Antibody conjugates targeting b7-h3

Abstract

The invention relates to antibody conjugates comprising an antibody or antigen-binding fragment thereof that specifically binds to B7-H3, and compositions comprising said antibody conjugates. Methods of using the antibody-drug conjugates and compositions, including for the treatment of cancer, are also provided.

Description

[0001] ANTIBODY CONJUGATES

[0002] FIELD OF THE INVENTION

[0003] The invention relates to antibody conjugates comprising an antibody or antigen-binding fragment thereof that specifically binds to B7 Homolog 3 (B7-H3), and compositions comprising said antibody conjugates. Methods of using the antibody-drug conjugates and compositions, including for the treatment of cancer, are also provided.

[0004] BACKGROUND TO THE INVENTION

[0005] B7 Homolog 3 (B7-H3, also known as CD276) is a transmembrane protein that is expressed at a low level on normal tissues but is over-expressed in a variety of cancers, including non- small-cell lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma, ovarian cancer, and pancreatic cancer. B7-H3 plays a role in promoting tumour progression and metastasis, as well as immune escape. In addition, B7-H3 is expressed on tumour-associated stromal cells within the tumour microenvironment, including tumour- associated vasculature, and plays an important role in angiogenesis. B7-H3 therefore represents an attractive target for cancer immunotherapies.

[0006] Antibody-drug conjugates (ADCs) are a class of therapeutics that exploit the specificity of monoclonal antibodies (mAbs) to enable targeted delivery of a cytotoxic payload. Typically, the payload by itself is highly toxic and so unsuitable for direct administration. ADCs function by using the antibody to target a specific antigen associated with cancerous cells and then releasing the drug payload under selected conditions to induce cell death. This enables the targeted delivery of the highly potent drug directly into the tumour, thereby reducing systemic exposure and toxicity to normal tissues. Accordingly, ADCs have significant potential to improve the treatment and survival of patients suffering from diseases, such as cancer.

[0007] The efficacy of an ADC is thus largely determined by the antigen-binding portion and cytotoxic payload conjugated to the antibody. There remains a need to identify and develop improved ADCs, for example having increased efficacy and reduced off-target effects.

[0008] SUMMARY OF THE INVENTION

[0009] The present inventors have identified novel antibodies that bind B7-H3 with high affinity. Antibody drug conjugates comprising said antibodies demonstrate potent cytotoxicity in vitro and substantial inhibition of tumour growth in vivo. Thus, in a first aspect, the invention provides an antibody conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising an antibody, or an antigen-binding fragment thereof, covalently linked to one or more payload moieties; wherein the antibody, or antigenbinding fragment thereof, comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7, optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0010] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0011] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0012] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

[0013] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0014] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto.

[0015] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto.

[0016] The antibody, or antigen-binding fragment thereof, may comprise an Fc region. The antibody, or antigen-binding fragment thereof, may comprise a modified Fc region. The Fc region may comprise an amino acid sequence according to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42. The antibody conjugate may comprise an amino acid sequence according to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO 90.

[0017] The antibody conjugate may comprise an antibody comprising a heavy chain (HC) and a light chain (LC).

[0018] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 44, and the LC comprises an amino acid sequence according to SEQ ID NO: 43.

[0019] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 46, and the LC comprises an amino acid sequence according to SEQ ID NO: 45.

[0020] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 48, and the LC comprises an amino acid sequence according to SEQ ID NO: 47.

[0021] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 50, and the LC comprises an amino acid sequence according to SEQ ID NO: 49.

[0022] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 56, and the LC comprises an amino acid sequence according to SEQ ID NO: 55. In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO:

[0023] 58, and the LC comprises an amino acid sequence according to SEQ ID NO: 57.

[0024] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 60, and the LC comprises an amino acid sequence according to SEQ ID NO: 59.

[0025] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 62, and the LC comprises an amino acid sequence according to SEQ ID NO: 61.

[0026] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 64, and the LC comprises an amino acid sequence according to SEQ ID NO: 63.

[0027] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 66, and the LC comprises an amino acid sequence according to SEQ ID NO: 65.

[0028] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 68, and the LC comprises an amino acid sequence according to SEQ ID NO: 67.

[0029] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 70, and the LC comprises an amino acid sequence according to SEQ ID NO: 69.

[0030] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 72, and the LC comprises an amino acid sequence according to SEQ ID NO: 71.

[0031] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 74, and the LC comprises an amino acid sequence according to SEQ ID NO: 73.

[0032] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 76, and the LC comprises an amino acid sequence according to SEQ ID NO: 75.

[0033] In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 78, and the LC comprises an amino acid sequence according to SEQ ID NO: 77.

[0034] The antibody, or antigen-binding fragment thereof, may be covalently linked to the payload moiety by a linker. The linker may be an enzyme cleavable linker.

[0035] In some embodiments, the linker comprises a p-D-glucuronide linker or a p-D-galactoside linker. The payload moiety may be selected from a prodrug, a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a colored agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, or another active agent that inhibits tumour cell growth, promotes tumour cell apoptosis or necrosis.

[0036] In some embodiments, the payload moiety is a prodrug. In some embodiments, the payload moiety is a drug.

[0037] The drug may be a topoisomerase I inhibitor, for example exatecan. The drug may be a topoisomerase II inhibitor, for example PNU-159682. The drug may be a tubulin inhibitor, for example monomethyl auristatin E (MMAE). The drug may be a DNA binding payload, for example PBD SG3249.

[0038] In some embodiments, the antibody conjugate comprises a structure represented by the formula: wherein: the wavy line indicates the covalent attachment to the antibody or antigen-binding fragment thereof via linker, L;

[0039] R is H or an optionally substituted C1-C4 alkyl group, an optionally substituted C1-C4 alkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C1-C4 alkyl carboxy C1-C4 alkyl group, F, Cl, Br, or I, CN, an optionally substituted C1-C4 alkylsulfonyl group, an optionally substituted arylsulfonyl group, or an NRz group, where NRz is selected from H, optionally substituted C1-C4 alkyl group or optionally substituted C1-C4 acyl; Ri is H, a C1-C4 alkyl group, or a C1-C4 alkoxy group;

[0040] Y is selected from an optionally substituted C1-C10 alkyl group, or a group having the formula:

[0041] O[C(RA)2] — X3— [C(RA)2]Pwhere o and p are independently from one another selected from an integer of 1 to 20, whereby o and p may be the same integer or a different integer, X3 is i) N, S or O, or ii) an aryl group or a heteroaryl group, wherein [C(RA)2]O and [C(RA)2]Pare present in the meta position of said aryl group or said heteroaryl group, and each RA is independently from one another selected from H or an optionally substituted C1-C4 alkyl group or an optionally substituted C1-C4 acyl group;

[0042] X is O or S; m is an integer from 1 to 20;

[0043] R2 and R3 are independently selected from H, C1-C20 alkyl, and -Ci-Cs heteroalkyl;

[0044] R4 is selected from H or an electron-withdrawing group, and G is a sugar moiety selected from p-D-galactoside, p-D-glucuronide, p-D-glucoside, a-

[0045] D-mannoside, or fucoside.

[0046] In some embodiments, the antibody conjugate comprises a structure having the formula:

[0047] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0048] In another aspect, the invention provides a pharmaceutical composition comprising the antibody conjugate according to the invention, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0049] The invention provides for the antibody conjugate or pharmaceutical composition according to the invention for use as a medicament.

[0050] The invention provides for the antibody conjugate or pharmaceutical composition according to the invention for use in the treatment of cancer. The invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody conjugate or the pharmaceutical composition according to the invention.

[0051] The cancer may be selected from melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T- cell lymphoma, and B-cell lymphoma.

[0052] DESCRIPTION OF THE FIGURES

[0053] Figure 1 - Shows a schematic for the generation of the scFv phage library and solid phase selection strategy for isolation of anti-human B7-H3 (4lg) antibodies.

[0054] Figure 2 - Shows enrichment of phage expressing hB7-H34lg binders after sequential rounds of library selection on immobilized hB7-H3 protein.

[0055] Figure 3 - Shows that most human B7-H3 binders isolated from phage library selections also bind to cynomolgus B7-H3 but none cross react with murine B7-H3.

[0056] Figure 4 - Cell binding of individual soluble scFvs in bacterial supernatants. ScFvs that bind to B7-H3+ HeLa cells are on the right of the black line.

[0057] Figure 5 - Binding of B7C18 and B7C23 chimeric antibodies to the surface of human cancer cells or murine 3T3 cells.

[0058] Figure 6 - Shows lack of B7C18 and B7C23 chimeric antibody cross- reactivity with other members of the B7 family.

[0059] Figure 7 - Binding evaluation to B7-H3 by ELISA for humanized antibodies. All 16 humanized B7C18 variants bound recombinant hB7-H3 4lg protein.

[0060] Figure 8 - Shows superior thermal stability of some humanized antibodies compared with parental chimeric IgG.

[0061] Figure 9 - Shows the thermal stability of humanized B7C23 versions cAb10375Y, cAb10381Y and cAb10383Y. Figure 10 - Shows the thermal stability of 4 lead humanized antibodies.

[0062] Figure 11 - Shows the binding affinity of chimeric anti-B7-H3 antibodies: B7C18-LALA, B7C23, BH6 and humanized anti-B7-H3 antibodies cAb10375-LALA, cAb10381-LALA, cAb10383-LALA and cAb10383Y-LALA to NCI-H1703 cells. Vobramitamab and Ifinatamab were used as comparators.

[0063] Figure 12 - Shows a PLRP chromatogram (A214 nm) of B7C18-Compound 1 ADC with a DAR of 4.0. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0064] Figure 13 - Shows a PLRP chromatogram (A214 nm) of B7C18-LALA-Compound 1 ADC with a DAR of 4.1. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0065] Figure 14 - Shows a PLRP chromatogram (A214 nm) of B7C18-LALA-Compound 1 ADC with a DAR of 4.4. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0066] Figure 15 - Shows a PLRP chromatogram (A214 nm) of cAb10375-LALA-Compound 1 ADC with a DAR of 4.6. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0067] Figure 16 - Shows a PLRP chromatogram (A214 nm) of cAb10375-LALA-Compound 1 ADC with a DAR of 4.1. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0068] Figure 17 - Shows a PLRP chromatogram (A214 nm) of cAb10381-LALA-Compound 1 ADC with a DAR of 4.5. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0069] Figure 18 - Shows a PLRP chromatogram (A214 nm) of cAb10381-LALA-Compound 1 ADC with a DAR of 4.2. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0070] Figure 19 - Shows a PLRP chromatogram (A214 nm) of cAb10383-LALA-Compound 1 ADC with a DAR of 4.7. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components. Figure 20 - Shows a PLRP chromatogram (A214 nm) of cAb10383Y-LALA-Compound 1 ADC with a DAR of 4.7. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0071] Figure 21 - Shows a PLRP chromatogram (A214 nm) of cAb10383Y-LALA-Compound 1 ADC with a DAR of 4.2. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0072] Figure 22 - Shows a PLRP chromatogram (A214 nm) of B7C23-Compound 1 ADC with a DAR of 4.2. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0073] Figure 23 - Shows a PLRP chromatogram (A214 nm) of a BH6-Compound 1 ADC with a DAR of 4.7. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0074] Figure 24 - Shows a SEC chromatogram (A214 nm) of a B7C18-Compound 1 ADC with a DAR of 4.0.#Monomer (>98.7%), * buffer components.

[0075] Figure 25 - Shows a SEC chromatogram (A214 nm) of a B7C18-LALA-Compound 1 ADC with a DAR of 4.4.#Monomer (>99%), * buffer components.

[0076] Figure 26 - Shows a SEC chromatogram (A214 nm) of cAb10375-LALA-Compound 1 ADC with a DAR of 4.6.#Monomer (>97.6%), * buffer components.

[0077] Figure 27 - Shows a SEC chromatogram (A214 nm) of cAb10375-LALA-Compound 1 ADC with a DAR of 4.1.#Monomer (>99.9%), * buffer components.

[0078] Figure 28 - Shows a SEC chromatogram (A214 nm) of cAb10381-LALA-Compound 1 ADC with a DAR of 4.5. * Monomer (>96.8%), * buffer components.

[0079] Figure 29 - Shows a SEC chromatogram (A214 nm) of cAb10381-LALA-Compound 1 ADC with a DAR of 4.2.#Monomer (>99.9%), * buffer components.

[0080] Figure 30 - Shows a SEC chromatogram (A214 nm) of cAb10383-LALA-Compound 1 ADC with a DAR of 4.7.#Monomer (>99.9%), * buffer components.

[0081] Figure 31 - Shows a SEC chromatogram (A214 nm) of cAb10383Y-LALA-Compound 1 ADC with a DAR of 4.7.#Monomer (>98.4%), * buffer components. Figure 32 - Shows a SEC chromatogram (A214 nm) of cAb10383Y-LALA-Compound 1 ADC with a DAR of 4.2. * Monomer (>99.9%), * buffer components.

[0082] Figure 33 - Shows a SEC chromatogram (A214 nm) of B7C23-Compound 1 ADC with a DAR of 4.2.#Monomer (>99%), * buffer components.

[0083] Figure 34 - Shows a SEC chromatogram (A214 nm) of a BH6-Compound 1 ADC with a DAR of 4.7.#Monomer (>99%), * buffer components.

[0084] Figure 35 - Shows a PLRP chromatogram (A214 nm) of B7C18-LALA-Compound 3 with a DAR of 6.8. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0085] Figure 36 - Shows a HIC chromatogram (A214 nm) of a B7C18s-Compound 4 ADC with a DAR of 2.0. * Buffer components.

[0086] Figure 37 - Shows a HIC chromatogram (A214 nm) of a BH6s-Compound 4 ADC with a DAR of 2.2. * Buffer components.

[0087] Figure 38 - Shows a HIC chromatogram (A214 nm) of an MOPC21s-Compound 4 ADC with a DAR of 2.2. * Buffer components.

[0088] Figure 39 - Shows a SEC chromatogram (A214 nm) of a B7C18s-Compound 4 ADC with a DAR of 2.0.#Monomer (97%).

[0089] Figure 40 - Shows a SEC chromatogram (A214 nm) of a BH6s-Compound 4 ADC with a DAR of 2.2.#Monomer (97.2%).

[0090] Figure 41 - Shows a SEC chromatogram (A214 nm) of a MOPC21s -Compound 4 ADC with a DAR of 2.2.#Monomer (98.3%).

[0091] Figure 42 - Shows a HIC chromatogram (A214 nm) of a B7C18s-Compound 5 ADC with a DAR of 3.8.

[0092] Figure 43 - Shows a HIC chromatogram (A214 nm) of a BH6s-Compound 5 ADC with a DAR of 3.9.

[0093] Figure 44 - Shows a HIC chromatogram (A214 nm) of an MOPC21s-Compound 5 ADC with a DAR of 4.1. Figure 45 - Shows a SEC chromatogram (A214 nm) of a B7C18s-Compound 5 ADC with a DAR of 3.8. * Monomer (97%).

[0094] Figure 46 - Shows a SEC chromatogram (A214 nm) of a BH6s-Compound 5 ADC with a DAR of 3.9.#Monomer (98.2%).

[0095] Figure 47 - Shows a SEC chromatogram (A214 nm) of a MOPC21s-Compound 5 ADC with a DAR of 4.1.#Monomer (99.3%).

[0096] Figure 48 - Shows a PLRP chromatogram (A214 nm) of an Ifinatamab-Compound 6 ADC with a DAR of 4.8. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain.

[0097] Figure 49 - Shows a SEC chromatogram (A214 nm) of an Ifinatamab-Compound 6 ADC with a DAR of 4.8.#Monomer (>97.2%).

[0098] Figure 50 - Shows a PLRP chromatogram (A214 nm) of Ifinatamab-Compound 1 ADC with a DAR of 4.1. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0099] Figure 51 - Shows a PLRP chromatogram (A214 nm) of Vobramitamab-Compound 1 ADC with a DAR of 4.3. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0100] Figure 52 - Shows a PLRP chromatogram (A214 nm) of Isotype-LALA-Compound 1 ADC with a DAR of 4.0. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0101] Figure 53 - Shows a PLRP chromatogram (A214 nm) of Vobramitamab-Compound 9 ADC with a DAR of 2.6. Numbers designate the amount of drug conjugated to light (L) or heavy (H) chain. * buffer components.

[0102] Figure 54 - Shows OVCAR-3 (A) and BxPC-3 (B) cell viability results of B7C18-Compound 1 , B7C23-Compound 1 and BH6-Compound 1.

[0103] Figure 55 - Shows B7C18-LALA-Compound 1 potency against Calu-6, A375.S2, NCI-H1703, DMS79 and MDA-MB-231 cell lines.

[0104] Figure 56 - Shows NCI-H1703 cell viability results of humanized anti-B7-H3 ADCs conjugated to Compound 1 Figure 57 - Shows NCI-H1703 cell viability results of cAb10383-LALA-Compound 1, cAb10381-LALA-Compound 1, cAb10383Y-LALA-Compound 1 , Ifinatamab-Compound 1 , Vobramitamab-Compound 1 and Isotype-Compound 1.

[0105] Figure 58- Shows Calu-6 cell viability results of cAb10383-LALA-Compound 1 , cAb10381- LALA-Compound 1, cAb10383Y-LALA-Compound 1, Ifinatamab-Compound 1, Vobramitamab-Compound 1 and Isotype-Compound 1.

[0106] Figure 59 - Shows Calu-6 and NCI-H1703 cell viability results of Ifinatamab-Compound 6.

[0107] Figure 60- Shows NCI-H1703 viability results of Vobramitamab-Compound 9 in the absence and presence of unconjugated Vobramitamab antibody.

[0108] Figure 61 - Shows RH30 rhabdomyosarcoma, (B7-H3 positive (A) B7-H3 negative (B)) and ME180 cervical cancer model (C) cell viability results of humanized anti-B7-H3 ADCs conjugated to Compound 1.

[0109] Figure 62 - Shows IGROV1 ovarian cancer model cell viability results of humanized anti-B7- H3 ADCs conjugated to Compound 1.

[0110] Figure 63 - Shows SKOV-3 ovarian cancer model cell viability results of humanized anti-B7- H3 ADCs conjugated to Compound 1.

[0111] Figure 64 - Shows MEL202 uveal melanoma cancer model cell viability results of humanized anti-B7-H3 ADCs conjugated to Compound 1.

[0112] Figure 65 - Shows RH30 rhabdomyosarcoma, (B7-H3 positive (A) B7-H3 negative (B)) and ME180 cervical cancer model (C) cell viability results of anti-B7-H3 ADCs conjugated to Compound 1 and Compound 4.

[0113] Figure 66 - B7C18s-Compound 4, BH6s-Compound 4 and MOPC21s (isotype control)- Compound 4 potency against HeLa, BxPC-3, MDA-MB-231 , A549, LS147T, and SW1222.

[0114] Figure 67 - Shows B7C18-LALA antibody and B7C18-LALA-Compound 1 binding to NCI- H1703 cells.

[0115] Figure 68 - Shows Calu-6 CDX in vivo efficacy results after treatment with B7C18-LALA- Compound 1.

[0116] Figure 69 - Shows Calu-6 CDX in vivo body weight measurements after treatment with B7C18-LALA-Compound 1. Figure 70 - Shows NCI-H1703 CDX in vivo efficacy results after treatment with B7C18-LALA- Compound 1.

[0117] Figure 71 - Shows NCI-H1703 CDX in vivo body weight measurements after treatment with B7C18-LALA-Compound 1.

[0118] Figure 72 - Shows A375 CDX in vivo efficacy results of B7C18-LALA-Compound 1.

[0119] Figure 73 - Shows A375 CDX in vivo body weight measurements after treatment with B7C18- LALA-Compound 1.

[0120] Figure 74 - Shows H1703 CDX in vivo efficacy results of cAb10383-LALA-ACTK-Compound 1 and Ifinatamab-Compound 6.

[0121] Figure 75 - Shows Calu-6 CDX in vivo efficacy results of B7C18-LALA-Compound 1, cAb10383-LALA-Compound 1 and Ifinatamab-Compound 6.

[0122] Figure 76 -Shows DMS79 CDX in vivo efficacy results of B7C18-LALA-Compound 1, cAb10383-LALA-Compound 1 and Ifinatamab-Compound 6.

[0123] Figure 77 -Shows MEL202 and IGROV-1 CDX in vivo efficacy results of cAb10383-LALA- Compound 1.

[0124] Figure 78 - Shows Scheme 4: Synthesis of Int 9 - Int 19.

[0125] Figure 79 - Shows Scheme 5: Synthesis of Int 20 - Int 25.

[0126] Figure 80 - Shows Scheme 6: Synthesis of Compound 1 from Int 19 and Int 25.

[0127] Figure 81 - Shows Scheme 7: Synthesis of Int 27 - Int 30.

[0128] Figure 82 - Shows Scheme 8: Synthesis of Int 31 - Int 36.

[0129] Figure 83 - Shows Scheme 9: Synthesis of Compound 2.

[0130] Figure 84 - Shows Scheme 10: Synthesis of Compound 3.

[0131] Figure 85 - Shows Scheme 11 : Synthesis of Int 42 - Int 45.

[0132] Figure 86 - Shows Scheme 12: Synthesis of Compound 7.

[0133] Figure 87 - Shows Scheme 13: Synthesis of Compound 8. DETAILED DESCRIPTION OF THE INVENTION

[0134] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0135] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0136] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0137] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0138] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0139] All publications mentioned in the specification are herein incorporated by reference.

[0140] B7-H3

[0141] The antibody conjugate according to the invention comprises an antibody or antigen-binding fragment thereof that specifically binds to B7 Homolog 3 (B7-H3).

[0142] In some embodiments, B7-H3 is considered the antigen of the antibody or antigen-binding fragment thereof according to the invention.

[0143] B7-H3 (also known as CD276), is a member of the B7 / CD28 immunoglobulin superfamily. B7- H3 is a single-pass transmembrane protein, existing in two isoforms. The 2lg form of B7-H3 contains a single pair of IgV-like and IgC-like immunoglobulin domains, a transmembrane region, and a cytoplasmic tail. The dominantly expressed form of human 4lgB7-H3 contains tandemly duplicated VC domains with four Ig-like domains.

[0144] Exemplary human B7-H3 sequences are provided in SEQ ID NO: 85 and 86.

[0145] 4lg-B7-H3 (SEQ ID NO: 85)

[0146] MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQ LTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSL QVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDV HSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSP EPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTC FVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNV TTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVC LIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA

[0147] 2lg-B7-H3 (SEQ ID NO: 86)

[0148] MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQ LTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSL QVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDV HSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWR KIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA

[0149] The antibody, or an antigen-binding fragment thereof, may "specifically bind" to an epitope or antigenic molecule, which means that the antibody interacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing to an epitope or antigenic molecule than alternative substances, including unrelated proteins. In specific embodiments, "specifically binds" means, for instance, that an antibody binds to a protein with a KD of approximately 0.1 mM or less, but more usually, less than about 1 pM. In specific embodiments, "specifically binds" means that an antibody binds to a protein at times with a KD of approximately 0.1 pM or less, and at other times, with a KD of approximately 0.01 pM or less, or with a KD of approximately 1 nM or less.

[0150] Suitable assays and techniques for measuring / quantifying binding activity of an antibody may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art.

[0151] Antibody or antigen binding fragment

[0152] The antibody conjugate according to the invention comprises an antibody or antigen-binding fragment thereof. The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof. The term “antibody” or “antigen-binding fragment thereof’ includes monoclonal antibodies and fragments or derivatives of antibodies, including, without limitation, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, e.g., scFvs and antigen binding antibody fragments such as Fab and Fab' fragments and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein.

[0153] Within an antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, and each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.

[0154] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0155] A CDR refers to one of three hypervariable regions (H1 , H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH p-sheet framework, or one of three hypervariable regions (L1 , L2 or L3) within the non-framework region of the antibody VL p- sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1977, J. Biol. Chem. 252:6609-6616; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved p-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). Alternatively, IMGT or EU numbering may be used. These terminologies are well recognized in the art. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 25 273:927-948; Morea et al., 2000, Methods 20:267-279). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those skilled in the art.

[0156] Suitably, the antibody or antigen binding fragment may be defined by the presence of HCDRs and LCDRs determined according to CDR numbering schemes which are known in the art. For example, the CDRs may be defined according to the IMGT, Chothia and / or Kabat numbering schemes. CDRs defined according to each of the IMGT, Chothia and Kabat numbering schemes are described herein.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 3 or

[0158] SEQ ID NO: 4, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 3, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 4, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 7. Table 1. CDR sequences according to IMGT numbering scheme

[0160] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 21 or

[0161] SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23 or SEQ ID NO: 24, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 25 or SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27 or SEQ ID NO: 28, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29; optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 21, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 25, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 21, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 24, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 25, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0165] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 28, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0166] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 24, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0167] Table 2. CDR sequences according to Kabat numbering scheme

[0168] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32 or

[0169] SEQ ID NO: 33, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 34 or SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36 or SEQ ID NO: 37, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38; optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 34, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 33, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 34, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38. In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 37, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 33, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0174] Table 3. CDR sequences according to Chothia numbering scheme

[0175] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations. In some embodiments, HCDR1 may comprise one, two or three amino acid mutations. In some embodiments, HCDR2 may comprise one, two or three amino acid mutations. In some embodiments, HCDR3 may comprise one, two or three amino acid mutations. In some embodiments, LCDR1 may comprise one, two or three amino acid mutations. In some embodiments, LCDR2 may comprise one, two or three amino acid mutations. In some embodiments, LCDR3 may comprise one, two or three amino acid mutations.

[0176] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the antibody or antigen-binding fragment thereof from binding to B7-H3. In other words, an antibody or antigen-binding fragment thereof comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to B7-H3. In some embodiments, the mutation suitably maintains the same capacity (e.g. affinity) to bind to B7-H3 as the parent antibody or antigen-binding fragment thereof. The term “parent” in this context refers to an antibody or antigen-binding fragment thereof without the mutation in question.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0179] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto.

[0180] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0190] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto.

[0191] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto.

[0192] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto.

[0193] Table 4. VH / VL sequences

[0194] It will be understood that a VH and / or VL having a percentage identity to the SEQ ID NO of any VH and / or VL defined herein may have an equivalent function to the VH and / or VL having the sequence set forth in the SEQ I D NO defined herein and may suitably maintain the capacity to bind to B7-H3, e.g. the same capacity as the VH and / or VL domain having the sequence set forth in the SEQ ID NO defined herein.

[0195] In some embodiments, the VH has at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0196] In some embodiments, the VL has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0197] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more immunoglobulin constant domains. In some embodiments, the immunoglobulin constant domains comprise a constant light chain domain (CL). In some embodiments, the immunoglobulin constant domains comprise a constant heavy 1 (CH1) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 2 (CH2) domain. In some embodiments, the immunoglobulin constant domains comprise a CH2 and a CH3 domain. In some embodiments, the CH2 and CH3 domains are considered to be an Fc (fragment crystallisable) region. In some embodiments, the immunoglobulin constant domains comprise a CL, CH1 , CH2 and CH3 domain.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 39 or SEQ ID NO: 40.

[0199] In some embodiments, the antibody or antigen-binding fragment thereof, e.g. via the Fc region, binds to one or more or all of the Fc receptors. The Fc receptors may comprise one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16), C1q and FcRn. In some embodiments, the antibody or antigen-binding fragment thereof, e.g. via the Fc region, binds to FcyRI. It will be understood that the Fc region may interact with Fc receptors presented on the surface of a cell and / or may interact with proteins of the complement system. The Fc receptors may be Fc gamma receptors, e.g. FcyRI. The proteins of the complement system may include C1q.

[0200] In other embodiments the Fc region of the antibody or antigen-binding fragment thereof is silenced to reduce, negate or abolish one or more Fc receptor binding and / or functionalities. In some embodiments, the Fc region of the antibody or antigen-binding fragment thereof is modified to negate one or more Fc receptor functionalities. In some embodiments, the Fc region of the antibody or antigen-binding fragment thereof is silenced in respect of one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16) and C1q functionality. Thus, in some embodiments, the Fc region of the antibody or antigen-binding fragment thereof is a modified Fc region. Such silencing mutations and / or modifications are well known to those skilled in the art.

[0201] In some embodiments, the antibody or antigen-binding fragment thereof comprises a modified Fc region. In some embodiments, the binding of the modified Fc region to FcyRI may be reduced compared to a wild-type Fc region.

[0202] In some such embodiments, the Fc region of the antibody or antigen-binding fragment thereof comprises a silencing modification selected from the LALA mutation and the LALA-dCTK mutation, as defined herein.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 41 or SEQ ID NO: 42.

[0204] Table 5. Fc and constant region sequences

[0205] In one embodiment the antibody conjugate according to the invention comprises an antibody.

[0206] The antibody conjugate according to the invention may comprise an antibody comprising a heavy chain(s) and a light chain(s). The term “heavy chain” refers to a large protein subunit of an immunoglobulin. Heavy chains can be of any immunoglobulin isotype (for example IgG, IgE, IgM, IgD, IgA or IgY), subtype (for example lgG1 , lgG2, lgG2a, lgG2b, lgG2c, lgG3, lgG4, lgA1 or lgA2) or allotype. The term “light chain” refers to a small protein subunit of an immunoglobulin. Light chains can be of any type (for example kappa or lambda), subtype or allotype. Antibodies described herein include polyclonal and monoclonal antibodies and include IgA such as lgA1 or lgA2, IgG such as lgG1 , lgG2, lgG3, or lgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an lgG1 antibody, more particularly an lgG1 , kappa or lgG1 , lambda isotype (i.e. lgG1 , K, A), an lgG2a antibody (e.g. lgG2a, K, A), an lgG2b antibody (e.g. lgG2b, K, A), an lgG3 antibody (e.g. lgG3, K, A) or an lgG4 antibody (e.g. lgG4, K, A). In preferred embodiments the antibody is an lgG1 , preferably lgG1 , kappa. The antibody may be of any species (for example human, monkey, camel, llama, goat, sheep, rabbit, mouse, rat, mouse, hamster or chicken) or it may be a hybrid derived from more than one species. It may be naturally occurring or it may be non-naturally occurring (i.e. an isolated antibody). The antibody may be created by genetic engineering (for example a chimeric antibody, humanized antibody, camelised antibody, intrabody, bispecific antibody).

[0207] In one embodiment the antibody conjugate according to the invention comprises a monoclonal antibody. In one embodiment the antibody conjugate according to the invention comprises a humanized antibody. In one embodiment the antibody conjugate according to the invention comprises a chimeric antibody.

[0208] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a variant having at least 80% identity thereto.

[0209] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 44, or a variant having at least 80% identity thereto.

[0210] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 44, or a variant having at least 80% identity thereto.

[0211] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 45, or a variant having at least 80% identity thereto.

[0212] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 46, or a variant having at least 80% identity thereto.

[0213] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 45, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 46, or a variant having at least 80% identity thereto.

[0214] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 47, or a variant having at least 80% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 48, or a variant having at least 80% identity thereto.

[0215] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 47, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 48, or a variant having at least 80% identity thereto.

[0216] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 49, or a variant having at least 80% identity thereto.

[0217] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 50, or a variant having at least 80% identity thereto.

[0218] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 49, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 50, or a variant having at least 80% identity thereto.

[0219] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 55, or a variant having at least 80% identity thereto.

[0220] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 56, or a variant having at least 80% identity thereto.

[0221] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 55, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 56, or a variant having at least 80% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 57, or a variant having at least 80% identity thereto.

[0222] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 58, or a variant having at least 80% identity thereto.

[0223] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 57, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 58, or a variant having at least 80% identity thereto.

[0224] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 59, or a variant having at least 80% identity thereto.

[0225] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 60, or a variant having at least 80% identity thereto.

[0226] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 59, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 60, or a variant having at least 80% identity thereto.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 61 , or a variant having at least 80% identity thereto.

[0228] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 62, or a variant having at least 80% identity thereto.

[0229] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 61 , or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 62, or a variant having at least 80% identity thereto.

[0230] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 63, or a variant having at least 80% identity thereto.

[0231] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 64, or a variant having at least 80% identity thereto.

[0232] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 63, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 64, or a variant having at least 80% identity thereto.

[0233] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 65, or a variant having at least 80% identity thereto.

[0234] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 66, or a variant having at least 80% identity thereto.

[0235] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 65, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 66, or a variant having at least 80% identity thereto.

[0236] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 67, or a variant having at least 80% identity thereto.

[0237] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 68, or a variant having at least 80% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 67, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 68, or a variant having at least 80% identity thereto.

[0238] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 69, or a variant having at least 80% identity thereto.

[0239] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 70, or a variant having at least 80% identity thereto.

[0240] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 69, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 70, or a variant having at least 80% identity thereto.

[0241] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 71 , or a variant having at least 80% identity thereto.

[0242] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 72, or a variant having at least 80% identity thereto.

[0243] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 71 , or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 72, or a variant having at least 80% identity thereto.

[0244] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 73, or a variant having at least 80% identity thereto. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 74, or a variant having at least 80% identity thereto.

[0245] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 73, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 74, or a variant having at least 80% identity thereto.

[0246] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 75, or a variant having at least 80% identity thereto.

[0247] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 76, or a variant having at least 80% identity thereto.

[0248] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 75, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 76, or a variant having at least 80% identity thereto.

[0249] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 77, or a variant having at least 80% identity thereto.

[0250] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 78, or a variant having at least 80% identity thereto.

[0251] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 77, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 78, or a variant having at least 80% identity thereto. Table 6. Full-length heavy and light chains

[0252] It will be understood that a heavy chain and / or light chain having a percentage identity to the SEQ ID NO of any heavy chain and / or light chain defined herein may have an equivalent function to the heavy chain and / or light chain having the sequence set forth in the SEQ ID NO defined herein and may suitably maintain the capacity to bind to B7-H3, e.g. the same capacity as the heavy chain and / or light chain having the sequence set forth in the SEQ ID NO defined herein.

[0253] In some embodiments, the heavy chain has at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0254] In some embodiments, the light chain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0255] The antibody or antigen-binding fragment thereof may be a single-chain variable fragment (scFv).

[0256] Thus, in some embodiments, the antibody conjugate according to the invention comprises a scFv. The scFv may comprise the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a short linker peptide.

[0257] In some embodiments, the antibody or antigen-binding fragment thereof comprises an scFv comprising or consisting of an amino acid sequence according to SEQ ID NO: 83, or a variant having at least 80% identity thereto.

[0258] In some embodiments, the antibody or antigen-binding fragment thereof comprises an scFv comprising or consisting of an amino acid sequence according to SEQ ID NO: 84, or a variant having at least 80% identity thereto.

[0259] In some embodiments, the scFv has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO 83 or 84 defined herein.

[0260] Table 7. ScFv sequences (with Chothia and Kabat CDR annotation).

[0261] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0262] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0263] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0264] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0265] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1): 187-8).

[0266] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0267] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0268] “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

[0269] Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally- occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0270] Linker

[0271] The antibody conjugate according to the invention comprises an antibody, or an antigenbinding fragment thereof, covalently linked to one or more payload moieties.

[0272] In some embodiments, the antibody, or antigen-binding fragment thereof, is covalently linked to the one or more payload moieties by a linker. The payload moieties may be the same or different, and / or the linkers may be the same or different.

[0273] The linker is a bifunctional compound which can be used to link the drug and the antibody or antigen-binding fragment thereof. Various examples have been described in the art for linking the antibody moiety to the payload. These linker systems can generally be categorised as either cleavable or non-cleavable. For cleavable linker systems, the release mechanism is typically enzymatically driven, although chemically labile cleavable systems are also known. In non-cleavable linker systems, drug release is effected by degradation within the cell after internalisation of the antibody conjugate.

[0274] In some embodiments, the linker is an enzyme cleavable linker.

[0275] In some embodiments, the linker is a peptide linker or glycan linker. In some embodiments, the linker is an enzyme cleavable peptide linker.

[0276] In some embodiments, the linker is a glycan linker. In some embodiments, the linker is an enzyme cleavable glycan linker.

[0277] In some embodiments, the linker (L) has the formula:

[0278] LA-LB-LC-LDwherein:

[0279] LAis connecting group or bond linking the antibody or antigen binding fragment to LB;

[0280] LBis an enzyme cleavable linker;

[0281] Lcis a self-immolative spacer or absent; and LDis spacer group covalently bound to the payload moiety.

[0282] The connecting group LAmay comprise a reactive site e.g., an electrophilic group, that is reactive to a nucleophilic group present on the antibody. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on the connecting group and forms a covalent bond thereto. Useful electrophilic groups include, but are not limited to, vinylpyridine, maleimide and haloacetamide groups. Alternatively, connecting group LAmay comprise a reactive site which has a nucleophilic group that is reactive to an electrophilic group present on the antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a linker unit can react with an electrophilic group on an antibody and form a covalent bond to the antibody. Useful nucleophilic groups on a linker unit include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. Alternatively, the antibody may be modified to include an azide group (-N3) and the connecting group may incorporate an azide reactive moiety, such as a cyclooctyne group, for production of the antibody-drug conjugate according to the present invention. In this way, the linker can be bound to the antibody through a click-reaction between the azide group in the antibody and the cyclooctyne group in the linker.

[0283] Optionally, LBmay be selected from a p-D-glucuronide linker or a p-D-galactoside linker and Lcis absent.

[0284] As used herein, a p-D-glucuronide linker comprises the formula:

[0285] As used herein, a p-D-galactoside linker comprises the formula:

[0286]

[0287] Alternatively, LBmay be amino acids or a peptide, such as a dipeptide. For example, the dipeptide linker may be selected from -Valine-Citrulline- (-Val-Cit-), -Valine-Alanine-(-Val-Ala- ), -Valine-Lysine-(-Val-Lys-), -Valine-Arginine-(-Val-Arg-), -Phenylalanine-Citrulline-(-Phe-Cit- ), -Phenylalanine-Lysine-(-Phe-Lys-), or -Phenylalanine-Arginine-(-Phe-Arg-). A linker including such dipeptides may be cleavable, for example, by cathepsin B.

[0288] Where LBis a dipeptide linker as described above, Lcmay be para-aminobenzyl, paraaminobenzyloxycarbonyl.

[0289] LDmay be a bisamine cyclization spacer. For example, LDmay be selected from - C(O)N(RB)(RC) or -C(S)N(RB)(RC), wherein LD is bonded to the payload moiety at the carbon of the C(O) or C(S) group, wherein RB is -C1-C20 alkylN(Ro)-, and wherein Rc and RD are independently selected from H, C1-C20 alkyl, and -Ci-Cs heteroalkyl. Optionally, Rc and RD may be independently selected from -C1-C20 alkyl and -(CFkC WJrH, where r is an integer from 1 to 4.

[0290] Optionally, the electron-withdrawing group is selected from the group consisting of a nitro group, a cyano group, -CFs or a halide (e.g. F, Cl, Br, I). Optionally, the electron withdrawing group is a nitro group.

[0291] Optionally, the antibody conjugate may comprise a structure selected from:

[0292] wherein the wavy line indicates the site of attachment to the phenolic oxygen of the first CBI unit.

[0293] Optionally, LAmay be represented by Formula I:

[0294] Formula I wherein: the wavy line indicates a point of attachment to the antibody or antigen fragment thereof; W is a coupling group for connecting LAwith LB; and q is an integer from 1 to 20.

[0295] For example, W may be an amide group (i.e. -NH-C(O)-) formed by coupling reactions well known in the art.

[0296] Linker stability contributes to the efficacy and toxicity of an antibody-drug conjugate. An unstable linker can release the cytotoxic payload during blood circulation before it reaches the target site, leading to undesirable systemic toxicities. For example, some linkers, such as the widely-used maleimide attachment method, can suffer from non-specific release of payloads in non-tumourous tissues, leading to off-target toxicity and a limited therapeutic window. Advantageously, connecting groups LAaccording to Formula I provide a stable connection between the antibody and the drug while allowing efficient cleavage of the drug in tumour cells.

[0297] As shown in Formula I, the connecting group LAmay include a hydrophilic poly(ethylene glycol) (PEG) spacer. The use of PEG-linkers is known in the art to improve pharmacokinetic profiles. As will be appreciated by the skilled person, the PEG spacer may have different lengths to keep the drug moiety or payload closer or further away from the antibody. For example, n may be 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. It will also be appreciated by the skilled person that the techniques disclosed herein may be used in conjunction with other poly(alkylene glycol) molecules, such as polypropylene glycol or polyethylene-polypropylene glycol copolymers. Branched or multi-arm poly(alkylene glycol) molecules, including branched or multi-arm PEG molecules, may also be used. Poly(alkylene glycol) molecules that may be used in accordance with the present invention are well known in the art and publicly available, for example from commercially available sources such as Sigma Aldrich.

[0298] Optionally, LAmay be covalently bound to the antibody by a thioether bond. For example, the thioether bond may comprise a sulfur atom of cysteine of the antibody. Cysteine-based conjugation methods offer greater control of drug loading, i.e. the drug-to-antibody ratio (DAR) and homogeneity, compared to lysine conjugation methods. Greater antibody conjugate homogeneity is known to be associated with improved pharmacokinetics and efficacy and reduced off-target toxicity. The covalent thioether bond may be formed using existing thiol groups or by introducing thiol groups in a precursor step, for example by reacting one or more functional groups of the antibody to produce a thiol group, or by introducing a thiol group or a precursor thereof into the antibody. By way of example, this may involve the step of introducing a cysteine residue into the antibody at a site where it is desired to bind the linker to the antibody. This may be useful in situations where a convenient cysteine residue for reaction is not present in a starting or wild-type antibody. Conveniently, this may be achieved using site directed mutagenesis of the antibody, the use of which is well established in the art. Advantageously, this may also enable attachment of a drug or other active agent to the antibody in a site-specific manner. This may allow for the preparation of homogeneous antibody conjugates having a defined number of drugs, which is known to improve pharmacokinetics and efficacy and is more desirable from a regulatory perspective. “Self-immolative spacer” refers to a moiety that spaces and covalently links together two or more components and degrades spontaneously in response to specific stimuli.

[0299] Payload

[0300] The antibody conjugate according to the invention comprises one or more payload moieties.

[0301] Upon binding of an antibody conjugate to a target antigen present on the surface of a cell, the antibody conjugate may become internalised and trafficked to intracellular compartments (e.g. a lysosome) where the payload is released from the antibody conjugate. When the payload is a drug, the release of the drug from the antibody conjugate may allow the drug to exert its effect on the cell. The payload may be released from the antibody conjugate by proteolysis or enzymatic cleavage of a cleavable linker (if present) or by degradation of the antibody of the antibody conjugate.

[0302] The payload may be selected from a prodrug, a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a coloured agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumour cell growth, promotes tumour cell apoptosis or necrosis.

[0303] In some embodiments, the payload is a prodrug.

[0304] As used herein, “prodrug” refers to a compound that is metabolised, for example hydrolysed, in the host after administration to form a biologically active molecule. Typical examples of prodrugs include compounds that have biologically labile or cleavable protecting groups on a functional moiety of the active compound.

[0305] In some embodiments, the payload is a drug.

[0306] The drug may be a cytotoxic drug or chemotherapeutic drug. By “cytotoxic drug” is meant a drug that is destructive to a cell, induces apoptosis in a cell, inhibits or prevents the function of a cell, inhibits or prevents proliferation of a cell, and / or reduces the viability of a cell.

[0307] In some embodiments, the cytotoxic drug is a topoisomerase I inhibitor, a topoisomerase II inhibitor, or a tubulin inhibitor.

[0308] In some embodiments, the antibody conjugate comprises a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is exatecan. In some embodiments, the antibody conjugate comprises a topoisomerase II inhibitor. In some embodiments, the topoisomerase II is PNU-159682.

[0309] In some embodiments, the antibody conjugate comprises a tubulin inhibitor. In some embodiments, the tubulin inhibitor is monomethyl auristatin E (MMAE).

[0310] In some embodiments, the antibody conjugate comprises a DNA binding payload. In some embodiments, the DNA binding payload is a PBD. In some embodiments, the DNA binding payload is PBD SG3249.

[0311] In some embodiments, the antibody conjugate comprises a cyclopropylbenzoindole (CBI) dimer drug moiety.

[0312] Cyclopropylbenzoindole (CBI)-based dimers are a class of DNA minor groove alkylators that are reported to be highly cytotoxic (see Tietze et al., Angew. Chem. Int. Ed. Engl. 2010, 49, 7336-7339) and have been developed into prodrugs for cancer therapy. For instance, halogen-containing seco derivatives of CBI are generally believed to require a ring closure to their cyclopropyl containing spiro analogues to become cytotoxic. Thus, by trapping the CBI unit in its seco form through the protection of the hydroxyl group by a prodrug functional group, the drug can be substantially inactivated until the prodrug function group is removed such that ring closure to the spiro form can occur.

[0313] In some embodiments, the antibody conjugate comprises a structure represented by the formula: wherein: the wavy line indicates the covalent attachment to the linker; R is H or an optionally substituted C1-C4 alkyl group, an optionally substituted C1-C4 alkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C1-C4 alkyl carboxy C1-C4 alkyl group, F, Cl, Br, or I, CN, an optionally substituted C1-C4 alkylsulfonyl group, an optionally substituted arylsulfonyl group, or an NRz group, where NRz is selected from H, optionally substituted C1-C4 alkyl group or optionally substituted Ci-C4 acyl;

[0314] R1 is H, a C1-C4 alkyl group, or a C1-C4 alkoxy group;

[0315] Y is selected from an optionally substituted C1-C10 alkyl group, or a group having the formula:

[0316] O[C(RA)2] — X3— [C(RA)2]Pwhere o and p are independently from one another selected from an integer of 1 to 20, whereby o and p may be the same integer or a different integer, X3 is i) N, S or O, or ii) an aryl group or a heteroaryl group, wherein [C(RA)2]O and [C(RA)2]Pare present in the meta position of said aryl group or said heteroaryl group, and each RA is independently from one another selected from H or an optionally substituted C1-C4 alkyl group or an optionally substituted Ci- 04 acyl group;

[0317] X is O or S; m is an integer from 1 to 20;

[0318] Rz and R3are independently selected from H, C1-C20 alkyl, and -Ci-Cs heteroalkyl;

[0319] R4 is selected from H or an electron-withdrawing group, and

[0320] G is a sugar moiety selected from p-D-galactoside, p-D-glucuronide, p-D-glucoside, a- D-mannoside, or fucose.

[0321] “Electron-withdrawing group” refers to a group that draws electron density from neighbouring atoms towards itself, typically by resonance or inductive effects.

[0322] Accordingly, an antibody conjugate of the present invention may comprise a CBI dimer prodrug whereby a first CBI unit of the CBI dimer incorporates a prodrug functional group and a second unit of the CBI dimer is attached to the antibody via linker. Thus, an antibody conjugate of the present invention may incorporate a two-step activation mechanism, whereby the prodrug functional group must be cleaved in addition to the linker before the active cytotoxic drug is released. Advantageously, the CBI dimer prodrugs according to the present invention are believed to be more stable in circulation than the active form of the drug, thereby reducing the occurrence of side effects caused by the premature decomposition of the linker in normal cells.

[0323] The prodrug functional group comprises a sugar moiety represented by G that is coupled to a self-immolative linker via a glycosidic bond. The self-immolative linker provides a stable linkage between the sugar moiety and the first CBI unit and collapses upon the cleavage of glycosidic bond to liberate a free phenol at the first CBI unit. For example, the self-immolative linker may comprise para-hydroxybenzyloxycarbonyl or derivative thereof and a bisamine cyclization spacer. Advantageously, the inclusion of a self-immolative linker may improve the efficacy of the antibody conjugate compared to direct attachment of sugar moiety to the first CBI unit.

[0324] The prodrug functional group may be cleaved by enzymes that are overexpressed in tumours, resulting in higher tumour selectivity of the active form of the drug. An example of this is the P-D-glucuronidase, which has been detected in increased concentrations in tumour tissue. Thus, in one example, G may be p-D-glucuronide. In another example, G may be p-D- galactoside.

[0325] Optionally, R2 and Rs may be independently selected from -C1-C20 alkyl and -(CH2CH2O)rH, where r is an integer from 1 to 4. The hydrophobicity of some cleavable linker systems can lead to the aggregation of drug conjugates, particularly with strongly hydrophobic drugs. Advantageously, the incorporation of one or more hydrophilic substituents in these positions may reduce linker-drug hydrophobicity, which may decrease aggregation of the antibody conjugate.

[0326] In some embodiments, the antibody conjugate comprises a structure represented by the formula: [Compound 1] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0327] In some embodiments, the antibody conjugate comprises a structure represented by the formula:

[0328] [Compound 2] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0329] In some embodiments, the antibody conjugate comprises a structure represented by the formula: [Compound 3] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. In some embodiments, the antibody conjugate comprises a structure represented by the formula:

[0330] [Compound 4] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0331] SG3249 (Tesirine), is a pyrrolobenzodiazepine (PBD) dimer payload. SG3199, the released warhead of SG3249 is a DNA minor groove cross-linking agent with potent cytotoxicity. The SG3249 (Linker-PBD) structure is shown above the SG3199 released PBD Toxin:

[0332]

[0333] In some embodiments, the antibody conjugate comprises a structure represented by the formula:

[0334] [Compound 5] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0335] In some embodiments, the antibody conjugate comprises a structure represented by the formula:

[0336] [Compound 6] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. In some embodiments, the antibody conjugate comprises a structure represented by the formula:

[0337] [Compound 7] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0338] In some embodiments, the antibody conjugate comprises a structure represented by the formula: [Compound 8] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. In some embodiments, the antibody conjugate comprises a structure represented by the formula:

[0339] [Compound 9] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0340] The “drug-antibody ratio” (DAR) in an antibody conjugate or composition of the invention is defined as the molar ratio between the drug moieties in the conjugate or composition and the antibodies in the conjugate or composition. Where an antibody has more than one site of attachment, more than one drug moiety may be linked to each antibody. In some instances, a mixture is obtained comprising more than one antibody-drug conjugate (ADC) molecules. The drug-antibody ratios of the antibody drug conjugates can be measured by analytical methods known in the art, for example, as described below. In some embodiments, the antibody conjugates have an average DAR of about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 6, or about 2 to about 4.

[0341] Composition

[0342] The present invention also provides a composition which comprises one or more antibody conjugates according to the invention.

[0343] The present invention also provides a pharmaceutical composition which comprises one or more antibody conjugates according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0344] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0345] The pharmaceutical composition may be formulated to be suitable for administration to a patient in order to prevent and / or treat disease. Pharmaceutical compositions can be formulated for administration by different routes, for example, for oral, parenteral, topical, inhalative, intravenous, intramuscular, rectal, sublingual, transdermal, subcutaneous, intratumoural application routes, according to their chemical and physical properties.

[0346] As indicated above, the drug-antibody ratio (DAR) of the antibody conjugates according to the invention may vary. Consequently, the composition may comprise a mixture of antibody conjugates having a number of different DARs, and may therefore have an average DAR which is non-integral.

[0347] Method of treatment

[0348] The invention provides a method of treating or diagnosing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody conjugate according to the invention. The invention provides a method of treating or diagnosing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to the invention. In some embodiments, the disease is cancer. The invention provides an antibody conjugate according to the invention for use as a medicament. The invention provides a pharmaceutical composition according to the invention for use as a medicament.

[0349] The invention provides an antibody conjugate according to the invention for use in a method of treatment and / or diagnosis. The invention provides a pharmaceutical composition according to the invention for use in a method of treatment and / or diagnosis. In some embodiments, the antibody conjugate or pharmaceutical composition according to the invention is for use in a method of treating cancer.

[0350] The invention provides for the use of an antibody conjugate according to the invention for the manufacture of a medicament. The invention provides for the use of pharmaceutical composition according to the invention for the manufacture of a medicament.

[0351] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterised by unregulated cell growth.

[0352] “Tumour” refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions.

[0353] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0354] An "effective amount" as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0355] The term "therapeutically effective amount" refers to an amount of an antibody conjugate, ADC or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumour size; inhibit (i.e. , slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumour metastasis; inhibit, to some extent, tumour growth; inhibit tumour regrowth or recurrence and / or relieve to some extent one or more of the symptoms associated with cancer. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0356] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumour size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumour metastasis; inhibition or an absence of tumour growth; relief of one or more symptoms associated with specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumourigenicity, tumourigenic frequency, or tumourigenic capacity, of a tumour; reduction in the number or frequency of cancer stem cells in a tumour; differentiation of tumourigenic cells to a non-tumourigenic state; or some combination of effects.

[0357] The cancer may express B7-H3. In some embodiments, the expression of B7-H3 is increased compared to the expression of B7-H3 by the same non-cancerous tissue or cells. In some embodiments, B7-H3 is expressed on tumour-associated cells such as vasculature or immune cells.

[0358] An increase refers to an increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is only found in a diseased tissue, while expression in a corresponding healthy tissue is repressed. According to the invention, diseases associated with cells expressing B7-H3 include cancer diseases. Furthermore, according to the invention, cancer diseases preferably are those wherein the cancer cells express B7-H3.

[0359] The cancer may be melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T-cell lymphoma, and 13- cell lymphoma.

[0360] Definitions and abbreviations

[0361] The term "alkyl" by itself or as part of another term refers to a straight chain or branched, saturated hydrocarbon having the indicated number of carbon atoms (e.g., "Ci-Cs" alkyl refers to an alkyl group having from 1 to 8 carbon atoms). Alkyl groups typically comprise from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, and more preferably from 1 to 4 carbon atoms. When the number of carbon atoms is not indicated, the alkyl group has from 1 to 8 carbon atoms. Representative straight chain Ci-Cs alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; while branched Ci-Cs alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tent-butyl, -isopentyl, and -2- methylbutyl; unsaturated C2-C8 alkyls include, but are not limited to, vinyl, allyl, 1-butenyl, 2- butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1 -butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1 -butynyl, 2-butynyl, 1- pentynyl, 2-pentynyl and 3-methyl-1 -butynyl. Reference to “alkyl” herein refers to unsubstituted and substituted moieties as described above.

[0362] The term "alkylene," by itself or as part of another term, refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1- 18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Examples include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene. Reference to “alkylene” herein refers to unsubstituted and substituted moieties as described above.

[0363] An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond.

[0364] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. Up to two heteroatoms may be consecutive. Heteroalkyl groups typically comprise from 1 to 15 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 8 carbon atoms, and most preferably from 1 to 4 carbon atoms. Reference to “heteroalkyl” herein refers to unsubstituted and substituted moieties as described above. Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Reference to “heteroalkylene” herein refers to unsubstituted and substituted moieties as described above.

[0365] An "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.

[0366] The term "aryl" or "aromatic ring" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g. 5 to 10, such as 5, 6 or 10) carbon atoms, more preferably 6 to 10 carbon atoms. These can be arranged in one ring, e.g. phenyl, or two or more condensed rings (e.g. naphthyl). Preferably aryl refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. In some embodiments, the aryl is unsubstituted in some embodiments the aryl is substituted.

[0367] The term "cycloalkyl" as used herein refers to saturated or unsaturated, non-aromatic cycloalkyl comprising 1 , 2 or more rings. Examples include cyclopropyl, cyclo- butyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexonyl, etc.

[0368] The term "acyl" as used herein refers to a functional group with the general formula Rac-C(O)H, wherein Racrefers to an optionally substituted hydrocarbon radical, in particular, a hydrocarbon chain having Ci - Cs carbon atoms.

[0369] The term "alkylsulfonyl" or "arylsulfonyl" refer to alkyl or aryl groups containing a SO2 residue.

[0370] The term "Nitro" refers to the -NO2 radical.

[0371] The following abbreviations are used herein and have the indicated definitions:

[0372] NUMBERED EMBODIMENTS

[0373] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs.

[0374] 1. An antibody conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising an antibody, or an antigen-binding fragment thereof, covalently linked to one or more payload moieties; wherein the antibody, or antigen-binding fragment thereof, comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7.

[0375] 2. The antibody conjugate according to paragraph 1 , wherein the HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3.

[0376] 3. The antibody conjugate according to paragraph 1 or paragraph 2, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto.

[0377] 4. The antibody conjugate according to any one of paragraphs 1 to 3, wherein the antibody, or antigen-binding fragment thereof, comprises a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0378] 5. The antibody conjugate according to paragraph 1 or paragraph 2, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto.

[0379] 6. The antibody conjugate according to any one of paragraphs 1 , 2 and 5, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0380] 7. The antibody conjugate according to paragraph 1 or paragraph 2, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto.

[0381] 8. The antibody conjugate according to any one of paragraphs 1 , 2 and 7, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto. The antibody conjugate according to paragraph 1 or paragraph 2, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto. The antibody conjugate according to any one of paragraphs 1, 2 and 9, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto. The antibody conjugate according to paragraph 1, wherein the HCDR3 comprises an amino acid sequence according to SEQ ID NO: 4. The antibody conjugate according to paragraph 1 or paragraph 11, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto. The antibody conjugate according to any one of paragraphs 1, 11 and 12, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto. The antibody conjugate according to paragraph 1 or paragraph 11, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto. The antibody conjugate according to any one of paragraphs 1, 11 and 14, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto. The antibody conjugate according to any one of the preceding paragraphs, wherein the antibody, or antigen-binding fragment thereof, comprises an Fc region, optionally wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 39 or SEQ ID NO: 40, or a modified Fc region, optionally wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 41 or SEQ ID NO: 42. The antibody conjugate according to any one of the preceding paragraphs, wherein the antibody conjugate comprises an amino acid sequence according to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO 90. The antibody conjugate according to any one of the preceding paragraphs, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein: i. the HC comprises an amino acid sequence according to SEQ ID NO: 44, and the LC comprises an amino acid sequence according to SEQ ID NO: 43; ii. the HC comprises an amino acid sequence according to SEQ ID NO: 46, and the LC comprises an amino acid sequence according to SEQ ID NO: 45; iii. the HC comprises an amino acid sequence according to SEQ ID NO: 48, and the LC comprises an amino acid sequence according to SEQ ID NO: 47; iv. the HC comprises an amino acid sequence according to SEQ ID NO: 50, and the LC comprises an amino acid sequence according to SEQ ID NO: 49; v. the HC comprises an amino acid sequence according to SEQ ID NO: 56, and the LC comprises an amino acid sequence according to SEQ ID NO: 55; vi. the HC comprises an amino acid sequence according to SEQ ID NO: 58, and the LC comprises an amino acid sequence according to SEQ ID NO: 57; vii. the HC comprises an amino acid sequence according to SEQ ID NO: 60, and the LC comprises an amino acid sequence according to SEQ ID NO: 59; viii. the HC comprises an amino acid sequence according to SEQ ID NO: 62, and the LC comprises an amino acid sequence according to SEQ ID NO: 61 ; ix. the HC comprises an amino acid sequence according to SEQ ID NO: 64, and the LC comprises an amino acid sequence according to SEQ ID NO: 63; x. the HC comprises an amino acid sequence according to SEQ ID NO: 66, and the LC comprises an amino acid sequence according to SEQ ID NO: 65; xi. the HC comprises an amino acid sequence according to SEQ ID NO: 68, and the LC comprises an amino acid sequence according to SEQ ID NO: 67; xii. the HC comprises an amino acid sequence according to SEQ ID NO: 70, and the LC comprises an amino acid sequence according to SEQ ID NO: 69; xiii. the HC comprises an amino acid sequence according to SEQ ID NO: 72, and the LC comprises an amino acid sequence according to SEQ ID NO: 71 ; xiv. the HC comprises an amino acid sequence according to SEQ ID NO: 74, and the LC comprises an amino acid sequence according to SEQ ID NO: 73; xv. the HC comprises an amino acid sequence according to SEQ ID NO: 76, and the LC comprises an amino acid sequence according to SEQ ID NO: 75; or xvi. the HC comprises an amino acid sequence according to SEQ ID NO: 78, and the LC comprises an amino acid sequence according to SEQ ID NO: 77.

[0382] 19. The antibody conjugate according to any one of the preceding paragraphs, wherein the antibody, or antigen-binding fragment thereof, is covalently linked to the payload moiety by a linker.

[0383] 20. The antibody conjugate according to paragraph 19, wherein the linker is an enzyme cleavable linker.

[0384] 21 . The antibody conjugate according to paragraph 19 or paragraph 20, wherein the linker comprises a p-D-glucuronide linker or a p-D-galactoside linker.

[0385] 22. The antibody conjugate according to any one of the preceding paragraphs, wherein the payload moiety is selected from a prodrug, a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a coloured agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumour cell growth, promotes tumour cell apoptosis or necrosis.

[0386] 23. The antibody conjugate according to paragraph 22, wherein the payload moiety is a prodrug or a drug.

[0387] 24. The antibody conjugate according to paragraph 22 or paragraph 23, wherein the drug is a topoisomerase I inhibitor, preferably exatecan.

[0388] 25. The antibody conjugate according to paragraph 22 or paragraph 23, wherein the drug is a topoisomerase II inhibitor, preferably PNU-159682.

[0389] 26. The antibody conjugate according to paragraph 22 or paragraph 23, wherein the drug is a tubulin inhibitor, preferably monomethyl auristatin E (MMAE). 27. The antibody conjugate according to paragraph 22 or paragraph 23, wherein the drug is a DNA binding payload, preferably PBD SG3249.

[0390] 28. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure represented by the formula: wherein: the wavy line indicates the covalent attachment to the antibody or antigen-binding fragment thereof via linker, L;

[0391] R is H or an optionally substituted C1-C4 alkyl group, an optionally substituted C1-C4 alkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C1-C4 alkyl carboxy C1-C4 alkyl group, F, Cl, Br, or I, CN, an optionally substituted C1-C4 alkylsulfonyl group, an optionally substituted arylsulfonyl group, or an NRz group, where NRz is selected from H, optionally substituted C1-C4 alkyl group or optionally substituted C1-C4 acyl;

[0392] R1 is H, a C1-C4 alkyl group, or a C1-C4 alkoxy group;

[0393] Y is selected from an optionally substituted C1-C10 alkyl group, or a group having the formula:

[0394] O[C(RA)2] — X3— [C(RA)2]Pwhere o and p are independently from one another selected from an integer of 1 to 20, whereby o and p may be the same integer or a different integer, X3 is i) N, S or O, or ii) an aryl group or a heteroaryl group, wherein [C(RA)2]O and [C(RA)2]Pare present in the meta position of said aryl group or said heteroaryl group, and each RA is independently from one another selected from H or an optionally substituted C1-C4 alkyl group or an optionally substituted C1-C4 acyl group;

[0395] X is O or S; m is an integer from 1 to 20; R2 and R3 are independently selected from H, C1-C20 alkyl, and -Ci-Cs heteroalkyl;

[0396] R4 is selected from H or an electron-withdrawing group, and

[0397] G is a sugar moiety selected from p-D-galactoside, p-D-glucuronide, p-D-glucoside, a- D-mannoside, or fucoside. The antibody conjugate according to paragraph 28, wherein the antibody conjugate comprises a structure having the formula: wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. The antibody conjugate according to paragraph 28, wherein the antibody conjugate comprises a structure having the formula: wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure having the formula:

[0398] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. 32. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure having the formula: wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0399] 33. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure having the formula: wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0400] 34. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure having the formula:

[0401] wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0402] 35. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure having the formula: wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

[0403] 36. The antibody conjugate according to any one of paragraphs 1 to 23, wherein the antibody conjugate comprises a structure having the formula: wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. 37. A pharmaceutical composition comprising the antibody conjugate according to any one of the preceding paragraphs, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0404] 38. The antibody conjugate according to any one of paragraphs 1-36, or the pharmaceutical composition according to paragraph 37, for use as a medicament.

[0405] 39. The antibody conjugate according to any one of paragraphs 1-36, or the pharmaceutical composition according to paragraph 37, for use in the treatment of cancer in a subject.

[0406] 40. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody conjugate according to any one of paragraphs 1-36, or the pharmaceutical composition according to paragraph 37.

[0407] 41. The antibody conjugate or pharmaceutical composition for use according to paragraph 39, or the method according to paragraph 40, wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T-cell lymphoma, and B-cell lymphoma.

[0408] The invention will now be further described by way of examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0409] EXAMPLES

[0410] Example 1 - Generation of anti-B7-H3 antibodies

[0411] BALB / c mice were vaccinated with human (h) 4lg B7-H3 (His-tag) protein to obtain antibody variable genes enriched for B7-H3 binders. Variable regions (VH and VL (kappa)) were amplified, using V-region framework-specific primers, by PCR from mRNA purified from splenic lymphocytes of the immunised mice (Figure 1A). Single chain Fv antibody fragments (scFv) were subsequently generated by overlap extension PCR to create random combinations of the amplified VH / VL genes, tethered by a flexible 3x(Gly4 Ser) linker (Figure 1 B). The amplified scFv genes were cloned into a pHEN phagemid vector and electroporated into TG1 E. coli to generate a library of 2.06 x 107individual clones (Figure 1C). M13KO7 helper phage was used to generate the scFv-expressing phage for serial rounds of selection (Figure 1 D). Two rounds of selection were carried out by panning on hB7-H3 coated immunotubes. After each round of selection, phages bound to B7-H3 protein were eluted and used to infect TG1 cells to generate new clones representing the selected specificities (Figure 1 E).

[0412] Specific enrichment of anti-B7-H3 phage isolated from round 1 and round 2 of library selection was evaluated by monoclonal phage ELISA, using plates coated with either recombinant human B7-H3 His tag protein or a negative control recombinant protein, hPD1-His tag. Absorbance values for binding to hB7-H3-His were plotted against binding to hPD-1-His tag. Anti-His-HRP was used to confirm the presence of the recombinant protein on the ELISA plates. (Figure 2). Cross reactivity of the selected phage-displayed scFvs was determined by monoclonal phage ELISA using plates coated with either recombinant hB7-H3 His tag protein, cynomolgus B7-H3 or mouse B7-H3. Some clones did not react to either protein, a proportion reacted only against human B7-H3, but most bound to both human and cyno B7-H3. No binding to mouse B7-H3 was observed. (Figure 3).

[0413] Example 2 - Selection of candidates

[0414] Bacterial clones shown to produce B7-H3-reactive phage were randomly selected and induced to express as monoclonal scFvs in bacterial supernatant. The crude supernatants were tested by flow cytometry for binding to B7-H3 on HeLa cells. Anti-B7-H3 TE9 scFv, derived from a previous library selection (WO2024 / 009075 A1) was used as a positive control for human B7-H3 specific binding. Staining with 2TY medium alone was used as omission (negative) control. The binding of the scFvs was detected using an anti-myc secondary antibody. ScFvs binding to B7-H3+ HeLa cells was identified by a shift from the omission control (Figure 4).

[0415] Candidates (B7C18 (SEQ ID NO: 83, clone #18) and B7C23 (SEQ ID NO: 84, clone #23)), were chosen from the 26 scFv clones by B7-H3 binding functionality and DNA sequence analysis. B7C18 and B7C23 had identical light chains and differed in only 1 CDR residue (F108Y) and 2 framework residues in VH. B7C18 and B7C23 were reformatted into human lgG1 chimeric monoclonal antibodies with mouse variable regions. Binding specificity of B7C18 and B7C23 chimeric antibodies was determined by flow cytometry, in a panel of B7- H3- expressing human tumour cell lines: A549 (lung adenocarcinoma), LS174T (colorectal), HeLa (cervix), MDA-MB-231 (breast). Goat anti-human IgG-Alexa Fluor 488 was used for detection. The B7C18 and B7C23 chimeric antibodies showed a positive shift in fluorescence as compared to isotype control (MOPC21). No binding was observed to (hB7-H3 negative) mouse 3T3 cells. (Figure 5).

[0416] Cross-reactivity of B7C18 and B7C23 chimeric antibodies to hB7-H3-related molecules was determined by ELISA. ELISAs were performed using recombinant human B7-H3 4lg (R&D 2318-B3), human B7-H3 2lg (V1C1) (R&D 1949-B3), mouse 2lg B7-H3 and cynomolgus B7- H3 (R&D 9426-B3) in addition to other human B7 family members: PD-L1 / B7-H1, PD-L2 / B7- DC, B7-H2 / ICOSL, B7-1 / CD80, B7-2 / CD86, B7-H4, B7-H6, VISTA / B7-H5 / PD-1H. The ELISAs demonstrated superior specificity of B7C18 / B7C23 chimeric antibodies to human B7- H3 above other family members tested (Figure 6).

[0417] Affinity of the B7C18 and B7C23 chimeric antibodies to human 4lg B7-H3 was assessed through SPR, demonstrating sub-nM affinity (Table 8).

[0418] Table 8 - SPR affinities of chimeric antibodies

[0419] Example 3 - Humanization of candidates

[0420] The antibodies were humanized by CDR grafting of murine B7C18 CDRs onto human germline sequences with the closest aligned human heavy chain and light chain frameworks. The humanization process was performed by an in-silico method based on CDR-grafting onto a curated selection of human germline sequences, with additional non-CDR residue back- mutations based on structural models. Parental CDRs were identified according to the IMGT definition of CDRs. At the start of the humanization process, a homology model of the parental variable heavy (VH) and variable light chains (VL) is built in a single chain variable fragment (scFv) format. The modelling is done in four stages: gathering homologous sequences; fold library scanning; loop modelling and finally side chain placement. The resulting model is used to guide the choice of donor or acceptor amino acids during the humanization process.

[0421] The parental VH and VL sequences are aligned with a panel of human germline sequences. The closest matching germlines from two different VH and VL families are selected. A humanization algorithm is then used to select CDR and framework amino acids to graft from the donor parental sequences onto the human acceptor germline sequence with four VH and four VL sequences produced leading to a possible sixteen variants if expressed in combination.

[0422] Sixteen humanized B7C18 lgG1 variants were generated using a combination of 4 heavy and 4 light chain human frameworks. The humanized antibodies were cloned for expression in HEK 293 (human embryonic kidney 293) mammalian cells. Antigen binding was confirmed for all humanized variants by ELISA against recombinant hB7-H34lg protein coated at 1 pg / ml (Figure 7).

[0423] Six clones were selected based on superior protein chemistry, and further triaged by robustness as measured by the ability to bind to antigen after heat treatment. Clones# cAb10375, cAb10381 and cAb10383 all showed superior thermal stability as compared to the parental chimeric IgG (Figure 8); cAb10383 was the best performer. Humanized versions of B7C23 were created by generating the 3 lead hB7C18 candidates as F108Y variants (cAb10375Y, cAb10381Y and cAb10383Y). Of these, cAb10383Y showed the best thermal stability (Figure 9).

[0424] Four humanized clones, cAb10375, cAb10381, cAb10383 and cAb10383Y, were taken forward as humanized lgG1 mAbs generated in CHO cells with LALA mutations to reduce unwanted FcyR interactions. cAb10383-LALA was the best performer, followed closely by its F108Y variant (Figure 10). The monomericity of the purified mAbs cAb10375-LALA, cAb10381-LALA, cAb10383-LALA and cAb10383Y-LALA were 97.2%, 95.1 %, 96.3% and 97.1%, respectively.

[0425] Affinity of the humanized mAbs to human 4lg B7-H3 was assessed through SPR, demonstrating sub-nM affinity (Table 9). Sub-nanomolar affinities were obtained for all humanized variants, comparable to that of Ifinatamab and up to 14-fold higher affinity than that of Vobramitamab. Table 9 - Affinities of humanized anti-B7-H3 antibodies by SPR

[0426] Example 4 - Determination of anti-B7-H3 antibody binding affinity to B7-H3 expressing cells.

[0427] NCI-H1703 cells (non-small cell lung cancer) were obtained from ATCC and cultured in-house as per supplier’s instructions using appropriate complete media (CM) (RPMI + 10% FBS).

[0428] Cells were detached with Accutase at 37 °C / 5% CO2. The reaction was quenched with appropriate pre-warmed CM and cells were harvested by centrifugation (300 ref / 5 min / 4 °C). The supernatant was aspirated, and pelleted cells were resuspended in cold (4 °C) FACS Buffer (PBS + 2% FBS). A cell count was taken by haemocytometer and Trypan Blue staining. The cells were diluted to the appropriate volume to achieve 3 x 105cells / mL using cold FACS buffer and 100 pL cells were added to pertinent wells to clear, Il-bottomed 96-well plates (ThermoFisher), for a seeding density of 3 x 104cells / well. Plates were stored at 4 °C until the addition of antibodies.

[0429] Chimeric anti-B7-H3 antibodies B7C18-LALA, B7C23 and BH6 and humanized anti-B7-H3 antibodies cAb10375-LALA, cAb10381-LALA, cAb10383-LALA and cAb10383Y-LALA were serially diluted in FACS buffer and 100 pL of each sample was added to cells in duplicate. For the negative control, 100 pL FACS buffer was added to cells instead. Assay plates were incubated for 1 h at 4 °C and cells were centrifuged (300 ref 1 5 min 14 °C) and supernatants were decanted. Pelleted cells were washed twice using FACS buffer.

[0430] Secondary antibody (goat anti-human IgG, AF488 - ThermoFisher) was diluted 1 :100 in FACS buffer and added to all wells. Assay plates were incubated for 1 h at 4 °C then cells were harvested by centrifugation (300 ref / 5 min 14 °C) and supernatants were decanted. Pelleted cells were washed twice before being resuspended in 200 pL FACS buffer.

[0431] Samples were analysed using a ThermoFisher Attune NxT flow cytometer. Using a flow rate of 200 pL / min, 5000 events were collected, and the following channels were used to measure cell size, granularity and fluorescence: FSC, SSC, BL-1.

[0432] Results were exported and analysed to obtain geometric means for each sample using FlowJo software. The geometric means of the negative control were subtracted from those of the samples, to remove background cell fluorescence. The data was processed using GraphPad Prism and a non-linear regression ([Agonist] vs. response - variable slope (four parameters)) was applied to obtain ECso values. (Figure 11 and Table 10).

[0433] The binding affinity of chimeric B7C18-LALA, B7C23 and BH6 were compared to humanized cAb 10375- LA LA, cAb10381-LALA, cAb10383-LALA and cAb10383Y-LALA antibodies and the comparator antibodies Ifinatamab (antibody component of Ifinatamab deruxtecan / DS- 7300) and Vobramitamab (antibody component of Vobramitamab duocarmazine / MGC018) against the NCI-H1703 cell line. (Figure 11 and Table 10). Chimeric B7C18-LALA, B7C23 and humanized cAb10375-LALA, cAb10381-LALA, cAb10383-LALA and cAb10383Y-LALA antibodies exhibit sub-nanomolar ECso for B7-H3 on NCI-H1703 cells. Chimeric BH6 and the comparator antibody Vobramitamab do not exhibit saturation on NCI-H1703 cells, so an accurate ECso could not be determined. The comparator antibody Ifinatamab resulted in an ECso above 1 nM on NCI-H1703 cells. Table 10 - Summary of EC50 values for NCI-H1703 cell binding affinity studies.

[0434] Example 5 - Preparation of antibody drug conjugates (ADC) composed of Compound 1

[0435] Chimeric anti-B7-H3 antibodies, B7C18, B7C23 and BH6 were partially reduced with 4, 2.3 or 3 molar equivalents of TCEP, respectively, and conjugated to Compound 1 , in a 6-fold molar excess in the presence 1.5% (v / v) dimethylacetamide (DMA), at pH 7.4 for >16 hours at 30 °C. The resulting ADCs were isolated using Sephadex G-25 resin.

[0436] Chimeric anti-B7-H3 antibody B7C18-LALA was partially reduced with 2.7 molar equivalents of TCEP and conjugated to Compound 1 , in a 20-fold molar excess in the presence of 10% (v / v) dimethylacetamide (DMA), at pH 7.4 for >16 hours at 30 °C. The resulting ADC was isolated using Superdex 200 pg chromatography.

[0437] Humanized anti-B7-H3 antibodies cAb10375-LALA, cAb10383-LALA, and cAb10383Y-LALA were partially reduced with 3.5 molar equivalents of TCEP, while cAb10381 -LALA was partially reduced with 3.0 molar equivalents of TCEP. All antibodies were then conjugated to Compound 1 in a 10-fold molar excess in the presence of 10% (v / v) dimethylacetamide (DMA), at pH 7.4 for >16 hours at 30 °C. The resulting ADCs were isolated using Superdex 200 pg chromatography.

[0438] Ifinatamab-Compound 1 , Vobramitamab-Compound 1 and Isotype-LALA-Compound 1 were partially reduced with 4.1 , 4.2, and 4.4 molar equivalents of TCEP, respectively. All antibodies were then conjugated to Compound 1 in a 10-fold molar excess in the presence of 10% (v / v) dimethylacetamide (DMA), at pH 7.4 for >16 hours at 30 °C. The resulting ADCs were isolated using Superdex 200 pg chromatography.

[0439] Example 6 - Determination of Drug Antibody Ratio (DAR) for ADCs generated from Compound 1.

[0440] Polymer-Linked Reverse-Phase (PLRP) chromatography was applied to characterize average drug-load for cysteine-linked ADCs. Determination of average drug-load is a crucial attribute as it affects the potency and pharmacokinetics of the ADC.

[0441] PLRP analysis of the ADCs was accomplished by chromatography with an Agilent PLRP-S (1000 A, 2.1 x 50 mm, 5 pm) column. Separation of dithiothreitol (DTT)-reduced conjugates via a PLRP column afforded peaks corresponding to unconjugated or drug conjugated antibody light and heavy chains. B7C18-Compound 1 , B7C23-Compound 1 , BH6-Compound 1 , B7C18-LALA-Compound 1 , cAb10375-LALA-Compound 1 , cAb10383-LALA-Compound 1 , cAb10383Y-LALA-Compound 1 were analysed with a 40 min (Buffer A - Water+0.1 % TFA; Buffer B - Acetonitrile + 0.1 % TFA; Gradient - 25% buffer B over 3 min, 25-50% buffer B from 3-28 min, 95% buffer B from 28-31 min, re-equilibration to 25% buffer B from 31-40). cAb10381-LALA-Compound 1 was analysed with a 40 min gradient and 20 min gradient (Buffer A - Water + 0.1 % TFA; Buffer B - Acetonitrile + 0.1 % TFA; 30% buffer B over 0.5 min, 30-50% buffer B from 0.5-15.5 min, 95% buffer B from 15.5-16.5 min, re-equilibration to 30% buffer B from 16.5-20.5 min). Figures 12 - 23 show examples of PLRP chromatograms of chimeric and humanized anti-B7-H3-Compound 1 conjugates.

[0442] Ifinatamab-Compound 1 and Vobramitamab-Compound 1 were analysed with a 20.5 minute method (Buffer A - Water+0.1 % TFA; Buffer B - Acetonitrile + 0.1% TFA; Gradient - 30% Buffer B over 0.5 min, 30-40% buffer B from 0.5-15.5 min, 95% buffer B 15.51-16.51 min, reequilibration to 30% buffer B from 16.52-20.5min. Isotype-LALA-Compound 1 was analysed with a 26.5 minute method (Buffer A - Water+0.1% TFA; Buffer B - Acetonitrile + 0.1% TFA; Gradient - 25% Buffer B over 0.5 min, 25-50% buffer B from 0.5-21.5 min, 95% buffer B 21. SI- 22.51 min, re-equilibration to 25% buffer B from 22.52-26.5min). Figures 50 - 52 show examples of PLRP chromatograms of Ifinatamab-Compound 1 , Vobramitamab-Compound 1 and Isotype-LALA-Compound 1.

[0443] Example 7 - Determination of the aggregation content of ADCs generated from Compound 1.

[0444] Size Exclusion chromatography (SEC) chromatography was used to quantify the amount of monomeric and aggregated species in ADCs. Quantification of the aggregated species is important as they can have a significant impact on the pharmacokinetics and biodistribution of an ADC.

[0445] Separation of aggregated and monomeric ADC species was achieved using a Thermo Scientific MAbPac SEC-1 (5 pm, 7.8 x 300 nm) column. B7C18-Compound 1 , B7C18-LALA- Compound 1 , B7C23-Compound 1 and BH6-Compound 1 were analysed on an Isocratic gradient with 20 mM MES, 150 mM NaCI, 5% (v / v) acetonitrile for 30 min at a flow rate of 0.5 mL / min. cAb10375-LALA-Compound 1 , cAb10381-LALA-Compound 1 , cAb 10383- LA LA- Compound 1 and cAb10383Y-LALA-Compound 1 were analysed on an Isocratic gradient with 20 mM MES, 150 mM NaCI, 5% (v / v) acetonitrile for 22 min at 0.8 mL / min. Figures 24-34 show examples of SEC chromatograms for chimeric and humanized anti-B7-H3-Compound 1 conjugates.

[0446] Example 8 - Preparation of antibody drug conjugate (ADC) composed of Compound 3.

[0447] B7C18-LALA was partially reduced with 4.5 molar equivalents of TCEP, then conjugated to Compound 3 in a 20-fold molar excess in the presence of 10% (v / v) dimethylacetamide (DMA), at pH 7.4 for >16 hours at 30 °C. The resulting ADCs were isolated using Superdex 200 pg chromatography

[0448] Example 9 - Determination of Drug Antibody Ratio (DAR) for ADCs comprised of Compound 3.

[0449] PLRP analysis of the ADCs was accomplished by chromatography with an Agilent PLRP-S (1000 A, 2.1 x 50 mm, 5 pm) column. Separation of dithiothreitol (DTT)-reduced conjugates via a PLRP column afforded peaks corresponding to unconjugated or drug conjugated antibody light and heavy chains. B7C18-LALA-Compound 3 was analysed with a 20.5-minute method (Buffer A - Water+0.1 % TFA; Buffer B - Acetonitrile + 0.1% TFA; Gradient - 30% Buffer B over 0.5 min, 30-40% buffer B from 0.5-15.5 min, 95% buffer B 15.51-16.51 min, Reequilibration to 30% buffer B from 16.52-20.5min. See Figure 35 for example of PLRP chromatogram of B7C18-LALA-Compound 3.

[0450] Example 10 - Preparation of antibody drug conjugates (ADC) composed of Compound 4.

[0451] Chimeric, sortase-tagged, anti-B7-H3 B7C18s and BH6s and a sortase-tagged Isotype antibody, MOPC21s were pH adjusted to pH 8.5 with 0.5 M Tris, 0.025 M EDTA, pH 8.5 and partially reduced with 1.1 , 1.2 and 1 .2 molar equivalents of TCEP, respectively and conjugated to Compound 4, in a 4-fold molar excess in the presence 5% (v / v) dimethylacetamide (DMA), for 1 hour at 20 °C. The conjugation reaction was quenched with 4 molar equivalents of N- acetylcysteine for 30 min at 20 °C. The resulting ADCs were isolated using diafiltration. Example 11 - Determination of Drug Antibody Ratio (DAR) for ADCs comprised of Compound

[0452] 4.

[0453] Hydrophobic interaction chromatography (HIC) was applied to characterize average DAR species for cysteine-linked ADCs. Determination of average drug-load is a crucial attribute as it affects the potency and pharmacokinetics of the ADC.

[0454] HIC analysis of the ADCs was accomplished by HPLC and afforded peaks corresponding to unconjugated antibody or conjugated antibody species from DAR 1 to DAR 8. The areas of the peaks were integrated to determine the average DAR for each ADC. Figures 36-38 show examples of HIC chromatograms of B7C18s-Compound 4, BH6s-Compound 4 and MOPC21s-Compound 4.

[0455] Example 12 - Determination of the aggregation content of ADCs composed of Compound 4.

[0456] Size Exclusion chromatography (SEC) chromatography was used to quantify the amount of monomeric and aggregated species in ADCs. Figures 39-41 show examples of SEC chromatograms for B7C18s-Compound 4, BH6s-Compound 4 and MOPC21s-Compound 4.

[0457] Example 13 - Preparation of antibody drug conjugates (ADC) composed of Compound 5.

[0458] Chimeric, sortase- tagged, anti-B7-H3 B7C18s and BH6s and the Isotype antibody MOPC21s were pH adjusted to pH 8.5 with 0.5 M Tris, 0.025 M EDTA, pH 8.5 and partially reduced with 2.1 , 2.5 or 2.4 molar equivalents of TCEP, respectively and conjugated to Compound 5, in an 8-fold molar excess in the presence 5% (v / v) dimethylacetamide (DMA), for 1 hour at 20 °C. The conjugation reaction was quenched with 8 molar equivalents of N-acetylcysteine for 30 min at 20 °C. The resulting ADCs were isolated using diafiltration.

[0459] Example 14 - Determination of Drug Antibody Ratio (DAR) for ADCs comprised of Compound 5.

[0460] HIC analysis of the ADCs was accomplished by HPLC and afforded peaks corresponding to unconjugated antibody or conjugated antibody species from DAR 1 to DAR 8. The areas of the peaks were integrated to determine the average DAR for each ADC. Figures 42-44 show examples of HIC chromatograms of B7C18s-Compound 5, BH6s-Compound 5 and MOPC21s-Compound 5. Example 15 - Determination of the aggregation content of ADCs composed of Compound 5.

[0461] Size Exclusion chromatography (SEC) chromatography was used to quantify the amount of monomeric and aggregated species in ADCs. Figures 45-47 show examples of SEC chromatograms for B7C18s-Compound 5, BH6s-Compound 5 and MOPC21s-Compound 5.

[0462] Example 16 - Preparation of Ifinatamab deruxtecan comparator (Ifinatamab-Compound 6).

[0463] Ifinatamab antibody was partially reduced with 2.6 molar equivalents of TCEP and conjugated to Compound 6 linker-drug, in a 4.5-fold molar excess in the presence of 20% (v / v) dimethylacetamide (DMA), at pH 7.4 for 1 hour at 30 °C. The resulting ADC (Ifinatamab- Compound 6) was isolated using Sephadex G-25 resin.

[0464] Example 17 - Determination of Drug Antibody Ratio (DAR) for Ifinatamab-Compound 6.

[0465] PLRP analysis of the ADC was accomplished by chromatography with an Agilent PLRP-S (1000 A, 2.1 x 50 mm, 5 pm) column. Separation of dithiothreitol (DTT) reduced conjugate via a PLRP column afforded peaks corresponding to unconjugated or drug conjugated antibody light and heavy chains. A 20 min gradient was used (Buffer A - Water + 0.1% TFA; Buffer B - Acetonitrile + 0.1 % TFA; 30% buffer B over 0.5 min, 30-50% buffer B from 0.5-15.5 min, 95% buffer B from 15.5-16.5 min, re-equilibration to 30% buffer B from 16.5-20.5 min. Figure 48 shows an example of PLRP chromatogram for Ifinatamab-Compound 6.

[0466] Example 18 - Determination of the aggregation content of Ifinatamab-Compound 6.

[0467] Separation of aggregated and monomeric ADC species was achieved using a Thermo Scientific MAbPac SEC-1 (5 pm, 7.8 x 300 nm) column. An Isocratic gradient with 20 mM MES, 150 mM NaCI, 5% (v / v) acetonitrile was applied for 22 min at 0.8 mL / min. Figure 49 shows an example of SEC chromatogram for Ifinatamab-Compound 6.

[0468] Example 19 - Preparation of Vobramitamab-Compound 9 comparator

[0469] Vobramitamab-Compound 9 was partially reduced with 1.5 molar equivalents of TCEP. The antibody was then conjugated to Compound 9 (MedChem Express, HY-128957) in a 20-fold molar excess in the presence of 20% (v / v) dimethylacetamide (DMA), at pH 7.4 for >16 hours at 30 °C. The resulting ADC was isolated using a NAP-5 column (Cytiva, 17-0853-01).

[0470] Example 20 - Determination of Drug Antibody Ratio (DAR) for Vobramitamab-Compound 9.

[0471] PLRP analysis of the ADCs was accomplished by chromatography with an Agilent PLRP-S (1000 A, 2.1 x 50 mm, 5 pm) column. Separation of dithiothreitol (DTT)-reduced conjugates via a PLRP column afforded peaks corresponding to unconjugated or drug conjugated antibody light and heavy chains. Vobramitamab-Compound 9 was analysed with a 20.5 minute method (Buffer A - Water+0.1 % TFA; Buffer B - Acetonitrile + 0.1% TFA; Gradient - 30% Buffer B over 0.5 min, 30-40% buffer B from 0.5-15.5 min, 95% buffer B 15.51-16.51 min, Reequilibration to 30% buffer B from 16.52-20.5min. Figure 53 shows an example of PLRP chromatogram for Vobramitamab-Compound 9.

[0472] Example 21 - Determination of B7-H3 expression levels on cancer cell lines using Flow Cytometry

[0473] The expression of B7-H3 in eight cancer cell lines was evaluated by flow cytometry using Phycoerythrin (PE)-conjugated anti-B7-H3 antibody. Phycoerythrin-conjugated B7C18-LALA antibody was prepared using the PE I R-Phycoerythrin Conjugation Kit - Lightning-Link® kit (Abeam, ab102918) following manufacturer’s instructions. The cell lines tested were A498 (kidney), OVCAR-3 (ovarian), Calu-6 (lung), A375.S2 (melanoma), NCI-H1703 (NSCLC), MDA-MB-231 (TNBC), BxPC-3 (pancreatic) and DMS79 (SCLC).

[0474] Cells were detached with Accutase at 37 °C I 5% CO2. The reaction was quenched with appropriate pre-warmed CM and cells were harvested by centrifugation (300 ref / 5 min I 37 °C). The supernatant was aspirated, and pelleted cells were re-suspended in FACS buffer. A cell count was taken by haemocytometer and Trypan Blue staining. Cell suspension was diluted with the appropriate volume of FACS buffer to achieve 3 x 105cells / mL. 100 pL cell suspension was added to pertinent wells of a clear, U-bottomed 96-well plate (ThermoFisher).

[0475] B7C18-LALA-PE was diluted to 40 pg / mL (x2 concentration) in FACS buffer and 100 pL was added in triplicate to pertinent wells at a final concentration of 20 pg / mL B7C18-LALA-PE. For the negative control, 100 pL FACS buffer was added to cells instead. Assay plates were incubated at 4 °C for 1 h, and afterwards were washed in FACS buffer twice. Cells were resuspended in 200 pL FACS buffer for flow cytometry analysis. BD Quantibrite™ PE beads (BD Biosciences, 340495) were reconstituted with 400 pL PBS and analysed following the manufacturer’s instructions.

[0476] The PE geometric means were exported and the Log10 values were calculated. Log10 values were also calculated for the number of PE molecules per bead, based on lot-specific values, provided by the manufacturer. A linear regression of Log10 values for PE geometric means against the number of PE molecules per bead was generated. To determine PE molecules per cell, Log10 PE geometric means were substituted into the equation and the anti-Log was determined. The expression of B7-H3 in 4 additional cancer cell lines: MEL202 (uveal melanoma), IGROV-

[0477] I (ovarian), SK-OV-3 (ovarian) and Caski (cervical), was evaluated using the BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation Kit, according to the manufacturer’s instructions. Cells were treated with a commercial anti-B7-H3-PE-labelled antibody, alongside an Isotype- PE control. Standard PE-labelled beads with known quantities of PE molecules were used to benchmark. Flow cytometric analysis was evaluated using a Novocyte Quanteon (Agilent), configured to PE fluorophore. The Geometric Mean Fluorescence Intensity (GMFI) was calculated using FlowJo™. A linear equation was extracted and extrapolated with sample GMFIs. The Isotype-PE value was subtracted from the anti-B7-H3-PE value to give the number of specific anti-B7-H3-PE-conjugated antibodies bound (antigen density). See Table

[0478] I I for a list of cell lines and estimated PE molecules / cell.

[0479] As shown in Table 11 , cell lines of a range of tumour indications express B7-H3 from 219,801 PE molecules per cell for A498, to 5,500 PE molecules per cell for DMS79, Calu-6, NCI- H1703, A375, MEL202 and IGROV-1 were used for in vitro and in vivo studies. Table 11 - PE molecules per cell on B7-H3 positive cancer cell lines. Example 22 - Determination of ADC in vitro activity

[0480] OVCAR-3 (ovarian), Calu-6 (lung), A375.S2 (melanoma), NCI-H1703 (NSCLC), MDA-MB-231 (TNBC), BxPC-3 (pancreatic) and DMS79 (SCLC) cells (obtained from the ATCC) were detached by trypsinisation at 37 °C / 5% CO2, diluted in appropriate pre-warmed complete media (CM) (RPMI + 10% FBS), and harvested by centrifugation (300 ref / 5 min i 37 °C).

[0481] The supernatant was aspirated, and pelleted cells were resuspended in appropriate CM. A cell count was taken by haemocytometer and T rypan Blue staining, and an appropriate volume of cells at 3 x 104cells / well (Calu-6, NCI-H1703, A375.S2, MDA-MB-231 and DMS79) or 5 x 104cells / well (OVCAR-3 and BxPC-3).

[0482] To white, clear-bottomed microplates (Corning), 100 pL of cells were added to an appropriate number of wells, resulting in a seeding density of 3000 cells / well (Calu-6, NCI-H1703, A375.S2, MDA-MB-231 and DMS79) or 5000 cells / well (OVCAR-3 and BxPC-3), and plates were incubated at 37 °C / 5% CO2 for 24 h. To three wells, only appropriate CM was added (negative control) for subsequent normalisation of data.

[0483] After 24 h, Ifinatamab-Compound 6 and Compound 1 conjugated ADCs comprised of chimeric anti-B7-H3 antibodies B7C18-LALA, B7C23 and BH6 and humanized anti-B7-H3 antibodies cAb 10375- LA LA, cAb10381-LALA, cAb10383-LALA, cAb10383Y-LALA, comparator antibodies Ifinatamab and Vobramitamab, and a non-binding isotype control were serially diluted in appropriate pre-warmed CM in a 96-well plate and 20 pL of each dilution added to appropriate wells in triplicate. Three wells of adhered cells had no compound added, only 20 pL CM, to act as a positive control for subsequent normalisation of data. 20 pL CM was added to negative control wells also.

[0484] After 24 h, for the evaluation of Vobramitamab-Compound 9 in the presence of unconjugated Vobramitamab antibody in H1703 cells, a fixed dilution of component antibody was prepared in appropriate warmed CM. 20 pL of antibody diluent was added to appropriate 96-well plate wells in triplicate and incubated (37 °C, 5 % CO2, 30 min). Vobramitamab-Compound 9 was serially diluted in appropriate pre-warmed CM in a 96-well plate. 20 pL of each dilution added to appropriate wells in triplicate in the presence or absence of unconjugated Vobramitamab antibody, following incubation. Three wells of adhered cells had no compound added, only 20 pL CM, to act as a positive control for subsequent normalisation of data. 20 pL CM was added to negative control wells also. Plates were incubated for 120 h at 37 °C I 5% (Calu-6, NCI-H1703, A375.S2, MDA-MB-231 and DMS79) or 144 h at 37 °C I 5% (OVCAR-3 and BxPC-3) before luminescence measurement using Cell Titre Gio reagent (Promega). The reagent was pre-warmed for 30 min at room temperature (RT) before use and was reconstituted as per manufacturer’s instructions. To wells of interest, 120 pL of reagent was added and covered with an adhesive seal. Plates were orbitally shaken for 1 min at 800 rpm and left to rest for 20 min, protected from light, before being orbitally shaken again and read on the luminometer.

[0485] Data were normalised to cells alone (100%) and CM alone (0%) using GraphPad Prism. IC50 values were estimated using non-linear regression ([Inhibitor] vs. response - Variable slope (four parameters)). (Figures 54-60 and Tables 12-16).

[0486] B7C18-Compound 1 , B7C23-Compound 1 and BH6-Compound 1 showed potent responses on cancer cells (Figure 54 and Table 12). B7C18-Compound 1 is ~2-fold more potent in vitro than B7C23-Compound 1 and between 24.6 and 86.8-fold more potent than BH6-Compound 1.

[0487] B7C18-LALA-Compound 1 showed potent response on cancer cells (Figure 55 and Table 13). Potency was greatest against NCI-H1703 (99k PE molecules / cell) at IC500.006 nM and lowest against MDA-MB-231 at IC50 0.587 nM (39.6k PE molecules / cell). cAb10375-LALA-Compound 1 , cAb10381-LALA-Compound 1 , cAb10383-LALA-Compound 1 , cAb10383Y-LALA-Compound 1 and B7C18-LALA-Compound 1 exhibited comparable potency on NCI-H1703 between IC50 values of 0.03 and 0.16 nM. (Figure 56 and Table 14).

[0488] Comparison of antibodies conjugated to Compound 1 showed that ADCs composed of humanized antibodies cAb10383-LALA and cAb10381-LALA showed improved cytotoxic activity over Ifinatamab-Compound 1 and Vobramitamab-Compound 1 comparators in H1703 and Calu 6 cells (see Figures 57-58 and Table 15). cAb10383Y-LALA-Compound 1 ADC composed of humanized cAb10383Y antibody also exhibits improved cytotoxic activity in comparison to Ifinatamab-Compound 1 and Vobramitamab-Compound 1 in Calu 6 cells (see Figure 58 and Table 15). Humanized antibodies showed improved delivery of cytotoxic Compound 1 to cancer cells over Ifinatamab and Vobramitamab antibodies.

[0489] Ifinatamab-Compound 6 exhibited no activity against Calu-6 and NCI-H1703 cell lines where chimeric B7C18-LALA-Compounfd 1 and humanized cAb10375-LALA-Compound 1 , cAb10381-LALA-Compound 1 , cAb10383-LALA-Compound 1 and cAb10383Y-LALA- Compound 1 exhibited sub-nanomolar IC50 against both models (Figure 59). Vobramitamab-Compound 9 has IC50 of 21.78 nM in H1703 cells and therefore shows lower cytotoxic activity than ADCs composed of humanized antibodies and Compound 1 (cAb10383-LALA and cAb 10381 -LA LA) in the same cell line see Figures 60 and 57, and Tables 15-16. Table 12 - IC50 values for OVCAR-3 and BxPC3 cell viability studies

[0490] Table 13 - IC50 values for B7C18-LALA-Compound 1 cell viability studies. Table 14 - IC50 values for humanized anti-B7-H3 ADCs conjugated to Compound 1.

[0491] Table 15 - IC50 values for anti-B7-H3 ADC conjugated to Compound 1 in H1703 and Calu-6 cells.

[0492] Table 16 - IC50 values for Vobramitamab-Compound 9 in H1703 cell viability study.

[0493] Example 23 - Determining ADC activity of anti- B7-H3-ADCs comprised of Compound 1 and Compound 3 in B7-H3 + / - rhabdomyosarcoma cells, cervical cancer cells and in ovarian cancer cells.

[0494] Target cells, RH30 (rhabdomyosarcoma cell line), RH30 KO (RH30 rendered B7-H3-negative by Crispr-Cas9 technology), ME180 (cervical squamous cell carcinoma cell line), IGROV-1 , (ovarian carcinoma cell line) and SK-OV-3 (ovarian carcinoma cell line) were evaluated. Cells were harvested and plated in appropriate medium with +10% FBS, in tissue culture treated 96 well plates, using conditions suitable for each cell line; RH30 and M E180 cells were trypsinised and seeded at 5,000 cells / well. IGROV-1 and SK-OV-3, were harvested with Accutase (Sigma-Aldrich) for 5 minutes at 37°C and seeded at 2,500 and 2,000 cells / well respectively.

[0495] Plates were incubated overnight at 37°C / 5% CO2 to allow attachment of the cells. Medium from the attached cells was aspirated and replaced with 100 pL of serially diluted ADC / Antibody in triplicate wells, starting from 10 ug / ml as top concentration. Cells were exposed to treatment for 6 days (-144 h) at 37 °C I 5% CO2. Medium from each well was aspirated and replaced with cell medium containing PrestoBlue® Cell Viability Reagent (ThermoFisher). For IGROV-1 and SK-OV-3, the medium was not removed prior to ADC treatment or prior to addition of PrestoBlue™. Instead, ADC (serially diluted) and PrestoBlue™ (for a final 1 in 10 dilution) was added directly to the plates. After incubation at 37°C 15% CO2 for 1 hour, fluorescence readings were measured in a VarioSkan plate reader Excitation (nm) 560, Emission (nm) 590, according to manufacturer’s recommendations. Percentage cell viability was determined relative to untreated cells alone (100%) and mean values ± standard deviations were plotted. Data were normalised to cells alone (100%) using GraphPad Prism. IC50 values were estimated using non-linear regression ([Inhibitor] vs. response - Variable slope (four parameters)).

[0496] Table 17 and Figure 61 show pM potency killing of B7-H3 positive cell lines with all the humanized and chimeric B7C18-derived antibodies when conjugated to Compound 1. Relative potency of killing RH30 / RH30KO cells by the 4 humanized antibodies was: 588:1 (cAb10375-LALA-Compound 1); 1 ,125:1 (cAb10381-LALA-Compound 1); 1 ,367:1 (cAb10383-LALA-Compound 1) and 1 ,133:1 (cAb10383Y-LALA-Compound 1). No killing was observed with free antibody.

[0497] The B7C18-LALA-Compound 3 showed much lower potency than B7C18-LALA-Compound 1 , having an IC50 of 3.9 nM vs 0.008 nM (conjugated payload), and was only effective on the ME180 cell line.

[0498] Table 17 - Summary of IC50 values for humanized anti-B7-H3 ADCs conjugated to Compound 1 and in B7-H3 + / - rhabdomyosarcoma and cervical cancer models.

[0499] Table 18 and Figure 62 show similar pM potency killing of IGROV-1 ovarian cancer cells with humanized (cAb10383-LALA-Compound 1) and chimeric (B7C18-LALA-Compound 1) B7C18- derived antibodies when conjugated to Compound 1.

[0500] Table 18 - Summary of IC50 values for humanized and chimeric anti-B7-H3 ADCs conjugated to Compound 1 in ovarian cancer model.

[0501] Table 19 and Figure 63 show pM potency killing of SK-OV-3 ovarian cancer cells with Compound 1 ADC (cAb10383-LALA-Compound 1), compared to little or no killing for Compound 3 (B7C18-LALA-Compound 3), and a respective IC50 of 0.013 nM vs >680 nM (conjugated payload). The limited activity of B7C18-LALA-Compound 3 is most likely due to lack of SK-OV-3 cell sensitivity to Compound 3 payload. B7C18-LALA-Compound 3 shows more potent activity with IC50 of 0.14 nM (conjugated payload) on MEL202 cells (see Figure 64 and Table 20).

[0502] Table 19 - Summary of IC50 values for humanized and chimeric anti-B7-H3 ADCs conjugated to Compound 1 and 3 in ovarian cancer model.

[0503] Example 24 - Determining ADC activity of anti- B7-H3-ADCs comprised of Compound 1 and Compound 3 in uveal melanoma.

[0504] ME202 (uveal melanoma) cells were harvested with Accutase (Sigma-Aldrich) for 5 minutes at 37°C and dispersed using a pipette to achieve a single-cell suspension. Cells were counted using the Countess Automated Cell Counter, and 5,000 cells / well (c / w) in 10OpI culture media were allowed to attach to a flat-bottom 96-well plate at 37°C for 24 hours. The plate was then treated with 1 / 10 serially diluted ADC in triplicate, starting from 10pg / ml as the highest concentration, with the final column left as blank (no treatment). Following incubation at 37°C for 144 hours, PrestoBlue™ Cell Viability Reagent (Invitrogen) was added directly to the plate to a final concentration of 10%. Next, the plates were incubated for 1 hour at 37°C, before fluorescence (Excitation: 560nm, Emission: 590nm) was measured, using a VarioSkan™ LUX multi-mode microplate reader.

[0505] Table 20 and Figure 64 show pM potency killing of MEL202 uveal melanoma cells with Compound 1 ADC (cAb10383-LALA-Compound 1) which was 10.8x more potent than Compound 3 ADC (B7C18-LALA-Compound 3) having a respective IC50 of 0.013 nM vs 0.14 nM (conjugated payload).

[0506] Table 20 - Summary of IC50 values for humanized and chimeric anti-B7-H3 ADCs conjugated to Compound 1 and 3 in uveal melanoma model.

[0507] Example 25 - Determining ADC activity of anti- B7-H3-ADCs comprised of Compound 1 and Compound 4 in cervical cancer cells.

[0508] Caski (cervical carcinoma) cells were harvested with Accutase (Sigma-Aldrich) for 5 minutes at 37°C and were dispersed using a pipette to achieve a single-cell suspension. Cells were counted using the Countess Automated Cell Counter, and 2,500 cells / well in 100pl culture media were allowed to attach to a flat-bottom 96-well plate at 37°C for 24 hours. The plate was then treated with serially diluted ADC in triplicate, starting from 10pg / ml as the highest concentration, with the final column left as blank (no treatment). Following incubation at 37°C for 144 hours, PrestoBlue™ Cell Viability Reagent (Invitrogen) was added directly to the plate to a final concentration of 10%. Next, the plates were incubated for 1 hour at 37°C, before fluorescence (Excitation: 560nm, Emission: 590nm) was measured, using a VarioSkan™ LUX multi-mode microplate reader.

[0509] Table 21 and Figure 65 show pM potency killing of Caski cervical cancer cells with the humanized Compound 1 ADC (cAb10383-LALA-Compound 1), which was 13.2 x more potent than the chimeric ADC (B7C18s-Compound 4), having a respective IC50 of 0.0094 nM vs 0.124 nM (conjugated payload).

[0510] Table 21 - Summary of IC50 values for humanized and chimeric anti-B7-H3 ADCs conjugated to Compound 1 and Compound 4 in cervical cancer model.

[0511] Example 26 - Determining ADC in vitro activity of anti-B7-H3 ADCs conjugated to Compound 4.

[0512] HeLa (cervical adenocarcinoma), BxPc-3 (pancreatic adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), A549 (lung adenocarcinoma), LS147T (colorectal adenocarcinoma) and SW1222 (colorectal adenocarcinoma) were trypsinised and seeded at 5,000 cells / well in appropriate medium with +10% FBS, in wells of tissue culture treated 96 well plates. Plates were incubated overnight at 37 °C / 5% CO2 to allow attachment of the cells. Medium from the attached cells was aspirated and replaced with 100 pL of serially diluted anti-human B7- H3 chimeric antibodies (B7C18s and BH6s) or isotype control (MOPC21s) or their derived ADCs bearing Compound 4 (PBD payload SG3249; Tesirine) in duplicate wells. Cells were exposed to treatment for 6 days (-144 h) at 37 °C / 5% CO2. Medium from each well was aspirated and replaced with cell medium containing PrestoBlue® Cell Viability Reagent (ThermoFisher) followed by incubation at 37 °C / 5% CO2 for 1 hour before fluorescence readings were measured in a plate reader according to manufacturer’s recommendations. Percentage cell viability was determined relative to untreated cells alone (100%) and mean values ± standard deviations were plotted. Data were normalised to cells alone (100%) using GraphPad Prism. IC50 values were estimated using non-linear regression ([Inhibitor] vs. response - Variable slope (four parameters)).

[0513] Superior killing by B7C18s-Compound 4 compared with BH6s-Compound 4 and MOPC21s- Compound 4 was observed on different cancer cell lines (HeLa, BxPC-3, MDA-MB-231 , A549, LS147T and SW1222). No cell killing was observed with unconjugated antibodies (see Figure 66 and Table 22).

[0514] Table 22 - IC50 values for chimeric anti-B7-H3 ADCs conjugated to Compound 4.

[0515] Und = Undetermined.

[0516] Example 27 - Determination of anti-B7-H3 ADC binding affinity to B7-H3 positive cells.

[0517] NCI-H1703 cells (NSCLC) were obtained from ATCC and cultured in-house as per supplier’s instructions using appropriate complete media (CM) (RPMI + 10% FBS). Cells were detached with Accutase at 37 °C / 5% CO2. The reaction was quenched with appropriate pre-warmed CM and cells were harvested by centrifugation (300 ref / 5 min / 4 °C). The supernatant was aspirated, and pelleted cells were resuspended in cold (4 °C) FACS Buffer (PBS + 2% FBS). A cell count was taken by haemocytometer and Trypan Blue staining. The cells were diluted to the appropriate volume to achieve 3 x 105cells / mL using cold FACS buffer and 100 pL cells were added to pertinent wells of clear, Il-bottomed 96-well plates (ThermoFisher), for a seeding density of 3 x 104cells / well. Plates were stored at 4 °C until the addition of antibodies.

[0518] B7C18-LALA and B7C18-LALA-Compound 1 were serially diluted in FACS buffer and 100 pL of each sample was added to cells. For the negative control, 100 pL FACS buffer was added to cells instead. Assay plates were incubated for 1 h at 4 °C then cells were harvested by centrifugation (300 ref / 5 min / 4 °C) and supernatants decanted. Pelleted cells were washed twice using FACS buffer.

[0519] Secondary antibody (goat anti-human IgG, AF488 - ThermoFisher) was diluted 1 :100 in FACS buffer and added to all wells. Assay plates were incubated for 1 h at 4 °C then cells were harvested by centrifugation (300 ref / 5 min / 4 °C) and supernatants decanted. Pelleted cells were washed twice, before being resuspended in 200 pL FACS buffer.

[0520] The assay plate was analysed on a ThermoFisher Attune NxT flow cytometer. Using a flow rate of 200 pL / min, 5000 events were collected, and the following channels were used to measure cell size, granularity and fluorescence: FSC, SSC, BL-1.

[0521] Results were exported from the Attune and analysed to obtain geometric means for each sample using FlowJo software. The geometric means of the negative control were subtracted from those of the samples, to remove background fluorescence from the cells. The data was processed using GraphPad Prism and a non-linear regression ([Agonist] vs. response - variable slope (four parameters)) was applied to obtain EC50 values. (Figure 67 and Table 23).

[0522] B7C18-LALA-Compound 1 and B7C18-LALA have comparable affinity against NCI-H1703 cell line (99k molecules / cell) indicating that conjugation of Compound 1 does not interfere with antigen recognition. (Figure 67 and Table 23).

[0523] Table 23 - shows a summary of EC50 values for NCI-H1703 cell affinity studies.

[0524] Example 28 - In vivo efficacy studies in Calu-6 xenograft model

[0525] B7C18-LALA-Compound 1 was evaluated in female CB17 SCID mice bearing Calu-6 xenografts. Mice were subcutaneously inoculated into the right flank with 1 x 107Calu-6 cells in 0.2 mL of DPBS mixed 1 :1 with BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution (30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v)) when the average tumour volume reached approximately 149 mm3. Conjugate doses of 0.1 , 0.3 or 1 mg / kg were used for the Calu-6 xenograft study. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (Figure 68). The tumour size was then used for calculations of TGI (%) values (Table24). TGI, representing antitumour effectiveness, was calculated using the formula TGI (%)=[1 -(Vtreat-t- Vtreat-i ) / (Vcontroi-t-Vcontroi-i)]x100, where Vtreat-i and Vcontroi-1 are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3. Body weight was also measured thrice weekly as a measure of compound toxicity (Figure 69).

[0526] The in vivo effect of B7C18-LALA-Compound 1 on Calu-6 tumour xenograft is shown in Figure 68 and Table 24. B7C18-LALA-Compound 1 induced substantial tumour growth inhibition (79.8, 107.43, 109.16% TGI, respectively) at 0.1 , 0.3 and 1 mg / kg with no observable toxicity. Comparator ADCs require 10 mg / kg Q2Wx 2 and 10 mg / kg single dose, respectively, to have a comparable response to the 1 mg / kg B7C18-LALA-Compound 1 dose in a Calu-6 CDX model (Yamato, et al., 2022 and Scribner, et al., 2020).

[0527] Table 24 - shows Tumour Growth Inhibition (TGI) of ADCs tested in Calu-6 xenograft model on day 24 of the study.

[0528] Example 29 - In vivo efficacy studies in NCI-H1703 xenograft model

[0529] B7C18-LALA-Compound 1 was evaluated in female CB17 SCID mice bearing NCI-H1703 xenograft. Mice were subcutaneously inoculated into the right flank with 1 x 107NCI-H1703 cells in 0.2 mL of DPBS containing 50% BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution comprising 30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v) when the average tumour volume reached approximately 157 mm3. Conjugate doses of 1 , 3 and 10 mg / kg were used. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (Figure 70). The tumour size was then used for calculations of TGI (%) values (Table 25). TGI, representing antitumour effectiveness, was calculated using the formula TGI (%)=[1-(Vtreat-t-Vtreat-i) / (Vcontroi-t-Vcontroi- i)]* 100, where Vtreat-i and Vcontroi-i are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3. Body weight was also measured thrice weekly as a measure of compound toxicity (Figure71).

[0530] The in vivo effect of B7C18-LALA-Compound 1 on NCI-H1703 tumour xenograft is shown in Figures 70 and Table 25. B7C18-LALA-Compound 1 induced substantial tumour growth inhibition (107.8, 109.1 and 109.6 TGI, respectively) at 1 , 3 and 10 mg / kg with no observable toxicity based on body weight. Table 25 - shows Tumour Growth Inhibition (TGI) of ADCs tested in NCI-H1703 xenograft model on day 22 of the study.

[0531] Example 30 - In vivo efficacy studies in A375 xenograft model

[0532] B7C18-LALA-Compound 1 was evaluated in female CB17 SCID mice bearing A375 xenograft. Mice were subcutaneously inoculated into the right flank with 5 x 106A375 cells in 0.2 mL of DPBS containing 50% BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution comprising 30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v) when the average tumour volume reached approximately 173 mm3. Conjugate doses of 1 , 3 and 10 mg / kg were used. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (Figure 72). The tumour size was then used for calculations of TGI (%) values (Table 26). TGI, representing antitumour effectiveness, was calculated using the formula TGI (%)=[1-(Vtreat-t-Vtreat-i) / (Vcontroi-t-Vcontroi-i)]x100, where Vtreat-i and Vcontroi-i are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3. Body weight was also measured thrice weekly as a measure of compound toxicity (Figure 73).

[0533] The in vivo effect of B7C18-LALA-Compound 1 on A375 tumour xenograft is shown in Figure 72 and Table 26. B7C18-LALA-Compound 1 induced substantial tumour growth inhibition (45.7, 84.9 and 98.6% TGI, respectively) at 1 , 3 and 10 mg / kg with no observable toxicity.

[0534] Table 26 - shows Tumour Growth Inhibition (TGI) of ADCs tested in A375 xenograft model on day 17 of the study. Example 31 - In vivo efficacy studies in NCI-H1703 xenograft model cAb10383-LALA-ACTK-Compound 1 and Ifinatamab-Compound 6 were evaluated in female CB17 SCID mice bearing NCI-H1703 xenograft. Mice were subcutaneously inoculated into the right flank with 1 x 107NCI-H1703 cells in 0.2 mL of DPBS containing 50% BD Matrigel. T umour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution comprising 30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v) when the average tumour volume reached approximately 141 mm3. Conjugate doses of 0.1 and 0.3 mg / kg were used for cAb10383-LALA-ACTK- Compound 1 and 3 mg / kg for Ifinatamab-Compound 6. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (Figure 74). The tumour size was then used for calculations of TGI (%) values (Table 27). TGI, representing antitumour effectiveness, was calculated using the formula TGI (%)=[1 -(Vtreat-t-Vtreat-i) / (Vcontroi-t-Vcontroi-i)]x100, where Vtreat-i and Vcontroi-1 are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3.

[0535] The in vivo effect of cAb10383-LALA-ACTK-Compound 1 and Ifinatamab-Compound 6 on NCI-H1703 tumour xenograft is shown in Figure 74 and Table 27. cAb10383-LALA-ACTK- Compound 1 induced substantial tumour growth inhibition (25.97% and 72.22% TGI, respectively) at 0.1 and 0.3 mg / kg doses. Comparator ADC Ifinatamab-Compound 6 required 3 mg / kg to reach comparative TGI% as 0.1 mg / kg cAb10383-LALA-ACTK-Compound 1 (29.68% TGI).

[0536] Table 27 - Shows Tumour Growth Inhibition (TGI) of ADCs tested in NCI-H1703 xenograft model on day 20 of the study.

[0537] Example 32 - In vivo efficacy studies in Calu-6 xenograft model

[0538] B7C18-LALA-Compound 1 , cAb10383-LALA-Compound 1 and Ifinatamab-Compound 6 were evaluated in female CB17 SCID mice bearing Calu-6 xenografts. Mice were subcutaneously inoculated into the right flank with 1 x 107Calu-6 cells in 0.2 mL of DPBS mixed 1 :1 with BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution (30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v)) when the average tumour volume reached approximately 168 mm3. Conjugate doses of 0.1 , 0.3 or 3 mg / kg were used for the Calu-6 xenograft study. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (Figure 75). The tumour size was then used for calculations of TGI (%) values (Table 28). TGI, representing antitumour effectiveness, was calculated using the formula TGI (%)=[1-(Vtreat-t-Vtreat-i) / (Vcontroi-t-Vcontroi-i)]x100, where Vtreat-i and Vcontroi-i are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3.

[0539] The in vivo effect of B7C18-LALA-Compound 1 , cAb10383-LALA-Compound 1 and Ifinatamab-Compound 6 on Calu-6 tumour xenograft is shown in Figure 75 and Table 28. B7C18-LALA-Compound 1 and cAb10383-LALA-Compound 1 induced substantial and comparative tumour growth inhibition at 0.1 and 0.3 mg / kg (95.44, 105% and 81.85 and 106.67%, respectively). Comparator ADC Ifinatamab-Compound 6 required 3 mg / kg single dose, respectively, to have a comparable response to the 0.3 mg / kg B7C18-LALA-Compound 1 and cAb10383-LALA-Compound 1 doses in a Calu-6 CDX model.

[0540] Table 28 - shows Tumour Growth Inhibition (TGI) of ADCs tested in Calu-6 xenograft model on day 27 of the study.

[0541] Example 33 - In vivo efficacy studies in DMS79 xenograft model

[0542] B7C18-LALA-Compound 1 , cAb10383-LALA-Compound 1 and Ifinatamab-Compound 6 were evaluated in female CB17 SCID mice bearing DMS79 xenograft. Mice were subcutaneously inoculated into the right flank with 1 x 107DMS79 cells in 0.2 mL of DPBS containing 50% BD Matrigel. Tumour-bearing mice were randomized into groups of 5 animals each and treated with a single intravenous dose of ADC or alternatively with a vehicle solution comprising 30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v) when the average tumour volume reached approximately 160 mm3. Conjugate doses of 0.3, 1 , 3 and 10 mg / kg were used. Tumour size was measured thrice weekly in two dimensions using a calliper, and the volume was expressed in mm3using the formula: V = 0.5 a x b2where a and b are the long and short diameters of the tumour, respectively (Figure 76). The tumour size was then used for calculations of TGI (%) values (Table 29). TGI, representing antitumour effectiveness, was calculated using the formula TGI (%)=[1-(Vtreat-t-Vtreat-i) / (Vcontroi-t-Vcontroi-i)]x100, where Vtreat-i and Vcontroi-i are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animals were euthanized when tumour volumes reached 2000 mm3.

[0543] The in vivo effect of B7C18-LALA-Compound 1 , cAb10383-LALA-Compound 1 and Ifinatamab-Compound 6 on DMS79 tumour xenograft is shown in Figure 76 and Table 29. B7C18-LALA-Compound 1 induced substantial tumour growth inhibition (45.57, 82.34, 93.33, 99.97% TGI, respectively) at 0.3, 1 , 3 and 10 mg / kg. cAb10383-LALA-Compound, at 3 mg / kg, induced substantial tumour growth inhibition (95.59%), comparable to B7C18-LALA- Compound 1 at the same dose level (93.33%). Comparator ADC Ifinatamab-Compound 6 at 3 mg / kg induced 79.06% TGI, comparable to B7C18-LALA-Compound 1 at 1 mg / kg. Table 29 - shows Tumour Growth Inhibition (TGI) of ADCs tested in DMS79 xenograft model on day 22 of the study.

[0544] Example 34 - In vivo efficacy studies in MEL202 and IGR0V1 xenograft models

[0545] The cAb10383-LALA-Compound 1 was evaluated in mice bearing MEL202 or IGROV-1 xenografts. For MEL202 xenografts, female NSG mice were subcutaneously inoculated into the right flank with 1 x 106MEL202 cells in 0.1 mL of DPBS containing 50% BD Matrigel on the right flank, and 2 x 106MEL202 cells in 0.1 ml of DPBS on the left flank. When combined tumour volumes (right and left flank) reached approximately 150 mm3, mice were randomised into two groups of 5 animals each. For IGROV-1 xenografts, female CD1 nu / nu mice were subcutaneously inoculated with 1 x 106IGROV-1 cells in 0.1 ml DPBS on the left flank. When tumour volume reached 70-100 mm3, mice were randomised into 3 groups of 5 animals each.

[0546] All NSG animals received an intravenous injected pre-dose of 10 mg / kg human intravenous immunoglobulin (hIVIG), 24 hours before randomisation into groups, to block FcR-mediated sequestration of cAB10383-LALA-Compound 1. All NSG and CD1 nu / nu tumour bearing mice received an intravenous single dose injection of cAB10383-LALA-Compound 1 or vehicle solution comprising 30 mM histidine, 250 mM sucrose, 0.02% PS20 (w / v). A conjugate dose of 3 mg / kg was used for NSG MEL202 tumour bearing mice and a dose of 1 mg / kg and 3 mg / kg was used for CD1 nu / nu IGROV-1 tumour bearing mice. Tumour size was measured using callipers with the volume expressed in mm3using the formula; V= I x w x d x TT / 6. The combined tumour volume of both flanks was used in NSG MEL202 mice. Tumour size was used to calculate TGI (%) using the formula TGI (%)=[1-(Vtreat-t-Vtreat-i) / (Vcontroi-t-Vcontroi-i)]x100, where Vtreat-i and Vcontroi-i are the mean volumes of the treated and control groups on grouping day; Vtreat-t and Vcontroi-t are the mean volumes of the treated and control groups on a given day. Animal body weight was recorded 2 times weekly. Mice were euthanized when tumour reached 1.5 cm in one direction or 1 cm3in volume.

[0547] The in vivo effect of cAB10383-LALA-Compound 1 on MEL202 and IGROV-1 tumour xenografts is shown in Figure 77. MEL202 treated tumours showed significantly lower tumour growth than controls from day 13 (p<0.05) with tumour inhibition lasting until day 36 where the tumours started to regrow providing a TGI of 28.34% at day 67. No difference in tumour growth inhibition was noted between tumours induced with or without Matrigel. Whereas IGROV-1 treated tumours showed a significant reduction in tumour volume from day 12 onwards for both 1 mg / kg and 3 mg / kg conjugated doses (P<0.001). 3 tumours from the 1 mg / kg conjugated dose and 2 tumours from the 3 mg / kg conjugated dose cohort exhibited complete tumour regression, but those with small palpable tumours started to increase in size at day 42 and 54 respectively. Thus, providing a TGI of 92.87 (1 mg / kg) and 108.52 (3 mg / kg) at day 54. No observable toxicity was seen in either mouse strain based on body weight. These data demonstrate that cAb10383-LALA-Compound 1 produced significant tumour growth inhibition and reductions in tumour size in mice bearing MEL202 or IGROV-1 xenografts.

[0548] Table 30 - shows Tumour Growth Inhibition (TGI) of ADCs tested in MEL202 and IGROV-1 xenograft model on day 67 and 54 of the study, respectively. Synthesis of Linker-Payloads

[0549] The compounds and conjugates of the invention can be made using the synthetic procedures outlined below. The following starting materials used for the synthesis of linker-drug compounds are commercially available, for example, from Sigma Aldrich: 4- hydroxybenzaldehyde (SM1), (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro- 2H-pyran-3,4,5-triyl triacetate (SM2), 4-hydroxy-3-nitrobenzaldehyde (SM3), 2-

[0550] (methylamino)ethan-l-ol (SM4), 2-(2-aminoethoxy)ethan-1-ol (SM5), terf-butyl methyl(2- (methylamino)ethyl)carbamate (SM7) and terf-butyl 1 -amino-3, 6,9,12-tetraoxapentadecan- 15-oate (SM10).

[0551] The linker precursor compounds listed below are known in the art and were synthesised according to the literature protocols specified in Table 26, the contents of which are hereby incorporated by reference in their entirety.

[0552] Table 31 : Literature protocols for synthesis of precursor compounds. Preparation of Precursors for Compounds 1 and 2

[0553] Payload precursor (CBI-OBn, CAS: 128300-11-8) was purchased (Arch Bioscience Company, Product#: c16760), and the synthetic intermediates specified below were prepared according to methods and procedures known in the art.

[0554] Steps 1 - 2: Method for synthesis of Int 1 and Int 2.

[0555] Scheme 1 : Synthesis of Int 2.

[0556] Steps 3 - 5: Methods for synthesis of glucuronide precursors Int 8 - Int 10, as reported in Ghosh et al., Tetrahedron Lett. (1997), 38, 8795-8798, the contents of which are hereby incorporated by reference in their entirety.

[0557] Scheme 2: Synthesis of Int 3 - Int 5. Steps 6 - 8: Methods for the synthesis of glucuronide precursors Int 6 - Int 8, as reported in Olesen et al., Angew. Chem. Int. Ed. (2020), 59, 7390-7396, the contents of which are hereby incorporated by reference in their entirety.

[0558] SM2QAc n

[0559] Scheme 3: Synthesis of Int 6 - Int 8.

[0560] Synthesis of Compound 1

[0561] Compound 1 was prepared according to Schemes 4 - 6.

[0562] Synthesis of Int 9 - Int 19

[0563] Scheme 4: Synthesis of Int 9 - Int 19 is shown in Figure 78.

[0564] To a solution of 2-(methylamino)ethan-1-ol (SM4) (50.0 g, 665.7 mmol, 1.0 eq) and N- (benzyloxycarbonyloxy)succinimide (165.9 g, 665.7 mmol, 1.0 eq) in THF (1.0 L) was added DI PEA (258.1 g, 2.0 mol, 3.0 eq). The mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with aq. citric acid (2 L) and extracted with CH2CI2 (4 x 200 mL). The combined organic extracts were washed with aq. NaHCOs (200 mL) and brine (200 mL), dried over Na2SO4 and concentrated in vacuo. The residue was triturated with PE to afford Int 9 (138.0 g, 99% yield) as yellow oil.

[0565] TLC: Rf= 0.2 (PE / EtOAc = 1 :1).

[0566] 1H NMR (400 MHz, DMSO-cfe): 6 7.39 - 7.27 (m, 5H), 5.08 (s, 2H), 4.82 - 4.68 (m, 1 H), 3.59 - 3.51 (m, 2H), 3.32 (t, J = 6.0 Hz, 2H), 2.92 (d, J = 7.6 Hz, 3H). To a solution of Int 9 (80.0 g, 0.38 mol, 1.0 eq) in CH2CI2 (1.0 L) was added DMSO (298.9 g, 3.82 mol, 10.0 eq), DIPEA (247.1 g, 333.0 mL, 1.91 mol, 5.0 eq) and pyridine sulfur trioxide (304.2 g, 1.91 mol, 5.0 eq). The mixture was stirred at -10°C for 2 h. The reaction mixture was diluted with 1 N HCI (2 L) and extracted with CH2CI2 (3 x 300 mL). The combined organic extracts were washed with aq. NaHCOs (150 mL) and brine (100 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc = 10:1 to 3:1) to afford Int 10 (60 g, 76% yield) as yellow oil.

[0567] TLC: Rr = 0.5 (PE / EtOAc = 1 :1).

[0568] LCMS: m / z 208.10 [M+H]+.

[0569] 1H NMR (400 MHz, DMSO-cfe): 6 9.51 (s, 1 H), 7.51 - 7.23 (m, 5H), 5.20 - 5.01 (m, 2H), 4.17 (d, J = 21.0 Hz, 2H), 2.89 (d, J = 18.0 Hz, 3H).

[0570] To a solution of 2-(2-aminoethoxy)ethan-1-ol (SM5) (20.00 g, 190 mmol, 1.0 eq) in CH2CI2 (300 mL) was added Int 10 (35.4 g, 171 mmol, 0.9 eq). The mixture was stirred at room temperature overnight. After cooling to 0 °C, NaBH(OAc)s (36.0 g, 170 mmol, 0.9 eq) was added and the mixture was warmed to room temperature and stirred for 3 h. To the crude reaction mixture of Int 11 was added di-terf-butyldicarbonate (38.3 g, 175 mmol, 0.9 eq) and DIPEA (49 g, 66 mL, 380 mmol, 2.0 eq). The mixture was stirred at room temperature for 5 h. The mixture was diluted with CH2CI2 (300 mL), washed with H2O (100 mL) and brine (100 mL). The organic extract was dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc = 2:1) to afford Int 12 (25.6 g, 38% yield) as a yellow oil.

[0571] TLC: Rf= 0.3 (PE / EtOAc = 1 :2).

[0572] LCMS: m / z 397.15 [M+H]+.

[0573] 1H NMR (400 MHz, Chloroform-d): 5 7.38 - 7.27 (m, 5H), 5.10 (s, 2H), 3.76 - 3.70 (m, 2H), 3.59 - 3.50 (m, 5H), 3.43 - 3.28 (m, 5H), 2.98 - 2.91 (m, 3H), 1.43 (s, 9H).

[0574] To solution of Int 12 (30.0 g, 75.7 mmol, 1.0 eq) in CH2CI2 (300 mL) was added AC2O (10.2 g, 9.4 mL, 99.9 mmol, 1.32 eq), TEA (15.22 g, 21.0 mL, 150.4 mmol, 2.0 eq) and DMAP (923 mg, 7.57 mmol, 0.1 eq). The mixture was stirred at room temperature for 5 h. The mixture was diluted with CH2CI2 (150 mL), washed with H2O (100 mL) and brine (100 mL). The organic extract was dried over Na2SC>4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc = 5:1) to afford Int 13 (8.0 g, 24% yield) as a yellow oil.

[0575] TLC: Rf= 0.3 (PE / EtOAc = 1 :2).

[0576] LCMS: m / z 439.2 [M+H]+.1H NMR (400 MHz, Chloroform-d): 5 7.40 - 7.27 (m, 5H), 5.11 (s, 2H), 4.24 - 4.14 (m, 2H), 3.65 - 3.50 (m, 4H), 3.43 - 3.23 (m, 6H), 3.01 - 2.90 (m, 3H), 2.03 (s, 3H), 1.43 (s, 9H).

[0577] To a solution of Int 13 (22.0 g, 50.1 mmol) in AcOH (3.0 mL) and MeOH (300.0 mL) was added 10% Pd / C (2.0 g). After stirring at 50 °C overnight under H2 atmosphere, the mixture was filtered. The filtrate was concentrated in vacuo to afford Int 14 (20 g, crude) as a colourless oil which was used directly in the next step without further purification.

[0578] LCMS: m / z = 305.16 [M+H]+.

[0579] 1H NMR (400 MHz, Chloroform-d): 5 4.21 (dq, J = 5.0, 2.4 Hz, 2H), 3.68 - 3.30 (m, 8H), 3.12 (s, 1 H), 2.64 (s, 2H), 2.07 (s, 3H), 2.01 (s, 4H), 1.44 (s, 9H).

[0580] To a solution of Int 5 (16.0 g, 26.4 mmol, 1.0 eq) and Int 14 (12.0 g, 39.6 mmol, 1.5 eq) in CH2CI2 (200 mL) was added DIPEA (5.1 g, 6.87mL, 39.6 mmol, 1.5 eq) and DMAP (645 mg, 5.28 mmol, 0.2 eq). The mixture was stirred at room temperature for 3 h. The mixture was poured into H2O (200 mL) and extracted with CH2CI2 (3 x 200 mL). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc / CH2Cl2 = 5:1 :1) and RP column chromatography (C18 column, MeCN / H2O containing 0.1% v / v HCOOH = 3:1) to afford Int 15 (9.8 g, 48% yield) as a pale-yellow solid.

[0581] TLC: Rf= 0.3 (CH2CI2 / MeOH = 20:1).

[0582] LCMS: m / z 771.25 [M+H]+.

[0583] 1H NMR (400 MHz, DMSO-cfe): 6 7.32 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 5.65 (d, J = 8.0 Hz, 1 H), 5.47 (t, J = 9.6 Hz, 1 H), 5.13 - 5.03 (m, 2H), 4.98 (s, 2H), 4.70 (d, J = 10.0 Hz, 1 H), 4.09 (s, 2H), 3.63 (s, 3H), 3.61 - 3.32 (m, 7H), 3.30 - 3.16 (m, 3H), 2.84 (d, J = 7.0 Hz, 3H), 2.04 - 1.97 (m, 12H), 1.35 (s, 9H).

[0584] To a solution of Int 15 (3.0 g, 3.89 mmol) in MeCN (20 mL) at 0 °C was added 4 N HCI in 1 ,4- dioxane (20.0 mL). The mixture was stirred at 0 °C for 1 h and concentrated in vacuo. The residue was triturated with Et20 to afford Int 16 as a white solid (2.6 g, crude), which was used in next step without further purification.

[0585] LCMS: m / z = 671.20 [M+H]+.

[0586] The crude residue of Int 2 (298 mg, 599 pmol, 1.0 eq) was dissolved into DMF (5 mL), then Int 16 (602 mg, 898 pmol, 1.5 eq) and DIPEA (231 mg, 1.79 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 3 h. The mixture was diluted with EtOAc (50 mL), washed with H2O (20 mL) and brine (20 mL), dried over Na2SC>4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, PE / EtOAc = 1 :1) and RP column chromatography (C18 column, MeCN / W = 7:3) to afford Int 17 (200 mg, 32% yield) as a pale-yellow oil.

[0587] LCMS: m / z 1052.55 [M+Na]+.

[0588] 1H NMR (400 MHz, DMSO-cfe): 6 7.96 - 7.89 (m, 1 H), 7.88 - 7.77 (m, 1 H), 7.59 - 7.52 (m, 1 H), 7.42 - 7.34 (m, 1 H), 7.32 - 6.91 (m, 3H), 5.66 - 5.54 (m, 1 H), 5.48 - 5.41 (m, 1 H), 5.11 - 4.92 (m, 4H), 4.72 - 4.62 (m, 1 H), 4.29 - 4.07 (m, 4H), 3.90 (s, 1 H), 3.80 - 3.36 (m, 14H), 2.94 - 2.83 (m, 3H), 2.03 - 1.95 (m, 12H), 1.51 (s, 9H).

[0589] To a solution of Int 17 (300 mg, 291 pmol) in MeCN (5 mL) at 0 °C was added 4 N HCI in 1 ,4- dioxane (5 mL). The mixture was stirred at 0 °C for 1 h and concentrated in vacuo. The residue was triturated with Et20 to afford Int 18 (300 mg, crude) as a white solid, which was used to next step directly.

[0590] LCMS: m / z = 930.20 [M+H]+.

[0591] To a solution of Int 18 (300 mg crude, 291 pmol, 1.0 eq) and dihydro-2 / 7-pyran-2,6(3 / 7)-dione (184 mg, 1.61 mmol, 5.5 eq) in CH2CI2 (5.0 mL) was added DIPEA (124 mg, 168 pL, 966 pmol, 3.3 eq) and DMAP (19.6 mg, 161 pmol, 0.55 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with CH2CI2 (50 mL), washed with H2O (20 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-TLC (C^Ch / MeOH = 15:1) to afford Int 19 (150 mg, 49% yield) as a pale-yellow oil.

[0592] LCMS: m / z 1044.60 [M+H]+.

[0593] 1H NMR (400 MHz, DMSO-cfe): 5 8.39 - 8.26 (m, 1 H), 7.95 - 7.68 (m, 2H), 7.52 (s, 1 H), 7.43 - 7.27 (m, 3H), 7.04 - 6.89 (m, 2H), 5.40 - 5.24 (m, 3H), 5.19 - 4.99 (m, 3H), 4.37 - 4.08 (m, 6H), 4.00 - 3.91 (m, 1 H), 3.80 (s, 1 H), 3.77 - 3.46 (m, 14H), 3.03- 2.91 (m, 3H), 2.75 - 2.35 (m, 5H), 2.06 - 2.02 (m, 12H).

[0594] Synthesis of Int 25

[0595] Scheme 5: Synthesis of Int 20 - Int 25 is shown in Figure 79.

[0596] To a solution of Int 8 (6.0 g, 9.2 mmol, 1.0 eq) dissolved in CH2CI2 (50 mL) was added Int 14 (4.2 g, 13.8 mmol, 1.5 eq) and DIPEA (2.4 g, 3.23 mL, 18.5 mmol, 2.0 eq). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (20 mL x 4). The combined organic extracts were washed with brine (100 mL), dried over Na2SC>4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc = 3:1 to 1 :1) to afford Int 20 (5.0 g, 66% yield) as a yellow oil. TLC: Rf= 0.4 (PE / EtOAc = 1 :2).

[0597] LCMS: m / z 816.25 [M+H]+.

[0598] 1H NMR (400 MHz, DMSO-cfe): 6 7.88 (s, 1 H), 7.73 - 7.64 (m, 1 H), 7.47 - 7.33 (m, 1 H), 5.79 - 5.73 (m, 1 H), 5.46 (t, J = 9.4 Hz, 1 H), 5.15 - 5.01 (m, 4H), 4.74 (d, J = 9.8 Hz, 1 H), 4.13 -4.03 (m, 2H), 3.64 (s, 3H), 3.59 - 3.41 (m, 4H), 3.39 - 3.33 (m, 2H), 3.31 - 3.15 (m, 4H), 2.91 - 2.79 (m, 3H), 2.05 - 1.94 (m, 12H), 1.35 (s, 9H).

[0599] To solution of Int 20 (4.5 g, 5.5 mmol) in EtOAc (30 mL) was added 10% Pd / C (900 mg). The reaction mixture was stirred under a H2 atmosphere for 24 h. The mixture was filtered and concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, PE / EtOAc = 5:1 to 1 :1) to afford Int 21 (3.5 g, 81% yield) as a white solid.

[0600] TLC: Rf= 0.3 (PE / EtOAc = 1 :2).

[0601] LCMS: m / z 786.25 [M+H]+.

[0602] 1H NMR (400 MHz, DMSO-cfe): 6 6.87 - 6.81 (m, 1 H), 6.66 (m, 1 H), 6.59 - 6.45 (m, 1 H), 5.54 - 5.38 (m, 2H), 5.16 - 5.01 (m, 2H), 4.86 (s, 2H), 4.74 - 4.61 (m, 3H), 4.15 - 4.04 (m, 2H), 3.64 (s, 3H), 3.60 - 3.40 (m, 4H), 3.31 - 3.19 (m, 4H), 2.84 (d, J = 8.6 Hz, 3H), 2.03 (s, 3H), 2.02 - 1.97 (m, 9H), 1.37 (s, 9H).

[0603] To a solution of SM6 (3.2 g, 4.90 mmol, 1.1 eq) in DMF (40 mL) was added HATU (2.54 g, 6.68 mmol, 1.5 eq), DIPEA (1.15 g, 1.55 mL, 8.91 mmol, 2.0 eq) and the mixture stirred at room temperature for 10 min, then Int 21 (3.5 g, 4.45 mmol, 1.0 eq) was added and the mixture was stirred at 30 °C overnight under N2 atmosphere. The mixture was diluted with H2O (100 mL) and extracted with EtOAc (4 x 20 mL). The combined organic extracts were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by reverse-phase column chromatography (C18 column, MeCN / H2O = 11 :9) to afford Int 22 (2.03 g, 33% yield) as a white solid.

[0604] TLC: Rf= 0.5 (CH2CI2 / MeOH = 10:1).

[0605] LCMS: m / z 1382.55 [M+H]+.

[0606] 1H NMR (400 MHz, DMSO-cfe): 6 8.69 (s, 1 H), 8.10 - 7.92 (m, 2H), 7.82 (d, J = 11.2 Hz, 2H), 7.18 - 7.00 (m, 2H), 6.88 (dd, J = 17.6, 10.8 Hz, 1 H), 6.47 (d, J = 17.6 Hz, 1 H), 5.91 (d, J = 10.8 Hz, 1 H), 5.60 (d, J = 7.8 Hz, 1 H), 5.50 (t, J = 9.6 Hz, 1 H), 5.25 - 5.14 (m, 1 H), 5.07 (t, J = 9.6 Hz, 1 H), 4.96 (s, 2H), 4.72 (d, J = 10.0 Hz, 1 H), 4.14 - 4.02 (m, 2H), 3.70 (t, J = 6.4 Hz, 2H), 3.64 (s, 3H), 3.61 - 3.39 (m, 33H), 3.37 (t, J = 5.6 Hz, 2H), 3.35 - 3.10 (m, 10H), 2.84 (d, J = 11 .2 Hz, 3H), 2.69 - 2.64 (m, 4H), 2.63 - 2.54 (m, 2H), 2.08 - 1.91 (m, 12H), 1.36 (s, 9H). To a solution of Int 22 (1.0 g, 724 pmol) in MeCN (20 mL) at 0 °C was added 4 N HCI in 1 ,4- dioxane (20 mL). The mixture was stirred at 0 °C for 1 h and concentrated in vacuo. The residue was triturated with Et20 to afford Int 23 (1.0 g, crude) as a white solid.

[0607] LCMS: m / z = 642.20 [M+2H]2+.

[0608] Int 23 (574 mg, 448 pmol, 1.5 eq) and DIPEA (115 mg, 156 pL, 897 mmol, 3.0 eq) were dissolved in DMF (5.0 mL) and added to crude mixture of Int 2 (148 mg, 299 pmol, 1.0 eq) in DMF (2.0 mL). The mixture was stirred at room temperature for 3 h. The mixture was diluted with EtOAc (50 mL), washed with H2O (20 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc = 1 :1) to afford Int 24 (150 mg, 30% yield) as a pale-yellow oil.

[0609] LCMS: m / z 1641.95 [M+H]+.

[0610] 1H NMR (400 MHz, DMSO-cfe): 6 8.73 - 8.60 (m, 1 H), 8.01 - 7.74 (m, 4H), 7.59 - 7.47 (m, 1 H), 7.43 - 7.31 (m, 1 H), 7.13 (d, J = 64.1 Hz, 4H), 6.68 (dd, J = 17.6, 10.9 Hz, 1 H), 6.11 (d, J = 17.7 Hz, 1 H), 5.64 - 5.42 (m, 3H), 5.24 - 4.90 (m, 4H), 4.74 - 4.65 (m, 1 H), 4.28 - 3.99 (m, 5H), 3.94 - 3.85 (m, 1 H), 3.78 - 3.55 (m, 14H), 3.54 - 3.43 (m, 36H), 3.22 - 3.15 (m, 3H), 2.95 - 2.84 (m, 5H), 2.61 - 2.55 (m, 2H), 2.45 (s, 3H), 2.05 - 1.96 (m, 12H), 1.56 - 1.44 (m, 8H).

[0611] To a solution of Int 24 (300 mg, 183 pmol) in MeCN (5 mL) at 0 °C was added 4 N HCI in 1 ,4- dioxane (5 mL). The mixture was stirred at 0 °C for 1 h and concentrated in vacuo. The residue was triturated with Et20 to afford Int 25 (300 mg, crude) as a white solid, which was used directly in the next step without further purification.

[0612] LCMS: m / z 772.20 [M+2H]2+.

[0613] Synthesis of Compound 1

[0614] Scheme 6: Synthesis of Compound 1 from Int 19 and Int 25 is shown in Figure 80.

[0615] To a solution of Int 19 (70 mg, 67.1 pmol, 1.0 eq) and Int 25 (154 mg, 100 pmol, 1.5 eq) in DMA (2.0 mL) was added EDCI (63 mg, 406 pmol, 6.0 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (50 mL), washed with H2O (20 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by prep-TLC (C^Ch / MeOH = 15:1) and prep-HPLC (C18 column, 5-80% MeCN in H2O containing 0.1 % HCOOH) to afford Int 26 (20 mg, 11.6% yield) as a white solid.

[0616] LCMS: m / z 1285.10 [M+2H]2+.1H NMR (400 MHz, DMSO-cfe): 6 8.73 - 8.45 (m, 2H), 8.26 - 8.20 (m, 2H), 8.05 - 7.75 (m, 7H), 7.62 - 7.52 (m, 2H), 7.41 (q, J = 7.6 Hz, 3H), 7.29 (s, 1 H), 7.22 - 6.91 (m, 6H), 6.65 (dd, J = 17.8, 10.8 Hz, 1 H), 6.04 (d, J = 17.6 Hz, 1 H), 5.60 (dd, J = 19.0, 7.8 Hz, 2H), 5.51 - 5.42 (m, 3H), 5.22 - 5.15 (m, 1 H), 5.11 - 4.93 (m, 8H), 4.73 - 4.63 (m, 2H), 4.47 - 3.84 (m, 17H), 3.75 - 3.60 (m, 22H), 3.51 - 3.44 (m, 34H), 3.21 - 3.15 (m, 2H), 2.97 - 2.82 (m, 9H), 2.60 - 2.55 (m, 3H), 2.48 - 2.44 (m, 2H), 2.41 (s, 2H), 2.03 - 1.96 (m, 24H).

[0617] To a solution of Int 26 (20 mg, 7.8 pmol, 1.0 eq) in MeCN (1.0 mL) was added a solution of UOH.H2O (13 mg, 312 pmol, 40 eq) in H2O (1 mL) at 0 °C. The mixture was stirred at 0 °C for 3 h. The pH was adjusted with 3M HCI to pH 2 - 3 and was purified by prep-HPLC (C18 column, 45% MeCN in H2O) to afford Compound 1 (4.7 mg, 27% yield) as a white solid.

[0618] LCMS: m / z 1102.15 [M+2H]2+.

[0619] 1H NMR (400 MHz, DMSO-cfe): 69.21 (s, 1 H), 8.27 -8.17 (m, 4H), 7.98 - 7.89 (m, 4H), 7.88 - 7.79 (m, 2H), 7.61 - 7.48 (m, 3H), 7.45 -7.32 (m, 3H), 7.26 - 7.22 (m, 1 H), 7.18 - 7.07 (m, 4H), 6.97 (t, J = 8.0 Hz, 2H), 6.05 (d, J = 17.8 Hz, 1 H), 5.79 (br s, 1 H), 5.46 (d, J = 10.8 Hz, 1 H), 5.11 - 4.86 (m, 7H), 4.48 - 4.17 (m, 8H), 4.10 - 3.99 (m, 3H), 3.96 - 3.87 (m, 3H), 3.80 - 3.59 (m, 21 H), 3.58 - 3.50 (m, 21 H), 3.37 (t, J = 5.8 Hz, 8H), 3.31 - 3.22 (m, 13H), 3.21 - 3.15 (m, 5H), 2.97 - 2.83 (m, 12H), 2.71 - 2.59 (m, 6H), 2.41 (s, 3H), 1.95 (br s, 3H).

[0620] Synthesis of Compound 2

[0621] Compound 2 was prepared according to Schemes 7 - 9.

[0622] Synthesis of Int 28 and Int 30

[0623] Scheme 7: Synthesis of Int 27 - Int 30 is shown in Figure 81.

[0624] To a solution of Int 5 (22.20 g, 36.66 mmol, 1.0 eq) in CH2CI2 (300 mL) was added terf-butyl methyl(2-(methylamino)ethyl)carbamate (SM7) (13.80 g, 73.30 mmol, 2.0 eq) and DMAP (448 mg, 3.67 mmol, 0.1 eq). The mixture was stirred at room temperature for 2 h. The mixture was diluted with H2O (1.5 L) and extracted with EtOAc (1.5 L). The organic extract was washed with brine (1 L) and dried over MgSCU. The filtrate was concentrated in vacuo and the crude residue purified by column chromatography on (SiC>2, PE / EtOAc = 10:1 to C^Ch / MeOH = 50:1) to afford Int 27 (13.5 g, 56% yield) as a pale-yellow solid.

[0625] TLC: Rf= 0.3 (PE / EtOAc = 1 :1).

[0626] LCMS: m / z 672.20 [M+NH4]+.1H NMR (400 MHz, Chloroform-d): 57.29 (d, J = 8.6 Hz, 2H), 6.99 - 6.94 (m, 2H), 5.36 - 5.30 (m, 2H), 5.26 (td, J = 7.0, 2.8 Hz, 1 H), 5.13 (d, J = 7.2 Hz, 1 H), 5.05 (s, 2H), 4.20 - 4.13 (m, 1 H), 3.72 (s, 3H), 3.37-3.30 (m, 4H), 2.93 (d, J = 6.7 Hz, 3H), 2.86- 2.78 (m, 3H), 2.06 - 2.02 (m, 9H), 1.43 (s, 9H).

[0627] A solution of Int 27 (2.0 g, 3.05 mmol, 1.0 eq) in MeCN (10 mL) at 0 °C was added 4 N HCI in 1 ,4-dioxane (10 mL, 40.0 mmol, 13.1 eq). The mixture was stirred at 0 °C for 1 h. The mixture was concentrated in vacuo and the residue was triturated with Et20 to afford Int 28 (1.8 g, crude) as a white solid.

[0628] LCMS: m / z 555.35 [M+H]+.

[0629] The crude residue of Int 2 (298 mg, 0.60 mmol, 1.0 eq) was dissolved in DMF (5 mL), then Int 28 (727 mg, 1.3 mmol, 2.2 eq) and DIPEA (233 mg, 314 pL, 1.8 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for overnight, diluted with H2O (200 mL) and extracted with EtOAc (3 x 60 mL). The combined organic extracts were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue obtained was purified by prep-TLC (C^Ch / MeOH = 15:1) to afford Int 29 (450 mg, 82% yield) as a white solid.

[0630] LCMS: m / z 931.6 [M+NH4]+.

[0631] 1H NMR (400 MHz, DMSO-cfe): 6 8.13 - 7.17 (m, 7H), 7.04 - 6.77 (m, 2H), 5.66- 5.52 (m, 1 H), 5.50 - 5.40 (m, 1 H), 5.13 - 4.94 (m, 4H), 4.73 - 4.62 (m, 1 H), 4.28 -4.14 (m, 2H), 4.12 - 3.99 (m, 2H), 3.95 - 3.84 (m, 1 H), 3.79 - 3.68 (m, 1 H), 3.63 (s, 3H), 3.59 - 3.40 (m, 3H), 3.20 - 3.10 (m, 1 H), 3.00 - 2.82 (m, 5H), 2.02 - 1.97 (m, 10H), 1.55 - 1.48 (m, 9H).

[0632] To a solution of Int 29 (450 mg, 0.49 mmol, 1.0 eq) in MeCN (5 mL) at 0 °C was added 4 N HCI in 1 ,4-dioxane (5 mL, 20 mmol, 40 eq). The mixture was stirred at 0 °C for 1 h. The mixture was concentrated in vacuo and the residue was triturated with Et20 to afford Int 30 (400 mg, crude) as a brown solid.

[0633] LCMS: m / z 814.45 [M+H]+.

[0634] Synthesis of Int 36

[0635] Scheme 8: Synthesis of Int 31 - Int 36 is shown in Figure 82.

[0636] To a solution of Int 8 (5.0 g, 7.7 mmol, 1.0 eq) in CH2CI2 (30 mL) was added terf-butyl methyl(2- (methylamino)ethyl)carbamate (SM7) (2.9 g, 15.4 mmol, 2.0 eq) and DMAP (94.0 mg, 0.77 mmol, 0.1 eq). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with CH2CI2 (200 mL), washed with 2 N HCI (70 mL x 2) and brine (50 mL). The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, PE / EtOAc = 5:1 to 1 :2) to afford Int 31 (4.0 g, 74% yield) as a yellow solid.

[0637] TLC: Rf= 0.3 (PE / EtOAc = 1 :2).

[0638] 1H NMR (400 MHz, DMSO-cfe): 6 7.95 - 7.85 (m, 1 H), 7.74 - 7.64 (m, 1 H), 7.47 - 7.37 (m, 1 H), 5.74 (d, J = 7.8 Hz, 1 H), 5.46 (t, J = 9.6 Hz, 1 H), 5.16 - 5.05 (m, 3H), 4.74 (d, J = 9.8 Hz, 1 H), 3.64 (s, 3H), 3.38 - 3.34 (m, 2H), 3.31 - 3.25 (m, 2H), 2.85 (d, J = 15.8 Hz, 3H), 2.79 - 2.68 (m, 3H), 2.03 - 2.00 (m, 6H), 2.00 - 1.99 (m, 2H), 1.38 - 1.29 (m, 9H).

[0639] To solution of Int 31 (4.0 g, 5.7 mmol, 1.0 eq) in EtOAc (30 mL) was added Pd / C (800 mg, 10% w / w). The mixture was stirred under a H2 atmosphere for 24 h. The mixture was filtered and concentrated in vacuo. The residue obtained was purified by column chromatography (SiC>2, PE / EtOAc = 2:1 to 1 :2) to afford Int 32 (2.5 g, 65% yield) as a white solid.

[0640] TLC: Rf= 0.3 (PE / EtOAc = 1 :2).

[0641] LCMS: m / z 670.30 [M+H]+.

[0642] 1H NMR (400 MHz, DMSO-cfe): 6 6.84 (d, J = 8.2 Hz, 1 H), 6.72 - 6.63 (m, 1 H), 6.59 - 6.45 (m, 1 H), 5.53 - 5.39 (m, 2H), 5.17 - 5.01 (m, 2H), 4.86 (s, 2H), 4.72 - 4.63 (m, 3H), 3.65 (s, 3H), 3.38 - 3.31 (m, 2H), 2.90 - 2.69 (m, 6H), 2.04 (s, 3H), 2.00 (s, 6H), 1.36 (s, 9H).

[0643] To a solution of SM6 (2.02 g, 3.10 mmol, 1.0 eq) in DMF (30 mL) was added HATU (1.70 g, 4.48 mmol, 1.5 eq), DIPEA (770.1 mg, 1038 pL, 5.97 mmol, 2.0 eq) and the mixture stirred at room temperature for 10 min, then Int 32 (2.0 g, 2.99 mmol, 1.0 eq) was added. The mixture was stirred at room temperature for 4 h under N2 atmosphere. The mixture was diluted with H2O (100 mL) and extracted with EtOAc (4 x 20 mL). The combined organic extracts were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO2, C^Ch / MeOH = 100:1 to 40:1) to afford Int 33 (2.06 g, 54% yield) as a white solid.

[0644] TLC: Rf= 0.5 (CH2CI2 / MeOH = 10:1).

[0645] LCMS: m / z 1266.65 [M+H]+.

[0646] 1H NMR (400 MHz, DMSO-cfe): 6 8.70 (s, 1 H), 8.02 - 7.92 (m, 2H), 7.51 - 7.38 (m, 1 H), 7.14 - 7.01 (m, 2H), 6.83 - 6.71 (m, 1 H), 6.32 - 6.18 (m, 1 H), 5.73 - 5.63 (m, 1 H), 5.60 (d, J = 7.8 Hz, 1 H), 5.50 (t, J = 9.6 Hz, 1 H), 5.23 - 5.14 (m, 1 H), 5.07 (t, J = 9.6 Hz, 1 H), 4.96 (s, 2H), 4.72 (d, J = 10.0 Hz, 1 H), 3.69 (t, J = 6.2 Hz, 2H), 3.63 (s, 3H), 3.37 (t, J = 5.8 Hz, 2H), 3.34 - 3.24 (m, 5H), 3.19 (q, J = 5.6 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 2.89 - 2.68 (m, 6H), 2.64 - 2.52 (m, 6H), 2.08 - 1.95 (m, 9H), 1.35 (s, 9H).

[0647] To a solution of Int 33 (1.2 g, 0.95 mmol, 1.0 eq) in MeCN (10.0 mL) at 0 °C was added 4 N HCI in 1 ,4-dioxane (10 mL, 40 mmol, 42 eq). The mixture was stirred at 0 °C for 1 h. The mixture was concentrated in vacuo and the residue was triturated with Et20 to afford Int 34 (1.0 g, crude) as a white solid.

[0648] LCMS: Theoretical: C55H83N5O221165.55, Observed: m / z 584.40 [M+2H]2+.

[0649] To a solution of Int 2 (300 mg, 0.60 mmol, 1.0 eq) in DMF (10.0 mL) was added Int 34 (1.0 g, 0.86 mmol, 1.4 eq) and DIPEA (233 mg, 314 pL, 1.8 mmol, 3.0 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with H2O (200 mL) and extracted with EtOAc (3 x 60 mL). The combined organic extracts were washed with brine (60 mL), dried over Na2SC>4, filtered and concentrated in vacuo. The residue was purified by prep-TLC (CH2Cl2 / MeOH = 15:1) to afford Int 35 (500 mg, 54% yield) as a yellow solid.

[0650] LCMS: m / z 763.80 [M+2H]2+.

[0651] To a solution of Int 35 (120 mg, 0.079 mmol) in MeCN (5 mL) at 0 °C was added 4 N HCI in 1 ,4-dioxane (5 mL, 20 mmol, 254 eq). The mixture was stirred at 0 °C for 1 h. The mixture was concentrated in vacuo and the residue was triturated with Et20 to afford Int 36 (100 mg, crude) as a red solid.

[0652] LCMS: m / z 714.55 [M+2H]2+.

[0653] Synthesis of Compound 2

[0654] Scheme 9: Synthesis of Compound 2 is shown in Figure 83.

[0655] To a solution of Int 30 (200 mg, 0.25 mmol, 1.0 eq) in CH2CI2 (10 mL) was added glutaric anhydride (140.1 mg, 1.23 mmol, 5.0 eq), DIPEA (158.7mg, 214 pL, 1.23 mmol, 5.0 eq) and DMAP (45.0 mg, 0.37 mmol, 1.5 eq). The mixture was stirred at room temperature for 48 h. The mixture was diluted with CH2CI2 (50 mL) and washed with H2O (20 mL) and brine (20 mL), dried over Na2SC>4, filtered and concentrated in vacuo. The residue was purified by prep-TLC (CH2Cl2 / MeOH = 15:1) to afford Int 37 (50 mg, 22% yield) as a yellow solid.

[0656] LCMS: m / z 950.55 [M+Na]+.1H NMR (400 MHz, DMSO-d6): 6 12.04 (s, 1 H), 8.20 (s, 1 H), 8.01 - 7.89 (m, 1 H), 7.88 -7.70 (m, 1 H), 7.65 - 7.54 (m, 1 H), 7.49 - 7.22 (m, 3H), 7.01 - 6.78 (m, 2H), 5.66 -

[0657] 5.55 (m, 1 H), 5.50 - 5.40 (m, 1 H), 5.13 - 4.92 (m, 4H), 4.72 - 4.61 (m, 1 H), 4.42 -

[0658] 4.28 (m, 2H), 4.24 - 4.16 (m, 1 H), 4.10 - 4.00 (m, 1 H), 3.95 - 3.88 (m, 1 H), 3.78 -

[0659] 3.57 (m, 6H), 3.51 - 3.46 (m, 2H), 3.21 - 3.09 (m, 1 H), 3.00 - 2.80 (m, 4H), 2.36 -

[0660] 2.31 (m, 2H), 2.28 - 2.21 (m, 2H), 2.00 (d, J = 5.4 Hz, 9H), 1.89 - 1.77 (m, 2H).

[0661] To a solution of Int 36 (95 mg, 0.067 mmol, 1.2 eq) and Int 37 (50 mg, 0.054 mmol, 1.0 eq) in DMA (5 mL) was added EDCI (42 mg, 0.271 mmol, 5.0 eq). The reaction mixture stirred at room temperature for 18 h. The mixture was diluted with H2O (100 mL) and extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine (30 mL), dried over Na2SC>4, filtered and concentrated in vacuo. The residue was purified by RP column chromatography (MeCN / H2O = 6:4) to afford Int 38 (30 mg, 24% yield) as a pale-yellow solid.

[0662] LCMS: m / z 1169.35 [M+2H]2+.

[0663] To a solution of Int 38 (30 mg, 0.013 mmol, 1.0 eq) in MeCN (1 mL) and H2O (1 mL) at 0 °C was added UOH.H2O (6.1 mg, 0.153 mmol, 11.3 eq). The mixture was stirred at 0 °C for 2 h, and then acidified with 2 M HCI to pH ~ 3. After concentration, the residue was purified by prep-HPLC (C18 column, 5-80% MeCN in H2O containing 0.1% HCOOH) to afford Compound 2 (4.6 mg, 17% yield) as a white solid.

[0664] LCMS: m / z 1028.95 [M+2H]2+.

[0665] HPLC: 95.6 % purity (abs 254nm)

[0666] Synthesis of Compound 3

[0667] Payload precursor (exatecan mesylate dihydrate, CAS: 197720-53-9) was purchased from Shanghai Jianchao Biotechnology Co. Ltd (Product #: WY052-S3-125-039).

[0668] Scheme 10: Synthesis of Compound 3 is shown in Figure 84.

[0669] To a solution of SM8 (1.0 g, 2.20 mmol, 1.0 eq) and SM6 (2.14 g, 3.29 mmol, 1.5 eq) in DMF (10 mL) was added HATU (1.25 g, 3.29 mmol, 1.5 eq) and DIPEA (1.42 g, 10.98 mmol, 5.0 eq). After stirring at room temperature for 4 h, the mixture was diluted with H2O (200 mL) and extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (SiO2, C^Ch / MeOH = 50:1 to 30:1) to afford Int 39 (1.56 g, 68 % yield) as a yellow oil.

[0670] TLC: Rf= 0.5 (SiO2, CH2CI2 / MeOH = 15:1 , v / v) LCMS: m / z = 526.9 [M+2H]2+.

[0671] 1H NMR (400 MHz, Chloroform-d): 58.29 (s, 1H), 8.23 (s, 1H), 7.21 - 6.90 (m, 5H), 6.67 - 6.57 (m, 1H), 6.07 - 5.98 (m, 1H), 5.56 - 5.51 (m, 1H), 5.43 - 5.37 (m, 1H), 5.29-5.22 (m, 2H), 5.09-5.01 (m, 1H), 4.61 (s, 2H), 4.20 (dd, J= 9.8, 1.6 Hz, 1H), 3.84 (t, J = 5.6 Hz, 2H), 3.74 (s, 3H), 3.69 - 3.59 (m, 28H), 3.52 (t, J = 5.3 Hz, 2H), 3.41 (t, J = 5.4 Hz, 2H), 3.07 (t, J = 7.5 Hz, 2H), 2.72 - 2.60 (m, 4H), 2.56 - 2.54 (m, 3H), 2.12-2.03 (m, 9H).

[0672] To a solution of Int 39 (1.56 g, 1.48 mmol, 1.0 eq) and DIPEA (574.9 mg, 4.44 mmol, 3.0 eq) in CH2CI2 (15 mL) at 0°C was added bis(4-nitrophenyl) carbonate (676.6 mg, 2.22 mmol, 1.5 eq). After stirring at room temperature for 4 h, the mixture was diluted with CH2CI2 (200 mL), washed with H2O and brine. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, CH2Cl2 / MeOH = 100:1 to 50:1) to afford Int 40 (1.1 g, 61% yield) as a yellow solid.

[0673] TLC: Rf= 0.6 (SiO2, CH2CI2 / MeOH = 15:1, v / v)

[0674] LCMS: m / z = 609.4 [M+2H]2+.

[0675] 1H NMR (400 MHz, Chloroform-d): 58.53 (d, J= 2.1 Hz, 1H), 8.25 (d, J= 9.2 Hz, 2H), 8.18 (s, 1H), 7.37 (d, J= 9.2 Hz, 2H), 7.09 (dd, J= 8.4, 2.1 Hz, 1H), 7.04-6.94 (m, 3H), 6.86 (s, br, 1H), 6.60 (dd, J= 17.6, 10.8 Hz, 1H), 5.94 (d, J= 17.6 Hz, 1H), 5.46 (d, J = 10.8 Hz, 1H), 5.39 (d, J = 9.4 Hz, 1H), 5.34 - 5.28 (m, 2H), 5.21 (s, 2H), 5.09 (d, J= 7.7 Hz, 1H), 4.19 (d, J= 9.5 Hz, 1H), 3.85 (t, J= 6.1 Hz, 2H), 3.75 (s, 3H), 3.68 - 3.56 (m, 28H), 3.49 (t, J = 5.1 Hz, 2H), 3.41 (t, J = 5.2 Hz, 2H), 3.09 (t, J = 7.4 Hz, 2H), 2.71 (t, J= 6.0 Hz, 2H), 2.65 (t, J= 7.4 Hz, 2H), 2.54 (s, 3H), 2.10-2.00 (m, 9H).

[0676] To a solution of exatecan mesylate dihydrate (615.5 mg, 1.08 mmol, 1.2 eq) and DI PEA (584.0 mg, 4.52 mmol, 5.0 eq) in DMF (10 mL) was added Int 40 (1.1 g, 0.90 mmol, 1.0 eq) portionwise. After stirring at room temperature for 4 h, the mixture was purified directly by RP column chromatography (C18 column, 50% MeCN in H2O containing 0.1% HCOOH) to afford Int 41 (601 mg, 44% yield) as a yellow solid.

[0677] LCMS: m / z = 757.5 [M+2H]2+.

[0678] 1H NMR (400 MHz, Chloroform-d): 58.38 (s, 1H), 8.09 (s, 1H), 7.55 - 7.44 (m, 2H), 7.14-7.01 (m, 4H), 6.85 (d, J= 7.8 Hz, 1H), 6.61 (dd, J= 17.6, 10.8 Hz, 1H), 5.99 (d, J= 17.6 Hz, 1H), 5.62 (d, J= 16.2 Hz, 1H), 5.52 (d, J= 10.8 Hz, 2H), 5.38 (t, J= 9.5 Hz, 2H), 5.28 - 5.19 (m, 4H), 5.19 - 5.08 (m, 3H), 5.00 - 4.89 (m, 1H), 4.15 (d, J = 9.5 Hz, 1 H), 3.80 - 3.75 (m, 2H), 3.73 (s, 3H), 3.64 - 3.53 (m, 30H), 3.49 - 3.46 (m, 3H), 3.40 - 3.35 (m, 2H), 3.12 (t, J = 7.2 Hz, 3H), 2.70 - 2.61 (m, 4H), 2.58 (s, 3H), 2.35 (s, 3H), 2.26 - 2.17 (m, 1 H), 2.13 - 1.99 (m, 9H), 1.96 - 1.88 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H).

[0679] To a solution of Int 41 (200 mg, 0.13 mmol, 1.0 eq) in DMF (2 mL) and H2O (2 mL) at 0°C was added LiOH H2O (55.5 mg, 1.32 mmol, 10.2 eq). After stirring at 0 °C for 30 min, the mixture was acidified to pH ~ 3-4 with AcOH. The resulting mixture was filtered and purified directly by prep-HPLC to afford Compound 3 (32 mg, 18% yield) as a yellow solid.

[0680] LCMS: m / z = 1373.5 [M+H]+.

[0681] HPLC: 96.7 % purity (abs 254nm)

[0682] 1H NMR (400 MHz, DMSO-d6): 5 9.11 (s, 1 H), 8.20 (s, 1 H), 8.07 (d, J = 8.7 Hz, 1 H), 7.91 (t, J = 5.3 Hz, 1 H), 7.77 (d, J = 10.9 Hz, 1 H), 7.31 (s, 1 H), 7.15 - 7.05 (m, 4H), 6.64 (dd, J = 17.6, 11.0 Hz, 1 H), 6.51 (s, 1 H), 6.05 (d, J = 17.6 Hz, 1 H), 5.86 (s, 1 H), 5.50 - 5.40 (m, 3H), 5.33 - 5.21 (m, 4H), 5.07 (s, 2H), 4.80 (d, J = 7.2 Hz, 1 H), 3.80 (d, J = 9.5 Hz, 1 H), 3.64 (t, J = 6.1 Hz, 2H), 3.52 - 3.44 (m, 33H), 3.40 - 3.36 (m, 5H), 3.18 (t, J = 5.8 Hz, 2H), 2.87 (t, J = 7.7 Hz, 2H), 2.70 - 2.54 (m, 2H), 2.48 - 2.44 (m, 1 H), 2.41 (s, 3H), 2.37 (s, 3H), 2.23 - 2.13 (m, 2H), 1.94 - 1.78 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0683] Synthesis of Int 43 and Int 45

[0684] Vinylpyridine linkers such as SM9 are known in the art (W02016067021A1).

[0685] Scheme 11 : Synthesis of Int 42 - Int 45 is shown in Figure 85.

[0686] To a solution of SM9 (550 mg, 2.79 mmol, 1.4 eq), DIPEA (721.2 mg, 5.58 mmol, 2.7 eq) and SM10 (663.6 mg, 2.06 mmol, 1.0 eq) in DMF (15 mL) was added T3P (50 % w / w in EtOAc (2.49 mL, 4.18 mmol, 2.0 eq). The reaction mixture was stirred at room temperature for 18 h. The mixture was diluted with H2O and extracted with EtOAc (3 x 50 mL). The combined organic phase was washed with brine and dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on (SiO2, PE / EtOAc = 5:1) to afford Int 42 (460 mg, 45% yield) as a yellow oil.

[0687] LCMS: m / z = 495.4 [M+H]+1H NMR (400 MHz, Chloroform-d): 5 6.97 (s, 1 H), 6.94 (s, 1 H), 6.87 (s, br, 1 H), 6.57 (dd, J = 17.6, 10.8 Hz, 1 H), 5.90 (d, J = 17.6 Hz, 1 H), 5.40 (d, J = 10.9 Hz, 1 H), 3.66 (t, J = 6.5 Hz, 2H), 3.62 - 3.53 (m, 12H), 3.47 (t, J = 5.0 Hz, 2H), 3.43 - 3.36 (m, 2H), 3.05 (t, J = 7.4 Hz, 2H), 2.61 (t, J = 7.4 Hz, 2H), 2.49 (s, 3H), 2.45 (t, J = 6.6 Hz, 2H), 1.41 (s, 9H). To a solution of Int 42 (300 mg, 0.61 mmol, 1.0 eq) in CH2CI2 (3 mL) was added HCI in 1 ,4- dioxane (4M, 2.4 mL, 9.60 mmol, 15.7 eq). The mixture was stirred at room temperature for 18 h. The mixture was concentrated in vacuo to afford Int 43 (360 mg, quantitative yield) as a crude pale-yellow oil, which was isolated as the HCI salt and used directly without further purification.

[0688] LCMS: m / z = 439.4 [M+H]+

[0689] HPLC: 96.1 % purity (abs 254nm)

[0690] 1H NMR (400 MHz, Chloroform-d): 5 7.92 (s, br, 1 H), 7.63 (s, 1 H), 7.32 (s, 1 H), 6.73 (dd, J = 17.5, 10.8 Hz, 1 H), 6.25 (d, J = 17.4 Hz, 1 H), 5.85 (d, J = 10.7 Hz, 1 H), 3.78 (t, J = 5.8 Hz, 2H), 3.64 - 3.63 (m, 10H), 3.61 - 3.58 (m, 2H), 3.53 (t, J = 5.0 Hz, 2H), 3.49 (t, J = 6.8 Hz, 2H), 3.42 - 3.38 (m, 2H), 2.96 - 2.91 (m, 2H), 2.90 (s, 3H), 2.61 (t, J = 5.8 Hz, 2H).

[0691] To a solution of SM8 (400 mg, 0.88 mmol, 1.0 eq) and Int 43 (577 mg, 1.21 mmol, 1.4 eq) in DMF (15 mL) was added DIPEA (567.6 mg, 4.39 mmol, 5.0 eq) and HATU (501 mg, 1.32 mmol, 1.5 eq). The reaction mixture was stirred at room temperature for 16 h. The mixture was diluted with H2O (100 mL) and extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, C^Ch / MeOH = 50:1 to 30:1) to afford Int 44 (500 mg, 65% yield) as a pale-yellow oil.

[0692] LCMS: m / z = 876.4 [M+H]+1H NMR (400 MHz, Chloroform-d): 5 8.35 (s, 1 H), 8.18 (s, 1 H), 7.08 - 6.94 (m, 6H), 6.60 (dd, J = 17.6, 10.8 Hz, 1 H), 5.94 (d, J = 17.6 Hz, 1 H), 5.46 - 5.32 (m, 4H), 5.05 (d, J = 8.2 Hz, 1 H), 4.63 (s, 2H), 4.17 (d, J = 9.6 Hz, 1 H), 3.84 (t, J = 6.0 Hz, 2H), 3.75 (s, 3H), 3.65 - 3.59 (m, 5H), 3.59 - 3.47 (m, 5H), 3.47 - 3.38 (m, 6H), 3.06 (t, J = 7.4 Hz, 2H), 2.68 (t, J = 6.4 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 2.51 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H).

[0693] To a solution of Int 44 (450 mg, 0.51 mmol, 1.0 eq) and DIPEA (199.2 mg, 1.54 mmol, 3.0 eq) in CH2CI2 (9 mL) was added bis(4-nitrophenyl) carbonate (234.4 mg, 0.77 mmol, 1.5 eq) at 0 °C. The mixture was stirred at room temperature for 18 h. The mixture concentrated in vacuo and purified by column chromatography (SiC>2, C^Ch / MeOH = 100:1 to 30:1) to afford Int 45 (140 mg, 26% yield) as a yellow oil.

[0694] LCMS: m / z = 1041.3 [M+H]+1H NMR (400 MHz, Chloroform-d): 5 8.25 (d, J = 9.2 Hz, 2H), 8.09 (d, J = 9.2 Hz, 2H), 7.39 - 7.36 (m, 2H), 7.00 - 6.94 (m, 5H), 6.58 (dd, J = 17.6, 10.8 Hz, 1 H), 5.92 (d, J = 17.6 Hz, 1 H), 5.43 (d, J = 10.8 Hz, 1 H), 5.21 (s, 2H), 5.09 (d, J = 7.6 Hz, 1 H), 4.20 (d, J = 10.0 Hz, 1 H), 3.89 - 3.81 (m, 2H), 3.75 (s, 2H), 3.66 - 3.57 (m, 14H), 3.52 - 3.45 (m, 2H), 3.43 - 3.37 (m, 2H), 3.14- 3.05 (m, 4H), 2.71 (s, 2H), 2.64 (t, J = 7.4 Hz, 2H), 2.50 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H).

[0695] Synthesis of Compound 7

[0696] Payload precursor (PNU-159682, CAS: 202350-68-3) was purchased from Shanghai Jainchao Biotechnology Co. Ltd (Product #: WY10706-230701).

[0697] Scheme 12: Synthesis of Compound 7 is shown in Figure 86.

[0698] To a solution of PNU-159682 (100 mg, 0.16 mmol, 1.0 eq) and DIPEA (60 mg, 0.46 mmol, 2.9 eq) in DMF (10 mL) was added bis(perfluorophenyl) carbonate (368 mg, 0.93 mmol, 5.8 eq) in DMF (4 mL) slowly and the mixture was stirred at 0 °C for 3 h. The mixture was diluted with H2O (60 mL) and extracted with EtOAc (3 x 40 mL). The organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by RP column chromatography (C18 column, MeCN with H2O containing 0.1% TFA) to afford Int 46 (70 mg, 53% yield) as a red solid.

[0699] LCMS: m / z = 852.0 [M+H]+1H NMR (400 MHz, Chloroform-d): 5 13.82 (s, 1 H), 13.23 (s, 1 H), 8.04 - 8.00 (m, 1 H), 7.78 (t, J = 8.1 Hz, 1 H), 7.39 (d, J = 8.5 Hz, 1 H), 5.66 - 5.61 (m, 1 H), 5.44 - 5.41 (m, 1 H), 5.36 - 5.32 (m, 2H), 4.94 (s, 1 H), 4.92 (s, 1 H), 4.42 - 4.32 (m, 6H), 4.09 - 4.06 (m, 5H), 3.91 - 3.84 (m, 2H), 3.71 - 3.61 (m, 3H), 3.45 (s, 3H), 1.43 (d, J = 6.6 Hz, 3H).

[0700] To a solution of Int 45 (10 mg, 9.61 pmol, 1.0 eq) in DMF (0.5 mL) was added HOBt (649.6 pg, 4.81 pmol, 0.5 eq) and / V, / V-dimethylethylenediamine (3 pL, 27.87 pmol, 2.9 eq). The mixture was stirred at room temperature for 10 min. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (3 x 10 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo to afford Int 47 (12 mg) as a crude colourless oil which was used directly without further purification.

[0701] LCMS: m / z = 990.7 [M+H]+

[0702] To a solution of Int 47 (133.2 mg, 0.13 mmol, 1.0 eq) and DIPEA (52.3 mg, 0.40 mmol, 3.1 eq) in DMF (2 mL) was added Int 46 (114.6 mg, 0.13 mmol, 1.0 eq). The mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (30 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude residue obtained was purified by prep-HPLC (C18 column, 55 - 100% MeCN with H2O containing 0.1% NH4HCO3) to afford Int 48 (20 mg, 9% yield) as an orange solid. LCMS m / z = 830.0 [M+2H]2+

[0703] To a solution of Int 48 (18.2 mg, 10.98 pmol, 1.0 eq) in MeCN (0.5 mL) and H2O (0.5 mL) was added LiOH.H2O (6.0 mg, 143.00 pmol, 13.0 eq). The mixture was stirred at 0 °C for 40 min and then quenched with HCOOH aq. solution and acidified to pH ~3. The resulting mixture was freeze-dried to afford the crude, which was dissolved in CH2CI2, filtered and the filtrate was lyophilised to afford Compound 7 (12 mg, 72% yield) as an orange solid.

[0704] LCMS: m / z = 759.6 [M+2H]2+

[0705] HPLC: 91.1 % purity (abs 254nm)

[0706] 1H NMR (400 MHz, DMSO-cfe): 6 14.05 (s, 1 H), 9.22 (d, J = 11.1 Hz, 1 H), 8.15 (d, J = 10.9 Hz, 1 H), 7.96 - 7.88 (m, 3H), 7.70 - 7.60 (m, 1 H), 7.22 - 7.06 (m, 3H), 7.03 - 6.88 (m, 1 H), 6.64 (dd, J = 17.7, 11.0 Hz, 1 H), 6.04 (d, J = 17.6 Hz, 1 H), 5.86 - 5.66 (m, 1 H), 5.45 (d, J = 10.9 Hz, 1 H), 5.32 (t, J = 4.9 Hz, 1 H), 5.24 - 5.18 (m, 1 H), 5.16 - 5.03 (m, 2H), 5.01 - 4.89 (m, 3H), 4.72 - 4.54 (m, 2H), 4.32 - 4.13 (m, 2H), 3.99 (s, 3H), 3.72 - 3.59 (m, 4H), 3.55 - 3.42 (m, 18H), 3.38 - 3.35 (m, 6H), 3.19 - 3.15 (m, 4H), 2.90 - 2.79 (m, 10H), 2.68 - 2.61 (m, 7H), 2.40 (s, 3H), 2.34 - 2.32 (m, 2H), 2.03 - 1.96 (m, 4H), 1.32 - 1.25 (m, 3H).

[0707] Synthesis of Compound 8

[0708] Payload precursor (Monomethyl auristatin E, CAS: 474645-27-7) was purchased from Shanghai Jianchao Biotechnology Co. Ltd (Product #: 20230316).

[0709] Scheme 13: Synthesis of Compound 8 is shown in Figure 87.

[0710] To a solution of Int 45 (140 mg, 0.13 mmol, 1.0 eq) in DMF (4 mL) were added pyridine (1 mL), MMAE (96.5 mg, 0.13 mmol, 1.0 eq), HOBt (36.4 mg, 0.27 mmol, 2.1 eq) and DIPEA (69.5 mg, 0.54 mmol, 4.2 eq). The mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with H2O (50 mL) and extracted with EtOAc (3 x 30 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by column chromatography (SiO2, CH2Cl2 / MeOH = 20: 1 to 10:1) to afford Int 49 (110 mg, 51 % yield) as a white solid.

[0711] LCMS: m / z = 1619.7 [M+H]+1H NMR (400 MHz, Chloroform-d): 5 8.42 (s, 1 H), 8.16 (s, 1 H), 7.38 - 7.28 (m, 6H), 7.26 - 7.22 (m, 1 H), 6.99 - 6.90 (m, 4H), 6.79 (s, 1 H), 6.62 - 6.50 (m, 3H), 5.92 (d, J = 17.6 Hz, 1 H), 5.43 (d, J = 10.9 Hz, 2H), 5.38 - 5.35 (m, 1 H), 5.31 - 5.26 (m, 3H), 5.06 (s, 3H), 4.94 (s, 1 H), 4.65 (t, J = 8.2 Hz, 1 H), 4.27 - 4.05 (m, 8H), 3.83 (t, J = 6.1 Hz, 3H), 3.74 (s, 3H), 3.69 - 3.56 (m, 18H), 3.49 - 3.47(m, 3H), 3.47 - 3.37 (m, 10H), 3.31 (s, 3H), 3.06 (t, J = 7.4 Hz, 3H), 3.00 (s, 3H), 2.87 (s, 3H), 2.69 (t, J = 5.8 Hz, 2H), 2.63 (t, J = 7.4 Hz, 3H), 2.51 (s, 4H), 2.42 - 2.36 (m, 2H), 2.21 - 2.12 (m, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 1.92 - 1.84 (m, 8H), 1.31 - 1.22 (m, 5H), 1.03 - 0.95 (m, 6H), 0.91 - 0.79 (m, 18H).

[0712] To a solution of Int 49 (100 mg, 0.06 mmol, 1.0 eq) in MeCN (5 mL) and H2O (5 mL) was added L1OH.H2O (26 mg, 0.62 mmol, 10.3 eq) at 0 °C. The mixture was stirred at 0 °C for 40 min, and then quenched with HCOOH (41 pL, 1.09 mmol, 18.2 eq). The mixture was concentrated in vacuo and purified by prep-HPLC (MeCN with H2O containing 0.1 % TFA) to afford Compound 8 as a TFA salt. The product was dissolved in H2O (10 mL), basified to pH 8 with sat. NaHCOs (aq.) and extracted with CHCh / IPA (3:1 v / v, 3 x 15 mL). The combined organic extracts were concentrated in vacuo to afford Compound 8 (50.1 mg, 55 % yield) as a white solid.

[0713] LCMS: m / z = 1479.7 [M+H]+

[0714] HPLC: Purity 95.9 % (abs 254nm)

[0715] 1H NMR (400 MHz, DMSO-d6): 5 9.16 (s, 1 H), 8.29 - 7.61 (m, 4H), 7.30 - 7.26 (m, 4H), 7.20 - 7.15 (m, 1 H), 7.10 - 7.01 (m, 3H), 6.65 (dd, J = 17.6, 10.8 Hz, 1H), 6.05 (d, J = 17.6 Hz, 1 H), 5.79 (s, 1 H), 5.46 (d, J = 10.8 Hz, 1 H), 5.41 - 5.21 (m, 2H), 5.09 - 4.93 (m, 2H), 4.73 (d, J = 7.8 Hz, 1 H), 4.50 - 4.39 (m, 2H), 4.26 (t, J = 11.0 Hz, 1 H), 4.04 - 3.93 (m, 2H), 3.79 - 3.68 (m, 4H), 3.59 - 3.47 (m, 14H), 3.37 - 3.34 (m, 4H), 3.24 - 3.11 (m, 9H), 2.97 (s, 1 H), 2.90 - 2.86 (m, 4H), 2.67 - 2.62 (m, 2H), 2.45 - 2.39 (m, 6H), 2.25 - 1.97 (m, 6H), 1.82 - 1.72 (m, 4H), 1.54 - 1.44 (m, 4H), 1.17 (s, 5H), 1 .05 - 0.97 (m, 6H), 0.85 - 0.77 (m, 18H).

Claims

CLAIMS1. An antibody conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising an antibody, or an antigen-binding fragment thereof, covalently linked to one or more payload moieties; wherein the antibody, or antigen-binding fragment thereof, comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7.

2. The antibody conjugate according to claim 1, wherein the HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3.

3. The antibody conjugate according to claim 1 or claim 2, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto.

4. The antibody conjugate according to any one of claims 1 to 3, wherein the antibody, or antigen-binding fragment thereof, comprises a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

5. The antibody conjugate according to claim 1 or claim 2, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto.

6. The antibody conjugate according to any one of claims 1, 2 and 5, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 11, or a variant having at least 80% identity thereto.

7. The antibody conjugate according to claim 1 or claim 2, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto.

8. The antibody conjugate according to any one of claims 1, 2 and 7, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

9. The antibody conjugate according to claim 1 or claim 2, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto.

10. The antibody conjugate according to any one of claims 1, 2 and 9, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

11. The antibody conjugate according to claim 1, wherein the HCDR3 comprises an amino acid sequence according to SEQ ID NO: 4.

12. The antibody conjugate according to claim 1 or claim 11, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto.

13. The antibody conjugate according to any one of claims 1, 11 and 12, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto.

14. The antibody conjugate according to claim 1 or claim 11, wherein the antibody, or antigen-binding fragment thereof, comprises a VH comprising an amino acidsequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto.

15. The antibody conjugate according to any one of claims 1 , 11 and 14, wherein the antibody, or antigen-binding fragment thereof, comprises a VL comprising an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto.

16. The antibody conjugate according to any one of the preceding claims, wherein the antibody, or antigen-binding fragment thereof, comprises an Fc region, optionally wherein the Fc region comprises an amino acid sequence according to SEQ ID NO: 39 or SEQ ID NO: 40, or a modified Fc region, optionally wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 41 or SEQ ID NO: 42.

17. The antibody conjugate according to any one of the preceding claims, wherein the antibody conjugate comprises an amino acid sequence according to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89 or SEQ ID NO 90.

18. The antibody conjugate according to any one of the preceding claims, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein: i. the HC comprises an amino acid sequence according to SEQ ID NO: 44, and the LC comprises an amino acid sequence according to SEQ ID NO: 43; ii. the HC comprises an amino acid sequence according to SEQ ID NO: 46, and the LC comprises an amino acid sequence according to SEQ ID NO: 45; iii. the HC comprises an amino acid sequence according to SEQ ID NO: 48, and the LC comprises an amino acid sequence according to SEQ ID NO: 47; iv. the HC comprises an amino acid sequence according to SEQ ID NO: 50, and the LC comprises an amino acid sequence according to SEQ ID NO: 49; v. the HC comprises an amino acid sequence according to SEQ ID NO: 56, and the LC comprises an amino acid sequence according to SEQ ID NO: 55; vi. the HC comprises an amino acid sequence according to SEQ ID NO: 58, and the LC comprises an amino acid sequence according to SEQ ID NO: 57; vii. the HC comprises an amino acid sequence according to SEQ ID NO: 60, and the LC comprises an amino acid sequence according to SEQ ID NO: 59; viii. the HC comprises an amino acid sequence according to SEQ ID NO: 62, and the LC comprises an amino acid sequence according to SEQ ID NO: 61 ;ix. the HC comprises an amino acid sequence according to SEQ ID NO: 64, and the LC comprises an amino acid sequence according to SEQ ID NO: 63; x. the HC comprises an amino acid sequence according to SEQ ID NO: 66, and the LC comprises an amino acid sequence according to SEQ ID NO: 65; xi. the HC comprises an amino acid sequence according to SEQ ID NO: 68, and the LC comprises an amino acid sequence according to SEQ ID NO: 67; xii. the HC comprises an amino acid sequence according to SEQ ID NO: 70, and the LC comprises an amino acid sequence according to SEQ ID NO: 69; xiii. the HC comprises an amino acid sequence according to SEQ ID NO: 72, and the LC comprises an amino acid sequence according to SEQ ID NO: 71 ; xiv. the HC comprises an amino acid sequence according to SEQ ID NO: 74, and the LC comprises an amino acid sequence according to SEQ ID NO: 73; xv. the HC comprises an amino acid sequence according to SEQ ID NO: 76, and the LC comprises an amino acid sequence according to SEQ ID NO: 75; or xvi. the HC comprises an amino acid sequence according to SEQ ID NO: 78, and the LC comprises an amino acid sequence according to SEQ ID NO: 77.

19. The antibody conjugate according to any one of the preceding claims, wherein the antibody, or antigen-binding fragment thereof, is covalently linked to the payload moiety by a linker.

20. The antibody conjugate according to claim 19, wherein the linker is an enzyme cleavable linker.

21. The antibody conjugate according to claim 19 or claim 20, wherein the linker comprises a p-D-glucuronide linker or a p-D-galactoside linker.

22. The antibody conjugate according to any one of the preceding claims, wherein the payload moiety is selected from a prodrug, a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a coloured agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumour cell growth, promotes tumour cell apoptosis or necrosis.

23. The antibody conjugate according to claim 22, wherein the payload moiety is a prodrug or a drug.

24. The antibody conjugate according to claim 22 or claim 23, wherein the drug is a topoisomerase I inhibitor, preferably exatecan.

25. The antibody conjugate according to claim 22 or claim 23, wherein the drug is a topoisomerase II inhibitor, preferably PNU- 159682.

26. The antibody conjugate according to claim 22 or claim 23, wherein the drug is a tubulin inhibitor, preferably monomethyl auristatin E (MMAE).

27. The antibody conjugate according to claim 22 or claim 23, wherein the drug is a DNA binding payload, preferably PBD SG3249.

28. The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure represented by the formula:wherein: the wavy line indicates the covalent attachment to the antibody or antigen-binding fragment thereof via linker, L;R is H or an optionally substituted C1-C4 alkyl group, an optionally substituted C1-C4 alkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C1-C4 alkyl carboxy C1-C4 alkyl group, F, Cl, Br, or I, CN, an optionally substituted C1-C4 alkylsulfonyl group, an optionally substituted arylsulfonyl group, or an NRz group, where NRz is selected from H, optionally substituted C1-C4 alkyl group or optionally substituted C1-C4 acyl;R1 is H, a C1-C4 alkyl group, or a C1-C4 alkoxy group;Y is selected from an optionally substituted C1-C10 alkyl group, or a group having the formula:O[C(RA)2] — X3— [C(RA)2]Pwhere o and p are independently from one another selected from an integer of 1 to 20, whereby o and p may be the same integer or a different integer, X3 is i) N, S or O, or ii) an aryl group or a heteroaryl group, wherein [C(RA)2]O and [C(RA)2]Pare present in the meta position of said aryl group or said heteroaryl group, and each RA is independently from one another selected from H or an optionally substituted C1-C4 alkyl group or an optionally substituted C1-C4 acyl group;X is O or S; m is an integer from 1 to 20;R2 and R3 are independently selected from H, C1-C20 alkyl, and -Ci-Cs heteroalkyl; R4 is selected from H or an electron-withdrawing group, andG is a sugar moiety selected from p-D-galactoside, p-D-glucuronide, p-D-glucoside, a- D-mannoside, or fucoside.

29. The antibody conjugate according to claim 28, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

30. The antibody conjugate according to claim 28, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof. 31 . The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

32. The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

33. The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

34. The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

35. The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

36. The antibody conjugate according to any one of claims 1 to 23, wherein the antibody conjugate comprises a structure having the formula:wherein the wavy line indicates attachment to the antibody or antigen binding fragment thereof.

37. A pharmaceutical composition comprising the antibody conjugate according to any one of the preceding claims, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

38. The antibody conjugate according to any one of claims 1-36, or the pharmaceutical composition according to claim 37, for use as a medicament.

39. The antibody conjugate according to any one of claims 1-36, or the pharmaceutical composition according to claim 37, for use in the treatment of cancer in a subject.

40. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody conjugate according to any one of claims 1-36, or the pharmaceutical composition according to claim 37.

41. The antibody conjugate or pharmaceutical composition for use according to claim 39, or the method according to claim 40, wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T-cell lymphoma, and B-cell lymphoma.

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