoter and signal sequences necessary for expression in host cells. Such sequences are known in the art. In addition to one or more nucleic acid sequences encoding such binding molecules, recombinant expression vectors may have further sequences, such as sequences that control vector replication within host cells (e.g., origin of replication) and selection marker genes. Another possible method is to express the gene sequences in plants (e.g., tobacco) or transgenic animals. Preferred methods for recombinant expression of the above binding molecules in plants or milk have already 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).

[0140] After recombinant expression of the anti-B7-H3 antibody hmAb-A, it can be purified from inside or outside host cells (e.g., from culture medium) by any method known in the art for the purification of polypeptides or polyproteins. Isolation and purification methods commonly used for antibody purification (e.g., antibody purification schemes based on antigen selectivity) can be used for the isolation and purification of such molecules, and the above methods are not limited to any particular method. Examples include column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Examples of chromatography include ion-exchange chromatography, particularly affinity chromatography based on affinity for specific antigens, 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. (Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0141] VI. Pharmaceutical Compositions The pharmaceutical compositions for formulating B7-H3-ADC described herein include bulk drug compositions useful in the manufacture of the pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions that can be used for the preparation of unit dosage forms (i.e., compositions suitable for administration to a subject or patient). These compositions include a prophylactic or therapeutically effective dose of B7-H3-ADC, or a combination of such an activator and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition includes a prophylactic or therapeutically effective dose of B7-H3-ADC and a pharmaceutically acceptable carrier. The pharmaceutical compositions may also further include a second therapeutic antibody (e.g., a tumor-specific monoclonal antibody) that is specific to a particular cancer antigen and a pharmaceutically acceptable carrier.

[0142] As used herein, the term “pharmaceutically acceptable carrier” means a diluent, solvent, dispersion, antimicrobial and antifungal agent, excipient, or vehicle that is approved by a federal or state regulatory agency as suitable for administration to animals, more particularly to humans, or that is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. Such pharmaceutically acceptable carriers may be sterile liquids such as water and oil, including petroleum, animal fats and oils, vegetable oils, or synthetic sources. Saline solutions, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Compositions may optionally contain trace amounts of wetting agents or emulsifiers, or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0143] Generally, the components of a composition are supplied separately or mixed in a dosage form, either as a freeze-dried powder or a water-free concentrate, or as an aqueous solution in an airtight container such as a vial, ampoule, or sachet, with the amount of the activator indicated. If the composition is administered by infusion, it can be prepared using an infusion bottle containing sterile pharmaceutical-grade water or saline solution. If the composition is administered by injection, ampoules of sterile water for injection, saline solution, or other diluents can be provided, allowing the components to be mixed before administration.

[0144] VII. Pharmaceutical Kits This disclosure also provides a pharmaceutical pack or kit comprising one or more containers containing one or more pharmaceutical compositions and explanatory materials (e.g., warnings, package inserts, instructions, etc.). Furthermore, one or more other prophylactic or therapeutic agents useful for the treatment of a disease may also be included in the pharmaceutical kit. The containers of such a pharmaceutical kit may comprise one or more airtight vials, ampoules, sachets, etc., which are indicated on the amount of active ingredient contained therein. If the composition is administered by infusion, the containers may be infusion bottles or bags containing sterile pharmaceutical-grade solutions (e.g., water, saline, buffer, etc.). If the composition is administered by injection, the pharmaceutical kit may contain ampoules of sterile water, saline, or other diluents for injection to facilitate mixing of the components of the pharmaceutical kit for administration to a subject (e.g., a human patient or other mammal). In several embodiments, the pharmaceutical pack or kit comprises the B7-H3-ADC pharmaceutical composition and explanatory materials.

[0145] In one embodiment, the B7-H3-ADC of such a kit is supplied as a lyophilized sterile powder or a water-free concentrate in an airtight container and can be reconstituted to a concentration suitable for administration to a subject using, for example, water, saline, or other diluents. In another embodiment, the B7-H3-ADC of such a kit is supplied as an aqueous solution in an airtight container and can be diluted to a concentration suitable for administration to a subject using, for example, water, saline, or other diluents. The kit may further contain, in one or more containers, one or more other prophylactic and / or therapeutic agents that can be used to treat cancer; and / or the kit may further contain one or more cytotoxic antibodies that bind to one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biotherapeutic agent or a hormonal therapy agent.

[0146] The explanatory materials included in the pharmaceutical kit may, for example, be in the content and format prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, and may indicate the approval of such agency for the manufacture, sale, or use of the pharmaceutical composition for administration to humans and / or for the treatment of humans. The explanatory materials may provide information such as the dosage of the pharmaceutical composition, the manner in which the pharmaceutical composition can be prepared (e.g., reconstituted), and the manner in which the pharmaceutical composition can be administered. Such explanatory materials may further provide information relating to the dosage and administration of one or more pharmaceutical compositions not provided in this kit.

[0147] VIII. Use of B7-H3-ADC The B7-H3-ADC described herein can be used to treat or prevent a variety of disorders, including cancer, for example, cancer that expresses B7-H3. Accordingly, this disclosure provides a method for treating cancer, the method comprising the step of administering B7-H3-ADC to a subject in need. In certain embodiments, this disclosure provides a method for treating cancer, the method comprising the step of administering MGC026 to a subject in need. Where used herein, the term “subject” means human (i.e., human patient) or other mammal. Non-exclusive drug regimens for administering such therapies to subjects in need are provided herein.

[0148] In several aspects, cancers that can be treated with B7-H3-ADC as described herein include cancers selected from the group consisting of: adrenal tumors, AIDS-related cancers, alveolar soft part sarcomas, astrocytic tumors, adrenal carcinomas, bladder cancers, bone cancers, brain and spinal cord cancers, metastatic brain tumors, B-cell carcinomas, breast cancers, carotid body tumors, cervical cancers, chondrosarcomas, chordomas, chromophobe renal cell carcinomas, clear cell carcinomas, colon cancers, colorectal cancers, benign fibrous histiocytomas, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancers, and gastric cancers. Cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, glioblastoma, hematopoietic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (e.g., acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), medulloblastoma, melanoma, meningioma, mesothelioma / pharyngeal cancer (mesothelioma pharyngeal cancer) Cancer), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastasis, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumors in childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN)), stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (e.g., metastatic thyroid cancer); and uterine cancer.

[0149] In multiple embodiments, B7-H3-ADC is effective against adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, gastroesophageal junction cancer, glioblastoma, kidney 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. It may be used to treat small cell carcinomas (e.g., head and neck squamous cell carcinoma (SCCHN), esophageal squamous cell carcinoma), testicular cancer, thyroid cancer (e.g., metastatic small cell carcinoma of the colon and rectum), endometrial cancer, cervical cancer, clear cell renal cell carcinoma, hepatocellular carcinoma, ovarian small cell carcinoma, colorectal small cell carcinoma, and uterine cancer.

[0150] In several embodiments, B7-H3-ADC may 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., head and neck squamous cell carcinoma (SCCHN)).

[0151] In several embodiments, B7-H3-ADC may be used for the treatment of melanoma.

[0152] In several embodiments, B7-H3-ADC may be used for the treatment of ovarian cancer.

[0153] In several embodiments, B7-H3-ADC may be used for the treatment of platinum-resistant ovarian cancer (PROC).

[0154] In several embodiments, B7-H3-ADC may be used for the treatment of pancreatic cancer.

[0155] In several embodiments, B7-H3-ADC may be used for the treatment of prostate cancer.

[0156] In several embodiments, B7-H3-ADC may be used to treat metastatic castration-resistant prostate cancer (mCRPC).

[0157] In several embodiments, B7-H3-ADC may be used to treat squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN)).

[0158] In several embodiments, B7-H3-ADC may be used for the treatment of non-small cell lung cancer (NSCLC).

[0159] In several embodiments, B7-H3-ADC may be used for the treatment of small cell lung cancer (SCLC).

[0160] In several embodiments, B7-H3-ADC may be used to treat advanced small cell lung cancer (ES-SCLC).

[0161] IX. Method of administration The B7-H3-ADC of this disclosure can be administered in a variety of ways to subjects, such as human patients, who require it. For many applications, the route of administration 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 forms of parenteral administration are also possible. Examples of such forms include: intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injections.

[0162] The B7-H3-ADCs of this disclosure may be administered as body weight-based doses or as uniform doses. The dose may also be selected to reduce or avoid the production of antibodies against the administered molecule. The dosing regimen is adjusted to provide a desired response, e.g., a therapeutic response or a combination therapeutic effect. In general, multiple doses of B7-H3-ADC (and additional agonists as optional) may be used to provide a bioavailable amount of the agonist to a subject. As used herein, the term “dose” refers to a specified amount of drug treatment administered in a single dose. The term “dosage” refers to the administration of a specific amount, number of times, and frequency of doses over a specified period, and therefore the term “dosage” includes chronological characteristics such as duration and periodicity.

[0163] As used herein, the term “weight-based dose” refers to an individual amount of the molecule administered per unit of the patient’s body weight, for example, milligrams of the drug per kilogram of the subject’s body weight (mg / kg body weight; abbreviated herein as “mg / kg”). As used herein, the term “flat dose” refers to a dose that is independent of the patient’s body weight and comprises physically distinct units of the molecule, suitable as a single dose for a subject under treatment, each unit containing a predetermined amount of the drug. Typically, a significant change (10% or more) in body weight from baseline or an established plateau body weight will generally prompt a recalculation of the dose. One or more doses may be administered. Compositions containing B7-H3-ADC may be administered by infusion to subjects requiring it.

[0164] As used herein, the term “fractionated dose” refers to two or more separate doses of a molecule administered to achieve a particular desired dose. A single fractionated dose provides the desired dose divided into two or more separate doses. Between such two or more doses, the dose may be divided evenly and / or unevenly. In certain embodiments, a fractionated dose may be two or more separate doses within a cycle (e.g., a three-week or four-week cycle). [Examples]

[0165] While several embodiments have been outlined above, the present invention will be more readily understood by referring to the following examples. The following examples illustrate various methods relating to compositions in the diagnostic or therapeutic methods of this disclosure. These examples are intended to illustrate the scope of the appended claims and are not intended to limit the scope of the appended claims in any way.

[0166] Example 1: In vitro cytotoxicity of MGC026 The ability of MGC026 to mediate cytotoxicity against B7-H3 expressing A375.S2 human melanoma cells in vitro was investigated. The MGC026 investigated was the same ADC as described above. The negative control ADC (Ctrl-SYNtecan E) is a humanized control ADC that does 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 detached using 0.05% trypsin-EDTA. The antibody and ADC were diluted based on a 9-point dose-response curve (including a control well without antibody) to a final peak concentration of 10 ug / ml (antibody concentration 67 nM), and then diluted 1:3 or 1:10 depending on the sensitivity of the cell line. The ADC was prepared to 5 times the final concentration and added to a 96-well tissue culture plate. Suspension cells were seeded at 5,000 cells / well and added to ADC wells at 80 ul / well (total volume 100 ul / well).

[0167] The plates were incubated at 37°C for 7 days. Cell viability was measured by color development using alamarBlue (Trek Diagnostics #00-100; 10 μl added to each well). The plates were read using a Gemini plate reader (Molecular Devices) according to the alamarBlue specifications.

[0168] We analyzed the data using Graph Pad Prism (4-parameter curve fitting analysis) and IC 50 The value was determined.

[0169] The cytotoxic curve obtained from this study is shown in Figure 1. MGC026 mediated dose-dependent cytotoxicity against the A375.S2 human melanoma strain in vitro. 50 The activity level was 31 pM. The negative control ADC (Ctrl-SYNtecan E) showed approximately 1000 times lower activity compared to MGC026, confirming the specificity of MGC026's cytotoxic activity.

[0170] Example 2 ADCC Assay The ability of unconjugated MGA017 (an unconjugated B7-H3 antibody equivalent to the antibody used for conjugation), conjugated MGC018 (a B7-H3-ADC containing the same antibody and a duocalmycin cytotoxic drug moiety; see International Publication No. 2017 / 180813A1), and conjugated MGC026 (mentioned above) to mediate antibody-dependent cytotoxicity (ADCC) was evaluated. In the ADCC assay, primary peripheral blood mononuclear cells were used as the source of natural killer (NK) cells. The ADCC assay tests the ability of the antibody to bridge the interaction between antigen-positive target cells and NK effector cells, and subsequently the killing of target cells by NK cells. Adherent target tumor cells grown in F-12 / DMEM containing 10% fetal bovine serum (FBS) were detached with 0.25% trypsin-EDTA solution and collected by centrifugation at 1000 rpm for 5 minutes. The harvested tumor cells were rinsed once with PBS, resuspended in assay medium (RPMI + 5% FBS without phenol red), and seeded at 20,000 cells / well in a 96-well U-bottom cell culture plate. The test antibody was serially diluted and triple-seeded onto the cells. Then, 600,000 fresh PBMCs were added to the wells (effector:target ratio, E:T = 30:1), and the plate was incubated overnight at 37°C / 5% CO2.

[0171] After incubation, 15 μL of 10X lysis solution (Promega #G182A) was added to the maximum release control well and allowed to stand for 10 minutes to completely lyse the target cells. The plate was then centrifuged at 1200 rpm for 5 minutes. 50 μL of supernatant was transferred from each assay plate 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 plate was incubated in the dark at room temperature for 5–10 minutes, after which 50 μL of stop solution was added. Optical density at 490 nm was measured within 1 hour using an Emax plate reader (Molecular Devices). Percent cytotoxicity was calculated as described below, and further analysis was performed using GraphPad Prism5 software.

[0172] Specific cell lysis was calculated from optical density (OD) data using the following formula, which incorporates maximum release (MR), non-antibody-dependent cytotoxicity (AICC), and spontaneous cell release (SR): Cytotoxicity (%) = 100 x (OD of sample - OD of AICC) / (OD of MR - OD of SR)

[0173] The results of the ADCC assay are shown in Figures 2A-2E. MGA017 and MGC018 mediated ADCC against the five B7-H3 expressing tumor cell lines tested. MGC026 did not mediated ADCC against these five tumor cell lines.

[0174] Example 3: Surface Plasmon Resonance Assay Binding to B7-H3 was analyzed using surface plasmon resonance (SPR). Anti-pentaHis tag mAbs were immobilized on SPR CM5 sensor chips according to the manufacturer's recommended procedure. Briefly, carboxyl groups on the sensor chip surface were activated by injecting a solution containing 0.2 M N-ethyl-N-(3-diethylaminopropyl)carbodiimide and 0.05 M N-hydroxysuccinimide. mAbs (5 μg / mL) in 10 mM sodium acetate (pH 5.0) were injected into the activated CM5 surface at a flow rate of 5 μL / min, followed by injection of 1 M ethanolamine to deactivate any remaining amine-reactive groups.

[0175] Injecting His-tagged human or cynomolgus monkey B7-H3(4Ig) extracellular domain protein at a flow rate of 20 μL / min for 10 seconds resulted in approximately 30 resonance units (RUs) of captured ligand, which is suitable for kinetic studies.

[0176] MGC026 or MGA017 (as described above) at concentrations of 0, 12.5, 25, 50, 100, and 200 nM were injected into HBS-EP buffer at a flow rate of 30 μL / min for 120 seconds (double injection). Regeneration of the immobilized anti-pentaHis mAb surface was performed by pulsed injection of 10 mM glycine (pH 1.5).

[0177] Reference curves were obtained by injecting each dilution of MGC026 or MGA017 into a treated surface that had not been immobilized by the protein. The binding curve at zero concentration was subtracted as a blank. The rate constant k for single-arm affinity interaction. a and k d This relates to the association / dissociation (k) of the divalent analyte interaction model. a / k d The dissociation equilibrium constant (K) was estimated by an overall analysis of the curve (BIA evaluation software v4.1). D ) is K D =k d / k a It was calculated as follows.

[0178] Both antibodies MGC026 and MGA017 showed equivalent single-arm affinity binding to captured human and cynomolgus monkey B7-H3(4Ig) (Figure 3A). The rate constants calculated by the bivalent analyte model of the single-arm interaction are shown in Table 1. The K values ​​determined for the interaction with captured human B7-H3(4Ig) are also shown. D The values ​​were comparable for all interactions, ranging from 16–18 nM and 6–8 nM for human and cynomolgus monkey B7-H3(4Ig), respectively. The difference in overall binding response intensity may be due to differences in the available binding sites of the two antigens captured on the sensor chip surface and does not reflect the kinetic parameters.

[0179] [Table 1]

[0180] By injecting His-tagged human CD16A (FcγRIIIA) extracellular domain protein at a flow rate of 20 μL / min for 10 seconds, the captured ligand reached approximately 120 resonance units (RU). MGC026 or MGA017 at concentrations of 0, 62.5, 125, 250, 500, and 1000 nM in HBS-EP buffer were injected at a flow rate of 30 μL / min for 120 seconds (dual). Regeneration of the immobilized anti-pentaHis mAb surface was performed by pulsed injection of 10 mM glycine (pH 1.5). The equilibrium binding response of the Fc portion of the antibody against the captured human CD16A allele, against mAb concentration, was fitted to a steady-state affinity model to determine K D The value was obtained.

[0181] The unconjugated antibody MGA017 bound to the captured human CD16 allele. As expected, MGA017 bound to the high-affinity CD16A 158V allele with higher affinity than the low-affinity CD16A 158F allele. The conjugated antibody MGC026 did not bind to either CD16A allele (Figure 3B). Therefore, MGC026 is not expected to mediate effector function via the Fcγ receptor CD16A, which is consistent with the inability of MGC026 to mediate ADCC in vitro (see Example 2). The lack of effector function is potentially advantageous for MGC026, as ADC binding to effector cells can lead to reduced tumor localization, inhibited internalization, and off-target toxicity (McDonagh, Mol Cancer Ther 2008; Perez, Drug Discovery Today 2013).

[0182] Furthermore, surface plasmon resonance was used to analyze the binding of recombinant B7-H3, including amino acids L29-G245 and the C-terminal 10xHis tag of human B7-H3 fused to HSV, to MGA017 and MGA017(D96E) (MGA017 with the D96E mutation in the heavy chain). Binding studies were performed in HBS-EP plus buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant). MGA017 or MGA017(D96E) was captured with polyclonal goat-anti-human Fc antibody immobilized on a CM4 chip. Binding of recombinant B7-H3 protein was analyzed at concentrations of 12.5, 50, and 200 nM. The equilibrium dissociation stationary state (K) is shown in Table 2. D ), association velocity (k a ), and dissociation rate (k d The value of ) was determined by fitting the binding curve to the Langmuir 1:1 binding model overall (BIAevaluation software, Version 4.1). MGA017(D96E) (Figure 3D) exhibited binding affinity to recombinant B7-H3 equivalent to that of MGA017 (Figure 3C).

[0183] [Table 2]

[0184] Example 4: MGC026 exhibits potent in vivo activity. To further demonstrate the antitumor activity of MGC026, the aforementioned MGC026 molecule was evaluated for in vivo toxicity in CD-1 nude mouse models using several different tumor cell lines. Briefly, approximately 5 x 10⁶ molecules suspended in 1:1 serum-free medium and Matrigel basement membrane matrix were evaluated. 6 Several surviving tumor cells were subcutaneously inoculated into the flanks of CD-1 nude mice (Charles River Laboratories). The average tumor volume was approximately 100-140 mm². 3 When the tumor volume reached a certain level, mice were randomly divided and administered either MGC026 or a vehicle control intravenously. Some studies included a non-targeted control ADC or MGC018 (described above). In these studies, one dose of MGC026, MGC018, a non-targeted control ADC, or a vehicle control was administered once weekly (QW). Tumors were measured twice a week using orthogonal measurements with electronic calipers, and tumor volume was calculated as (length x width x height) / 2. Animals were considered to have shown partial regression (PR) if the tumor volume decreased by 50% or more compared to the tumor volume on the day the first dose was administered. During the study period, the tumor volume of treated animals was 5 mm 3 A reduction to less than 42% was considered a complete response ("CR"). Tumor volume (relative to vehicle control) was determined ("T / C"). Antitumor activity was evaluated according to the National Cancer Institute (NCI) criteria; a T / C of 42% or less represents the lowest level of antitumor activity, while a T / C value greater than 42% is inactive. A T / C of less than 10% is considered highly active.

[0185] In vivo activity of Calu-6 against non-small cell lung cancer tumor cells The results of this study on Calu-6 lung adenocarcinoma tumor cells subcutaneously are presented in Table 3 and Figure 4, demonstrating the responsiveness to Calu-6 tumor cells.

[0186] [Table 3]

[0187] Female CD-1 nude (homozygous) mice (n=6 / group) were subjected to a study of Calu-6 (lung adenocarcinoma) tumor cells (5 × 10⁶ cells) suspended in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On the 22nd day, the tumor was approximately 100 mm. 3 (104±25mm 3 When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 22 (arrow), administered intravenously at the prescribed dose levels to form a single total dose, either with a vehicle control (1X PBS) or a targeted ADC (MGC026 or MGC018). Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at all three dose levels after treatment with MGC026, and at both dose levels after treatment with MGC018.

[0188] MGC026 reduced tumor volume by 99% at day 49 at all doses tested and was highly active at each dose according to NCI criteria (T / C=1%). MGC018 was highly active at 10 mg / kg (T / C=1%) and active at 3 mg / kg (T / C=22%). 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 at doses of 10 mg / kg, 6 mg / kg, and 3 mg / kg, respectively. MGC018 induced partial regression in 6 / 6 animals at a dose of 10 mg / kg and in 2 / 6 animals at a dose of 3 mg / kg, and complete regression in 4 / 6 animals at a dose of 10 mg / kg. The antitumor activity and tumor regression of MGC026 persisted to the end of the study at all dose levels tested, while tumor regression of MGC018 persisted at a dose of 10 mg / kg. Immunohistochemistry (IHC) determined the tumor H score to be 95.

[0189] In vivo activity at the minimum effective dose against Calu-6 lung adenocarcinoma tumor cells The results of this study on Calu-6 lung adenocarcinoma tumor cells subcutaneously are presented in Table 4 and Figure 5, demonstrating the responsiveness to Calu-6 tumor cells.

[0190] [Table 4]

[0191] Female CD-1 nude (homozygous) mice (n=5 / group) were subjected to a study of Calu-6 (lung adenocarcinoma) tumor cells (5 × 10⁶ cells) suspended in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On the 16th day, the tumor was approximately 100 mm. 3 (100±22mm 3When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 16 (arrow), administered intravenously at the prescribed dose level to form a total of one dose, either with a vehicle control (1X PBS) or a targeted ADC (MGC026 or MGC018). Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed after treatment with MGC026 and MGC018.

[0192] MGC026 reduced tumor volume by 87% on day 41 and was active at the tested dose level (3 mg / kg) according to NCI criteria (T / C = 13%). MGC018 was active at 3 mg / kg (T / C = 34%). MGC026 induced partial regression in 2 out of 5 animals but not complete regression. At 3 mg / kg, MGC018 did not induce partial or complete regression (0 out of 5 animals). The tumor H score was determined to be 210 by IHC.

[0193] In vivo activity against A375.S2 melanoma tumor cells The results of this study on subcutaneously inoculated A375.S2 melanoma tumor cells are presented in Table 5 and Figure 6, demonstrating the responsiveness to A375.S2 tumor cells.

[0194] [Table 5]

[0195] Female CD-1 nude (homozygous) mice (n=6 / group) were subjected to a suspension of A375.S2 (melanoma) tumor cells (5 × 10⁴ cells) in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On the 24th day, the tumor was approximately 100 mm. 3 (102±33mm 3When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 25 (arrow), administered intravenously at the prescribed dose levels to form a single total dose, either a vehicle control (1X PBS) or a targeted ADC (MGC026 or MGC018). Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at all three dose levels of MGC026 and at both dose levels of MGC018.

[0196] After treatment with 10 mg / kg of MGC026, a reduction in tumor volume was observed, with a 99% reduction by day 57. Treatment with 6 mg / kg and 3 mg / kg of MGC026 resulted in a 100% reduction in tumor volume. Based on NCI criteria, MGC026 was highly active at 10 mg / kg (T / C=1%), 6 mg / kg (T / C=0%), and 3 mg / kg (T / C=0%). MGC018 was highly active at both 10 mg / kg and 3 mg / kg (T / C=0%). 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. The antitumor activity and complete tumor regression of MGC026 and MGC018 persisted at all dose levels tested until the end of the study. The tumor H score was determined to be 180 by IHC.

[0197] In vivo activity at the minimum effective dose against A375.S2 melanoma tumor cells The results of this study on subcutaneously inoculated A375.S2 melanoma tumor cells are presented in Table 6 and Figures 7A-7C, demonstrating the responsiveness to A375.S2 tumor cells.

[0198] [Table 6]

[0199] Female CD-1 nude (homozygous) mice (n=5 / group) were subjected to a suspension of A375.S2 (melanoma) tumor cells (5 × 10⁶ cells) in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On the 26th day, the tumor was approximately 100 mm. 3 (87±21mm 3 When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 26 (arrow), administered intravenously with either a vehicle control (1X PBS) or a targeted ADC (MGC026 or MGC018) at the instructed dose levels (A, B, C) to form a single total dose. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed after treatment with MGC026 and MGC018 at the three doses tested.

[0200] MGC026 reduced tumor volume by 100% on day 80 and was highly active at 3 mg / kg (T / C=0%) and 1 mg / kg (T / C=7%), and active at 0.3 mg / kg (T / C=16%), according to NCI criteria. Treatment with MGC018 was highly active at 3 mg / kg (T / C=0%), active at 1 mg / kg (T / C=41%), and inactive at 0.3 mg / kg (T / C=54%). The untargeted control ADC showed antitumor activity at 3 mg / kg and 0.3 mg / kg, but the activity was limited and significantly weaker than that observed with targeted MGC026, and was considered inactive at 3 mg / kg and 0.3 mg / kg according to NCI criteria (T / C=74%, 50%). MGC026 induced partial regression in 5 out of 5 animals at dose levels of 3 mg / kg and 1 mg / kg, and in 4 out of 5 animals at a dose level of 0.3 mg / kg. Complete regression was observed in 5 out of 5 animals and 3 out of 5 animals at 3 mg / kg and 1 mg / kg, respectively. MGC018 induced partial regression in 5 out of 5 animals and 3 out of 5 animals at 3 mg / kg and 1 mg / kg, respectively, and also induced complete regression in 4 out of 5 animals at 3 mg / kg. Treatment with MGC026 and MGC018 maintained antitumor control at a dose level of 3 mg / kg. The tumor H score was determined to be 210 by IHC.

[0201] In vivo activity of FaDu against pharyngeal and head and neck squamous cell carcinoma tumor cells. The results of this study on subcutaneously inoculated FaDu pharyngeal and head and neck squamous cell carcinoma tumor cells are presented in Table 7 and Figure 8, demonstrating the responsiveness to FaDu tumor cells.

[0202] [Table 7]

[0203] Female CD-1 nude (homozygous) mice (n=7 / group) were given FaDu (pharyngeal, head and neck squamous cell carcinoma) tumor cells (5 × 10⁶ cells) suspended in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6(Number of cells) were subcutaneously transplanted. On the 15th day, the tumor was approximately 100 mm. 3 (125±25mm 3 When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 15 (arrow), administered intravenously at the prescribed dose levels to form a single total dose, either a vehicle control (1X PBS), a targeted ADC (MGC026), or a non-targeted control ADC. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Dose-responsive antitumor activity was observed at all three dose levels of MGC026.

[0204] After treatment with 10 mg / kg of MGC026, a reduction in tumor volume was observed, with an 88% reduction in tumor volume at day 35 compared to the vehicle control. Treatment with 6 mg / kg and 3 mg / kg of MGC026 resulted in a 93% and 86% reduction in tumor volume, respectively. Based on NCI criteria, MGC026 was highly active at 6 mg / kg (T / C=7%), and active at 10 mg / kg (T / C=12%) and 3 mg / kg (T / C=14%). Although non-targeted control ADCs exhibited antitumor activity, it 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 (T / C=60%, 74%, and 87%, respectively) based on NCI criteria. MGC026 induced partial regression in 6 / 7, 4 / 7, and 3 / 7 animals at doses of 10 mg / kg, 6 mg / kg, and 3 mg / kg, respectively, and induced complete regression in 1 / 7 animals at all three tested doses. In contrast, the non-targeted control ADC showed partial regression in 1 / 7 animals at doses of 6 mg / kg and 3 mg / kg, and complete regression in 1 / 7 animals at 6 mg / kg. The tumor H score was determined to be 200 by IHC.

[0205] In vivo activity against Hs700T pancreatic adenocarcinoma tumor cells The results of this study on subcutaneously inoculated Hs700T pancreatic adenocarcinoma tumor cells are presented in Table 8 and Figure 9, demonstrating the responsiveness to Hs700T tumor cells.

[0206] [Table 8]

[0207] Female CD-1 nude (homozygous) mice (n=7 / group) were given Hs700T (pancreatic adenocarcinoma) tumor cells (5 × 10⁶ cells) suspended in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On day 46, the tumor was approximately 100 mm. 3 (114±27mm 3 When the mean tumor volume (± standard deviation) was reached, mice were randomly divided and, on day 46 (arrow), administered intravenously at the prescribed dose levels to form a total of one dose, either vehicle control (1X PBS), targeted ADC (MGC026), or non-targeted control ADC. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. The dose levels of MGC026 differ from those of the non-targeted control ADC because the MGC026 test certificate was modified after the start of the study. Antitumor activity was observed at all three dose levels of MGC026 compared to the vehicle control.

[0208] Dose-response effects were observed at three dose levels of MGC026. After treatment with 7.4 mg / kg of MGC026, a reduction in tumor volume was observed, with a 96% reduction in tumor volume compared to the vehicle control at day 96. Treatment with 4.5 mg / kg and 2.2 mg / kg of MGC026 resulted in a 93% and 85% reduction in tumor volume, respectively, at day 96. Based on NCI criteria, MGC026 was highly active at 7.4 mg / kg (T / C=4%) and 4.5 mg / kg (T / C=7%), and active at 2.2 mg / kg (T / C=15%), while the non-targeted control ADC was inactive at 10 mg / kg, 6 mg / kg, and 3 mg / kg (T / C=70%, 69%, and 77%). MGC026 induced partial regression in 5 / 7, 4 / 7, and 2 / 7 animals at doses of 7.4 mg / kg, 4.5 mg / kg, and 2.2 mg / kg, respectively, and complete regression in 1 / 7 and 1 / 7 animals at doses of 7.4 mg / kg and 2.2 mg / kg, respectively. In contrast, the non-targeted control ADC group did not show partial or complete regression at any of the three dose levels. The tumor H score was determined to be 295 by IHC.

[0209] In vivo activity against 22Rv1 prostate cancer tumor cells The results of this study on 22Rv1 prostate cancer cells administered subcutaneously are presented in Table 9 and Figure 10, demonstrating the responsiveness to 22Rv1 tumor cells.

[0210] [Table 9]

[0211] Female CD-1 nude (homozygous) mice (n=7 / group) were subjected to a study of 22Rv1 (prostate cancer) tumor cells (5 × 10⁶ cells) suspended in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On day 31, the tumor was approximately 100 mm. 3 (126±26mm 3When the mean tumor volume (± standard deviation) was reached, mice were randomly divided and, on day 32 (arrow), administered intravenously at the prescribed dose levels to form a total of one dose, either vehicle control (1X PBS), targeted ADC (MGC026), or non-targeted control ADC. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. The dose levels of MGC026 differ from those of the non-targeted control ADC because the MGC026 test certificate was modified after the start of the study. Antitumor activity was observed at all three dose levels of MGC026.

[0212] Dose-response effects were observed at three dose levels of MGC026. After treatment with 7.4 mg / kg of MGC026, a reduction in tumor volume was observed, with a 78% reduction in tumor volume compared to the vehicle control at day 72. Treatment with 4.5 mg / kg and 2.2 mg / kg of MGC026 resulted in 77% and 65% reductions in tumor volume, respectively. Based on NCI criteria, MGC026 was active at 7.4 mg / kg (T / C=22%), 4.4 mg / kg (T / C=23%), and 2.2 mg / kg (T / C=35%), while the non-targeted control ADC was inactive at 10 mg / kg, 6 mg / kg, and 3 mg / kg (T / C=81%, 72%, and 78%). MGC026 induced partial regression in 5 / 7, 3 / 7, and 2 / 7 animals at doses of 7.4 mg / kg, 4.5 mg / kg, and 2.2 mg / kg, respectively, but did not induce complete regression. In contrast, the non-targeted control ADC group did not show partial or complete regression at any of the three dose levels. The tumor H score was determined to be 155 by IHC.

[0213] The results of these in vivo studies demonstrate that the tested MGC026 exhibited dose-dependent antitumor activity against B7-H3 positive tumors in mouse xenograft models of lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, prostate cancer, and melanoma.

[0214] Example 5: MGC026 exhibits enhanced efficacy in vivo. To further analyze the antitumor activity of MGC026, the antitumor activity of MGC026 or DS-mAb-Dxd (an ADC using a B7-H3-binding M30-H1-L4 antibody conjugated with deruxtecan (Dxd); see International Publication No. 2012147713) was evaluated using two different tumor cell lines.

[0215] In vivo activity of Calu-6 lung adenocarcinoma tumor cells The results of this study on Calu-6 lung adenocarcinoma tumor cells subcutaneously are presented in Table 10 and Figures 11A-11C, demonstrating the responsiveness to Calu-6 tumor cells.

[0216] [Table 10]

[0217] Female CD-1 nude (homozygous) mice (n=5 / group) were subjected to a study of Calu-6 (lung adenocarcinoma) tumor cells (5 × 10⁶ cells) suspended in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On the 16th day, the tumor was approximately 100 mm. 3 (100±22mm 3 When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 16 (arrow), administered intravenously with either a vehicle control (1X PBS) or a targeted ADC (MGC026 or DS-mAb-Dxd) at the prescribed dose levels (A, B, C) to form a single total dose. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. 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.

[0218] MGC026 reduced tumor volume by 87% at 3 mg / kg on day 41 and was active at this dose according to NCI criteria (T / C = 13%). DS-mAb-Dxd was inactive at 3 mg / kg. MGC026 induced partial regression in 2 out of 5 animals at a dose of 3 mg / kg, while DS-mAb-Dxd did not show partial or complete regression at 3 mg / kg. The tumor H score was determined to be 210 by immunohistochemistry (IHC).

[0219] In vivo activity against A375.S2 melanoma tumor cells The antitumor activity of conjugated MGC026 or DS-mAb-Dxd was evaluated using A375.S2 melanoma tumor cells. The results of this study on subcutaneously inoculated A375.S2 melanoma tumor cells are presented in Table 11 and Figures 12A-12C, showing the responsiveness to A375.S2 tumor cells.

[0220] [Table 11]

[0221] Female CD-1 nude (homozygous) mice (n=5 / group) were subjected to a suspension of A375.S2 (melanoma) tumor cells (5 × 10⁶ cells) in 0.1 mL of serum-free medium / Matrigel (1:1) per mouse. 6 (Number of cells) were subcutaneously transplanted. On the 26th day, the tumor was approximately 100 mm. 3 (87±21mm 3 When the mean tumor volume (± standard deviation) was reached, the mice were randomly divided and, on day 26 (arrow), administered intravenously with either a vehicle control (1X PBS) or a targeted ADC (MGC026 or DS-mAb-Dxd) at the prescribed dose levels (A, B, C) to form a single total dose. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed after treatment with MGC026 at the three dose levels tested, and also with 3 mg / kg of DS-mAb-Dxd.

[0222] MGC026 reduced tumor volume by 100% on day 80 and was highly active at 3 mg / kg (T / C=0%) and 1 mg / kg (T / C=7%), and active at 0.3 mg / kg (T / C=16%), according to NCI criteria. Treatment with DS-mAb-Dxd was active at 3 mg / kg (T / C=13%) and inactive at 1 mg / kg (T / C=84%) and 0.3 mg / kg (T / C=100%). MGC026 induced partial regression in 5 / 5 animals at dose levels of 3 mg / kg and 1 mg / kg, and in 4 / 5 animals at dose level of 0.3 mg / kg. Complete regression was observed in 5 / 5 animals and 3 / 5 animals at dose levels of 3 mg / kg and 1 mg / kg, respectively. D-mAb-Dxd at 3 mg / kg induced partial regression in 5 out of 5 animals and complete regression in 1 out of 5 animals. 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.

[0223] Example 6: Study on the tolerability of MGC026 in cynomolgus monkeys To assess the tolerability of MGC026 (mentioned above), preclinical animal studies were conducted in cynomolgus monkeys. These studies included both non-GLP and GLP (Good Laboratory Practice) studies.

[0224] In the non-GLP study, a total of six cynomolgus monkeys (Macaca fascicularis) received two doses of MGC026 at doses of 7.4, 22.2, and 44.4 mg / kg via intravenous (IV) infusion over 4 minutes, with a 2-week interval (Q2W) between doses, with two animals receiving each dose (Table 12).

[0225] [Table 12]

[0226] MGC026 was well-tolerated when administered at doses of 7.4, 22.2, or 44.4 mg / kg during Q2W (days 1 and 15 of the study). Study evaluations included: toxicological assessment, body weight, food intake, clinical observation, clinicopathology (hematology, clinical chemistry), and anatomical pathology. Findings were limited to anatomical pathology, and consisted of decreased thymic cell solidity (lymphocytes) observed in one of the two animals administered 7.4 mg / kg (mild) and in both of the two animals administered 44.4 mg / kg (mild and moderate).

[0227] In the GLP study, a total of 26 cynomolgus monkeys received a total of three doses of MGC026 at doses of 10, 30, and 50 mg / kg, administered by intravenous (IV) infusion over 15 minutes to 6, 10, and 10 animals, respectively, with a 3-week interval between doses (Q3W) (Table 13).

[0228] [Table 13]

[0229] MGC026 administered at doses of 10, 30, or 50 mg / kg / dose during Q3W (days 1, 22, and 43 of the study) was well-tolerated. Study evaluations included: toxicological assessment, body weight, food intake, clinical observation, clinicopathology (hematology, clinical chemistry, coagulation tests, or urinalysis), ECG, blood pressure, respiratory rate, heart rate, or neurological assessment (response to environmental stimuli, body temperature, eye movements, motor reflexes, and proprioceptive function), and anatomical pathology. maxBoth the parameter and AUC parameter increased with increasing dose levels of MGC026 and were approximately proportional to the dose. Findings in the study were limited to anatomical pathology. At the final autopsy (day 49 of the study), a decrease in thymic weight correlated with a decrease in thymic cell solidity (lymphocytes) in 3 out of 10 animals (mild to moderate) administered 30 mg / kg, and in 6 out of 10 animals (mild to moderate) administered 50 mg / kg. This finding was not considered harmful because a substantial amount of thymic tissue remained in animals with moderate severity, there was no evidence of opportunistic infection, and other components of the immune system (spleen, mesenteric lymph nodes, mandibular lymph nodes, gut-associated immune system) were normal. No findings were observed at the convalescent autopsy (day 91 of the study).

[0230] Example 7: Study on the tolerability of MGC018 in cynomolgus monkeys To assess the tolerability of MGC018 (mentioned above), preclinical animal studies were conducted in cynomolgus monkeys. These studies included both non-GLP and GLP (Good Laboratory Practice) studies.

[0231] Non-GLP studies included single-dose non-GLP PK studies in which 14 animals were administered 1, 3, 10, 20, or 27 mg / kg of MGC018, and repeated-dose non-GLP toxicological studies in which 6 animals (3 animals / group) were administered 6 or 20 mg / kg of MGC018 every two weeks (Q2W) (Table 14).

[0232] [Table 14]

[0233] Data collected from two non-GLP MGC018 studies showed that single doses up to 27 mg / kg were well tolerated based on body weight, food intake, clinical observation, clinicopathology (hematology, clinical chemistry), and anatomical pathology. However, repeated doses of 20 mg / kg at Q2W were not well tolerated, and animals required veterinary treatment for fever (possibly infection), skin changes (dryness, erythema, and / or exudative abrasions / skin erosions), and watery stools. Veterinary treatment did not affect the study data or interpretation because its therapeutic effect is well known and / or short-term. One animal administered 20 mg / kg on days 1 and 15 of the study presented with erythema in the left inguinal region, which progressed to dry, erythematous skin in both the inguinal and axillary regions, as well as skin erosions in the left inguinal area. This animal exhibited decreased activity, kyphosis, and loss of appetite from day 20. By day 22, skin lesions were observed in both the groin and axillae, as well as on the left hind limb. The skin lesions were washed with a topical disinfectant (chlorhexidine), and treated with a topical sedative (Douxo) and antibiotic (Neo-predef) as needed from day 14 to day 22. For pain and decreased motor function, a nonsteroidal anti-inflammatory drug (Metacam) was administered on days 21 and 22. On day 22, washing of the skin lesions resulted in deglove, and based on the size of these lesions, the animal was euthanized at the end of life. The remaining two animals, administered 20 mg / kg / dose, were also euthanized on day 22, and the degree of skin erosion, dryness, and pigmentation was evaluated.

[0234] Other MGC018-related clinical signs after repeated administration of 6 and 20 mg / kg / dose during Q2W included sporadic watery stools, increased frequency of dry skin (from day 8 or later), and skin discoloration (red, black, and brown). Further observations with MGC018 at 20 mg / kg / dose included green stools within 3 days of dose administration, decreased activity, loss of appetite, kyphosis, increased frequency of abrasions (mainly in the groin), and yellow discharge (from abrasions), which were most pronounced from day 18 or later.

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

[0236] In the 20 mg / kg / dosing regimen, a moderate decrease in red blood cell count became more pronounced from day 15, which was associated with a decrease in the absolute number of reticulocytes. These were related to MGC018, and the decrease or suppression of hematopoiesis correlated with decreased hematopoietic cell density in the sternal bone marrow in two of the three animals receiving the 20 mg / kg / dosing regimen. In the 20 mg / kg / dosing regimen, a decrease in lymphocytes (mild), neutropenia (moderate), and thrombocytopenia (mild) were also observed from day 15. In the 6 mg / kg / dosing regimen, similar levels of lymphocyte and thrombocytopenia occurred at later time points, but no decrease in neutropenia occurred. These decreases were related to MGC018 and correlated with thymic lymphocyte depletion in two of the three animals receiving the 20 mg / kg / dosing regimen.

[0237] The simultaneous occurrence of moderate increases in fibrinogen concentration, mild to moderate increases in CRP concentration, mild increases in globulin concentration, and moderate decreases in albumin concentration indicates an inflammatory response and was observed in animals after the second dose of 20 mg / kg / dose (day 22). These are thought to be related to MGC018 and are very likely to be associated with subcutaneous lesions in the forelimbs, hindlimbs, and ventral abdomen of these animals. Findings in animals administered 6 mg / kg / dose were limited to a moderate increase in CRP.

[0238] Animals administered MGC018 at doses of 6 or 20 mg / kg exhibited a variety of associated skin findings observed in multiple locations, including the face, forelimbs, hindlimbs, and ventral abdomen. While the severity of these findings varied across multiple locations within a single animal and between animals, the characteristics of these findings were generally similar within each dose group, were observed in all animals at these doses, and showed a clear dose-response in the severity and extent of the findings.

[0239] At a dose of 20 mg / kg, abrasions / crusts were observed on the forelimbs, hindlimbs, and ventral surface of the abdomen, which microscopically correlated with ulcers. Mild to moderate lymphocytic inflammation was present at the epidermal-dermal interface and surrounding superficial epidermal vessels, and edema was often present within the superficial epidermis in these areas. Mild to mild single-cell necrosis was present in the upper epidermis, ranging from vacuolation and degeneration of the basal layer of the epidermis to necrosis of individual epidermal cells. Mild to mild epidermal hyperplasia with increased keratin was occasionally present. Mild to moderate epidermal exfoliation occurred in inflamed areas, consisting of the epidermis separating from the underlying basement membrane and dermis. This separation occurred beneath the basal cell layer, forming multiple fused blisters of varying sizes. In cases of more extensive peeling, the epidermis was no longer present, leaving behind visible ulcers of varying sizes, ranging in severity from mild to severe, and correlating with visible abrasions / crusts. In one of the three animals, mild erosions / ulcers were present in the perianal skin and around the anus.

[0240] At a dose of 6 mg / kg, skin melanosis was observed on the face, forelimbs, hindlimbs, and / or ventral abdomen, which correlated microscopically with a slight increase in pigment present within the epidermis and sometimes extending into the superficial dermis. Mild to moderate lymphocytic inflammation was present at the epidermal-dermal interface and surrounding superficial epidermal vessels, with varying amounts of edema within the superficial epidermis in these areas. Mild single-cell necrosis was present in the upper epidermis and sometimes in hair follicles, ranging from vacuolation and degeneration of the basal layer of the epidermis to necrosis of individual epidermal cells. Mild epidermal hyperplasia with increased keratin was also occasionally present.

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

[0242] [Table 15]

[0243] The evaluation of the MGC018 GLP toxicological study included: toxicological assessment, body weight, food intake, clinical observation, clinicopathology (hematology, clinical chemistry, coagulation tests, or urinalysis), ECG, blood pressure, respiratory rate, heart rate, or neurological assessment (response to environmental stimuli, body temperature, eye movements, motor reflexes, and proprioceptive function), and anatomical pathology.

[0244] In cynomolgus monkeys, the pharmacokinetic (PK) of MGC018 was linear between 6 mg / kg / dose and 10 mg / kg / dose, but nonlinear at doses of 6 mg or less / kg. This is likely due to a target-dependent clearance mechanism at low doses and / or saturation of target-specific clearance at higher doses.

[0245] MGC018-related clinical signs were primarily dose-dependent and included skin findings, hyperpigmentation (males at doses of ≤1 mg / kg / and females at doses of ≤3 mg / kg / ), dry skin ± erythema at doses of ≥3 mg / kg / , increased incidence of loose stools / watery stools, mainly at doses of 3 and 6 mg / kg / , occasional loss of appetite, loose stools / watery stools, lean body shape (one female at doses greater than 6 mg / kg / ), and hair loss observed in some animals, particularly at doses of ≥6 mg / kg / .

[0246] MGC018-related changes in clinicopathological parameters included 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 volume and reticulocyte count), and transient increases in AST and / or ALT. However, all of these were dose-responsive, recovered before or during the recovery phase without microscopic correlation, and were not considered harmful because the changes often remained within or immediately after the historical control range of these parameters in cynomolgus monkeys.

[0247] Microscopically, diverse findings were present in the skin in typical sections (flanks), at the infusion site, and in further locations including the forelimbs, hindlimbs, and head. While the severity and / or presence of these findings varied within a single animal and across multiple animals, the characteristics of these findings were generally similar within a single dose group, showing a clear dose-response in severity and extent, generally more pronounced at doses of 6 mg or more / kg, and included increased pigmentation, lymphocytic inflammation, epidermal hyperplasia, and unicellular necrosis. Inflammation and unicellular necrosis resolved by the end of the recovery phase, while changes in pigmentation and hyperplasia were in progress towards the end of the recovery phase.

[0248] Example 8: Comparison of toxicological studies of MGC026 and MGC018 in cynomolgus monkeys Table 16 provides a comparison of toxicological studies of MGC026 and MGC018 conducted in cynomolgus monkeys, as described in Examples 6 and 7. This comparison demonstrates that MGC026 has a cleaner profile compared to MGC018, which causes pigmentation in animals, as well as lymphocytic inflammation, epidermal hyperplasia, and unicellular necrosis in the skin. These findings were more severe when the dosing interval was shortened to Q2W, and animals administered 20 mg / kg were euthanized.

[0249] [Table 16] JPEG2026529159000031.jpg249161JPEG2026529159000032.jpg250160JPEG2026529159000033.jpg111158

[0250] Example 9: MGC026 exhibits in vivo activity in patient-derived xenografts. To further demonstrate the antitumor activity of MGC026, the above-mentioned MGC026 molecule was used in athymic Nude-Foxn1 using tumor fragments from multiple different patients. nu (Envigo, Charles River Laboratories) In vivo toxicity in a mouse model was evaluated. Briefly, low-passage tumor fragments were used to treat athymic Nude-Foxn1 nu The tumor was transplanted into stock mice. The stock mice had tumors of 1000-1500 mm. 3 When the tumor reached a certain size, it was harvested, and the tumor fragment was re-transplanted to the left flank of preclinical study mice, but only on one side. 3When tumor volume was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer (FB)). Mice were assigned to each group and administered intravenously by tail vein injection (approximately 100 μL). In these studies, MGC026 or the vehicle control was administered twice, once every two weeks (Q2Wx2). Tumors were measured twice a week by orthogonal measurement using electronic calipers, and tumor volume was calculated as (width x 2) x length x 0.52. Animals were considered to have shown partial regression ("PR") if 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, the tumor volume of treated animals was 5 mm 3 A reduction below the specified threshold was considered a complete response ("CR"). Tumor volume (relative to vehicle control) was determined ("T / C"). Antitumor activity was evaluated according to the National Cancer Institute (NCI) criteria; a T / C of 42% or less represents the lowest level of antitumor activity, while a T / C value greater than 42% is inactive. A T / C of less than 10% is considered highly active.

[0251] In vivo activity against tumors derived from small cell lung cancer patients The results of this study regarding tumor fragments derived from small cell lung cancer patients inoculated subcutaneously from Model 1 are presented in Table 17 and Figure 13, demonstrating the responsiveness to tumor fragments derived from small cell lung cancer patients.

[0252] [Table 17]

[0253] Female Athymic Nude-Foxn1 nu Tumor fragments derived from small cell lung cancer patients were subcutaneously transplanted into mice (n=3 / group) from stock mice. The tumors averaged 150-300 mm. 3 (257±46mm 3When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0254] At a dose level of 10 mg / kg and Q2Wx2, MGC026 reduced tumor volume by 78% on day 10 and was active (T / C = 22%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 1 out of 3 animals but did not induce complete regression.

[0255] In vivo activity against tumors derived from small cell lung cancer patients The results of this study regarding tumor fragments derived from small cell lung cancer patients inoculated subcutaneously from Model 2 are presented in Table 18 and Figure 14, demonstrating the responsiveness to tumor fragments derived from small cell lung cancer patients.

[0256] [Table 18]

[0257] Female Athymic Nude-Foxn1 nu Tumor fragments derived from small cell lung cancer patients were subcutaneously transplanted into mice (n=3 / group) from stock mice. The tumors averaged 150-300 mm. 3 (268±10mm 3 When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0258] At a dose level of 10 mg / kg and Q2Wx2, MGC026 reduced tumor volume by 99% on day 52 and was highly active (T / C=1%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 3 out of 3 animals and complete regression in 1 out of 3 animals. Treatment with MGC026 at a dose level of 10 mg / kg and Q2Wx2 maintained antitumor control until the end of the study.

[0259] In vivo activity against tumors derived from small cell lung cancer patients The results of this study regarding tumor fragments derived from small cell lung cancer patients inoculated subcutaneously from Model 3 are presented in Table 19 and Figure 15, demonstrating the responsiveness to tumor fragments derived from small cell lung cancer patients.

[0260] [Table 19]

[0261] Female Athymic Nude-Foxn1 nu Tumor fragments derived from small cell lung cancer patients were subcutaneously transplanted into mice (n=3 / group) from stock mice. The tumors averaged 150-300 mm. 3 (236±80mm 3 When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0262] At a dose level of 10 mg / kg and Q2Wx2, MGC026 reduced tumor volume by 100% on day 23 and was highly active (T / C=0%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 3 out of 3 animals and complete regression in 2 out of 3 animals. Treatment with MGC026 at a dose level of 10 mg / kg and Q2Wx2 maintained antitumor control until the end of the study.

[0263] In vivo activity against tumors derived from ovarian cancer patients The results of this study on tumor fragments derived from ovarian cancer patients administered subcutaneously are presented in Table 20 and Figure 16, demonstrating the responsiveness to tumor fragments derived from ovarian cancer patients.

[0264] [Table 20]

[0265] Female Athymic Nude-Foxn1 nu Tumor fragments derived from ovarian cancer patients were subcutaneously transplanted into mice (n=3 / group) from stock mice. The tumors averaged 150-300 mm. 3 (211±82mm 3 When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0266] At a dose level of 10 mg / kg and Q2Wx2, MGC026 reduced tumor volume by 98% on day 48 and was highly active (T / C=2%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 3 out of 3 animals but not complete regression. Treatment with MGC026 at a dose level of 10 mg / kg and Q2Wx2 maintained antitumor control until the end of the study.

[0267] In vivo activity against tumors derived from melanoma patients The results of this study on tumor fragments derived from melanoma patients administered subcutaneously are presented in Table 21 and Figure 17, demonstrating the responsiveness to tumor fragments derived from melanoma patients.

[0268] [Table 21]

[0269] Female Athymic Nude-Foxn1 nu Mice (n=3 / group) were subcutaneously transplanted with tumor fragments derived from melanoma patients from stock mice. The tumors averaged 150-300 mm. 3 (202±78mm 3 When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0270] At a dose level of 10 mg / kg and Q2Wx2, MGC026 reduced tumor volume by 97% on day 30 and was highly active (T / C=3%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 2 out of 3 animals and complete regression in 1 out of 3 animals. Treatment with MGC026 at a dose level of 10 mg / kg and Q2Wx2 maintained antitumor control until the end of the study.

[0271] In vivo activity against tumors derived from colorectal cancer patients The results of this study on tumor fragments derived from colorectal cancer patients administered subcutaneously are presented in Table 22 and Figure 18, demonstrating the responsiveness to tumor fragments derived from colorectal cancer patients.

[0272] [Table 22]

[0273] Female Athymic Nude-Foxn1 nu Tumor fragments derived from colorectal cancer patients were subcutaneously transplanted into mice (n=3 / group) from stock mice. The tumors averaged 150-300 mm. 3 (248±93mm 3 When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0274] At a dose level of 10 mg / kg and Q2Wx2, MGC026 reduced tumor volume by 76% on day 29 and was active (T / C = 24%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 1 out of 3 animals but did not induce complete regression.

[0275] In vivo activity against tumors derived from patients with head and neck squamous cell carcinoma (SCCHN) The results of this study on tumor fragments derived from SCCHN patients administered subcutaneously are presented in Table 23 and Figure 19, demonstrating the responsiveness to tumor fragments derived from SCCHN cancer patients.

[0276] [Table 23]

[0277] Female Athymic Nude-Foxn1 nu Tumor fragments derived from SCCHN patients were subcutaneously transplanted into mice (n=3 / group). The tumors averaged 150-300 mm. 3 (258±50mm 3When the mean tumor volume (± standard deviation) was reached, preclinical study mice were randomly divided according to tumor volume and treated with either MGC026 or a vehicle control (formulation buffer) on days 0 and 14 (arrows) for a total of two doses at the indicated dose levels. Tumor volume is shown as the mean ± standard error of the mean (SEM) of the group, and the SEM bar at the top can be seen. Antitumor activity was observed at the tested dose levels after treatment with MGC026.

[0278] At a dose level of 10 mg / kg with a Q2Wx2 regimen, MGC026 reduced tumor volume by 92% on day 34 and was highly active (T / C = 8%) according to NCI criteria. At this dose level, MGC026 induced partial regression in 3 out of 3 animals but not complete regression.

[0279] All publications and patents referenced herein are incorporated by reference as if specifically and individually indicated that each of these individual publications or patent applications in their entirety is incorporated by reference. While several specific embodiments are described herein, it will be understood that further modifications are possible, and that this application is intended to encompass any of these modifications, uses, or alterations.

Claims

1. formula: Ab-(LM) m --(D) n An anti-B7-H3 antibody drug conjugate (B7-H3-ADC) comprising: Ab is bonded to B7-H3, and: (i) Within the variable light chain (VL) domain, CDR L 1. Sequence RASESIYSYLA (Sequence No. 16), CDR L Two sequences of NTKTLPE (sequence number 17), and CDR L 3-sequence QHHYGTPPWT (sequence number 18), (ii) Within the variable heavy chain (VH) domain, CDR H 1. Sequence SYGMS (Sequence ID 19), CDR H Two sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H 3. Sequence HDGGAMDY (SEQ ID NO: 21) or HEGGAMDY (SEQ ID NO: 26) A humanized B7-H3 antibody or its B7-H3 conjugated fragment, containing D is the camptothecin part; LM is a linker molecule that covalently bonds Ab and D; m is an integer from 1 to n, representing the number of linker molecules in B7-H3-ADC; n is an integer between 1 and 10, representing the number of cytotoxic camptothecin moieties covalently bound to the B7-H3-ADC molecule, in the anti-B7-H3 antibody-drug conjugate (B7-H3-ADC).

2. The aforementioned Ab is: (i) A humanized VL domain containing the amino acid sequence of SEQ ID NO: 12, and (ii) Humanized VH domain containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 22 The B7-H3-ADC according to claim 1, including the B7-H3-ADC.

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 to 3, wherein Ab is an antigen-binding fragment of an antibody.

5. The B7-H3-ADC according to any one of claims 1 to 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 Fc domain is a variant Fc domain: (a) One or more amino acid modifications that reduce the affinity of the variant Fc domain to FcγR: and / or (b) One or more amino acid modifications that increase the serum half-life of the variant Fc domain. B7-H3-ADC according to claim 5 or 6, which is a variant Fc domain containing

8. The B7-H3-ADC according to claim 7, wherein the modification for reducing the affinity of the variant Fc domain to FcγR includes substitutions of L234A;L235A; or L234A and L235A, and the numbering is EU index numbering similar to that in Kabat.

9. The B7-H3-ADC according to claim 7 or 8, wherein the modification that increases the serum half-life of the variant Fc domain includes substitutions of M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, and the numbering is EU index numbering similar to that in Kabat.

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

11. The B7-H3-ADC according to claim 1, wherein the LM includes a severable linker.

12. The aforementioned LM is formula (4a) or (4b), or a salt thereof: 【Chemistry 1】 Including, here: a is independently either 0 or 1; b is independently either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is an integer in the range of 1 to 150; g is either 0 or 1; i is either 0 or 1; D is the cytotoxic camptothecin moiety; Q 1 is an alkenyl group, (hetero)cycloalkenyl group, bicyclotriazole group, or cycloalkenyl group; Q 1 It is bound to the functional group of the antibody; Sp 1 , Sp 2 , Sp 3 , and Sp 4 These are independently linear or branched C 1 -C 200 Alkylene group, C 2 -C 200 Alkenylene group, C 2 -C 200 Alkynylene group, C 3 -C 200 Cycloalkylene group, C 5 -C 200 Cycloalkenylene group, C 8 -C 200 Cycloalkylene group, C 7 -C 200 Alkylalylene group, C 7 -C 200 Arylalkylene group, C 8 -C 200 Arylalkenylene group, and C 9 -C 200 Selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, arylalkylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylalylene group, arylalkylene group, arylalkenylene group, and arylalkylene group may be optionally substituted, and optionally O, S, and NR 3 It is interrupted by one or more heteroatoms selected from the group, where R 3 These are independently hydrogen and C 1 -C 24 Alkyl alkyl group, C 2 -C 24 Alkenyl group, C 2 -C 24 Alkynyl group and C 3 -C 24 Selected from the group consisting of cycloalkyl groups, wherein the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted; Z 1 Q 1 or Sp 3 Sp 2 , O or C(O) or N(R 1 It is a connecting group that connects to ); Z 2 is D or Sp 4 Sp 1 , N(R 1 A connecting group that connects to O, or C(O); Z 1 and Z 2 each independently is —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[[ID= June 1]] 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-NR 2 -S(O) 2 -O-.-O-NR<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ R 1 is hydrogen, C 1 -C 24 Alkyl alkyl group, C 3 -C 24 Cycloalkyl groups, C 2 -C 24 (hetero)aryl group, C 3 -C 24 Alkyl (hetero)aryl group, and C 3 -C 24 Selected from the group consisting of (hetero)arylalkyl groups, the C 1 -C 24 Alkyl alkyl group, C 3 -C 24 Cycloalkyl groups, C 2 -C 24 (hetero)aryl group, C 3 -C 24 Alkyl (hetero)aryl group, and C 3 -C 24 The (hetero)arylalkyl group may be optionally substituted, and optionally O, S, and NR 3 It is interrupted by one or more heteroatoms selected from, where R 3 These are independently hydrogen and C 1 -C 4 Selected from the group consisting of alkyl groups; or R 1 is D, -[(Sp 1 ) b (Z 2 ) e - (Sp 4 ) i -D], or -[(Sp 2 ) c - (Z 1 ) d - (Sp 3 ) g -Q 1 ] and here Sp 1 , Sp 2 , Sp 3 , Sp 4 Z 1 Z 2 , D, Q 1 B7-H3-ADC according to claim 1, wherein b, c, d, e, g, and i are as defined above.

13. Sp 1 , Sp 2 , Sp 3 , and Sp 4 If it exists, Sp 1 , Sp 2 , Sp 3 , and Sp 4 These are independently linear or branched C 1 -C 20 Selected from the group consisting of alkylene groups, the alkylene group may be optionally substituted, and may also be O, S, and NR 3 It is interrupted by one or more heteroatoms selected from the group consisting of R, where R 3 These are independently hydrogen and C 1 -C 4 B7-H3-ADC according to claim 12, selected from the group consisting of alkyl groups.

14. The B7-H3-ADC according to any one of claims 1 to 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 linker is a Val-Ala-PABC linker.

16. 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 called rubitecan), lulutotecan (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin), exatecan, berotecan, calenitecin, and homocamptothecin, as described in any one of claims 1 to 15.

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

18. LM and D together: 【Chemistry 2】 The B7-H3-ADC according to any one of claims 1 to 17, comprising the above.

19. formula: Ab-(LM) m --(D) n An anti-B7-H3 antibody drug conjugate (B7-H3-ADC) comprising: Ab is bonded to B7-H3, and: (i) Within the variable light chain (VL) domain, CDR L 1. Sequence RASESIYSYLA (Sequence No. 16), CDR L Two sequences of NTKTLPE (sequence number 17), and CDR L 3-sequence QHHYGTPPWT (sequence number 18), (ii) Within the variable heavy chain (VH) domain, CDR H 1. Sequence SYGMS (Sequence ID 19), CDR H Two sequences TINSGGSNTYY PDSLKG (Sequence ID 20), and CDR H 3. Sequence HDGGAMDY (SEQ ID NO: 21) or HEGGAMDY (SEQ ID NO: 26) A humanized B7-H3 antibody or its B7-H3 conjugated fragment, containing D and LM are both: 【Transformation 3】 Constitute, m is an integer from 0 to n, representing the number of linker molecules in B7-H3-ADC; n is an integer between 1 and 10, representing the number of cytotoxic camptothecin moieties covalently bound to the B7-H3-ADC molecule, in the anti-B7-H3 antibody-drug conjugate (B7-H3-ADC).

20. The aforementioned Ab is: (i) A humanized VL domain containing the amino acid sequence of SEQ ID NO: 12, and (ii) Humanized VH domain containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 22 The B7-H3-ADC according to claim 19, including the B7-H3-ADC.

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

22. Use of B7-H3-ADC according to any one of claims 1 to 20, or use of the pharmaceutical composition according to claim 21, in the treatment of a disease or condition related to or characterized by the expression of B7-H3.

23. A method for treating a disease or condition related to or characterized by the expression of B7-H3, comprising the step of administering to a subject B7-H3-ADC according to any one of claims 1 to 20 or the 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 the expression of B7-H3 is cancer.

25. The aforementioned cancers include: adrenal tumors, AIDS-related cancers, alveolar soft part sarcomas, astrocytic tumors, adrenal carcinomas, bladder cancers, bone cancers, cancers of the brain and spinal cord, metastatic brain tumors, B-cell carcinomas, breast cancers, carotid body tumors, cervical cancers, chondrosarcomas, chordomas, chromophobe renal cell carcinomas, clear cell carcinomas, colon cancers, colorectal cancers, benign fibrous histiocytomas, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancers, and gastric cancers. cancer), gestational trophoblastic disease, germ cell tumors, head and neck cancer, glioblastoma, hematopoietic malignancies, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (e.g., acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), medulloblastoma, melanoma, meningioma, mesothelioma / pharyngeal cancer (mesothelioma pharyngeal cancer) Use or method according to claim 24, selected from the group consisting of cancer), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastasis, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumors of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma (SCCHN)), stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer (e.g., metastatic thyroid cancer); and uterine cancer.

26. The aforementioned cancers include: adrenal cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer (NSCLC), acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic 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. The use or method according to claim 25, selected from the group consisting of 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), small round blue cell tumors of childhood (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.

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 head and neck squamous cell carcinoma (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 ovarian small cell carcinoma.

34. The use or method according to claim 24, wherein the cancer is small cell carcinoma of the colon and rectum.

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 esophagogastric junction cancer.

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

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

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