Linker moieties

Abstract

Provided here are linker moieties comprising (a) an amino group for conjugation to an antigen-binding moiety; (b) at least one click group for connecting a payload comprising moiety; (c) a urea group wherein RN is selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

Description

[0001] Linker Moieties

[0002] Cross-Reference to Related Applications

[0003] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 694,444, filed

[0004] September 13, 2024, the disclosure of which is hereby incorporated herein by reference in its entirety.

[0005] Reference to an Electronic Sequence Listing

[0006] The content of the electronic sequence listing (366592000740seqlist.xml; Size: 22,434 bytes; and Date of Creation: August 29, 2025) is herein incorporated by reference in its entirety.

[0007] Technical Field

[0008] The present disclosure relates to linker moieties, and their use in antibody drug-conjugates. The present disclosure also relates to methods of medical treatment and prophylaxis using the antibody drugconjugates.

[0009] Background

[0010] Cancers remain the leading cause of deaths worldwide. Chemotherapies have good clinical benefits, but due to their low specificity they have very significant side effects and low therapeutic indices. More targeted therapies, such as monoclonal antibody therapies, show good specificity but response rates are smaller. Antibody-drug conjugates (ADCs) are a therapeutic modality that harness an antibody’s target specificity to selectively deliver cytotoxic payloads to tumors and are proving increasingly effective in the clinic.

[0011] While ADCs have proven successful in both solid and haematological cancers, resistance and tumor heterogeneity are major causes of failure clinically (Yamazaki et al., Nat Commun (2021) 12 (1): 3528). Tumor heterogeneity is known to lead to recurrence, metastasis, and acquired resistance to ADCs and other therapeutic strategies. Heterogenous tumors with differential drug sensitivities result in aggressive tumor growth, high relapse rates, and poor survival.

[0012] To combat these challenges, the majority of chemotherapeutic regimens consist of a combination of drugs. Co-delivery of small molecules can overcome resistance, generate additive or synergistic effects, and enhance therapeutic efficacy.

[0013] Emergence of tumors refractory to current therapies has given impetus to the evaluation of new ADC formats. This challenge has led to the exploration of dual-drug ADCs capable of delivering two mechanistically distinct payloads simultaneously. Strategies for the construction of dual-drug ADCs involve attachment of both drugs to one linker or through the use of two different conjugation sites on the antibody, and existing approaches are reviewed by Nervig et al., J. ADC. (2023) (DOI: 10.14229 / jadc.2023.01 .05.001). The design of linkers which enable such approaches is important. Summary

[0014] In a first aspect, the present disclosure provides a linker moiety comprising:

[0015] (a) an amino group for conjugation to an antigen-binding moiety;

[0016] (b) at least one click group for connecting a payload comprising moiety;

[0017] (c) a urea group: wherein RNis selected from H and -(C1 5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

[0018] In some embodiments, one click group is linked to the urea group.

[0019] In some embodiments, two or more click groups are linked to the urea group. In some of these embodiments, two click groups are linked to the urea group.

[0020] In a second aspect, the present disclosure provides a linker between:

[0021] (a) at least one payload; and

[0022] (b) an antigen-binding moiety; comprising a moiety derived from a compound of the first aspect.

[0023] Thus, the linker comprises:

[0024] (a) an amino group conjugated to an antigen-binding moiety;

[0025] (b) at least one payload comprising moiety clicked to a click group;

[0026] (c) the urea group: wherein RNis selected from H and -(C1 5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

[0027] In a third aspect, the present disclosure provides a conjugate comprising:

[0028] (a) at least one payload;

[0029] (b) an antigen-binding moiety; wherein the linker between the payload and the antibody comprises a moiety derived from a compound of the first aspect. Thus, the conjugate comprises:

[0030] (a) an amino group conjugated to an antigen-binding moiety;

[0031] (b) at least one payload comprising moiety clicked to a click group;

[0032] (c) the urea group: wherein RNis selected from H and -(C1 5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

[0033] In a fourth aspect, the present disclosure provides the use of a conjugate of the third aspect in the manufacture of a medicament for treating cancer. The fourth aspect also provides a conjugate of the third aspect for use in the treatment of cancers. These methods encompass the use of the conjugates of the third aspect.

[0034] In a fifth aspect, the present disclosure provides a linker-payload molecule comprising at least a payload for conjugation to an antigen-binding moiety, wherein the linker for conjugation to the antibody comprises a moiety derived from a compound of the first aspect.

[0035] Thus, the linker-payload molecule comprises:

[0036] (a) an amino group for conjugation to an antigen-binding moiety;

[0037] (b) at least one payload comprising moiety clicked to a click group;

[0038] (c) the urea group: wherein RNis selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

[0039] In a sixth aspect, the present disclosure provides a modified antigen-binding moiety comprising a moiety derived from a compound of the first aspect.

[0040] Thus, the modified antigen-binding moiety comprises: (a) an amino group conjugated to an antigen-binding moiety;

[0041] (b) at least one click group for connecting a payload comprising moiety; and

[0042] (c) the urea group: wherein RNis selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; b indicates where the at least one click group is linked to the urea group.

[0043] The present disclosure also provides a composition comprising an antigen-binding molecule according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0044] Description

[0045] The use of click groups to attach payload containing moieties to the urea group allows for a wide range of payload types to be conjugated. The linking groups of the present disclosure comprise a hydrophilic urea group which may lead to a reduction in toxicities and an improvement in biophysical, stability and pharmacokinetic properties.

[0046] The present disclosure provides a linker moiety comprising:

[0047] (a) an amino group for conjugation to an antigen-binding moiety;

[0048] (b) at least one click group for connecting a payload comprising moiety;

[0049] (c) a urea group: wherein RNis selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

[0050] In some embodiments, RNis H.

[0051] In some embodiments, RNis -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-.

[0052] In some embodiments, RNis -(C1-5 alkylene)-C(O)OH.

[0053] In some embodiments, RNis -CH2CH2OCH2CH2C(O)OH.

[0054] In some embodiments, RNis CH2C(O)OH. The amino group may be linked to the urea group by a first spacer group. The first spacer group (A1) may comprise:

[0055] (i) a C1-7 alkylene group; and / or

[0056] (ii) a PEG 1 to 12 group.

[0057] A1 may be of the formula:

[0058] -(CH2)xa-(C2H4O)xb-(CH2)xc-urea, where xa is 0 or 1 , xb is 0-12, and xc is 0 to 6, wherein at least one of xa and xb is 1 .ureaindicates where A1 is bound to the urea group.

[0059] In some embodiments, xb is 0, and xa+xc are from 1 to 7, such as 5. In some embodiments, A1 is -(CH2)6-.

[0060] In other embodiments, xb is from 1 to 12. In some of these embodiments, xb is from 1 to 6. In some of these embodiments, xb is from 1 to 3, i.e. 1 , 2 or 3.

[0061] In some embodiments where xb is 1 to 12, xc is 1 to 6, or 1 to 2. In some of these embodiments, xc is 1 . In some of these embodiments, xc is 2.

[0062] In some embodiments, xa is 0, xb is 1 to 6 and xc is 2. In some of these embodiments, A1 is -(C2H4)-O-(C2H4)-. In some of these embodiments, A1 is -(C2H4O)3-(C2H )-.

[0063] The at least one click group may be selected from either member of the following click-group pairs:

[0064] In some embodiments, the at least one click group linked to the urea group of the linker moiety is selected from the first member of the click-group pairs (e.g. , azide, tetrazine, and tetrazine derivatives). In some embodiments, the at least one click group linked to the urea group of the linker moiety is selected from the second member of the click-group pairs (e g., alkyne, cyclooctyne, cyclooctyne derivatives and cyclooctyne analogues, and strained alkene).

[0065] Cyclooctyne, cyclooctyne derivatives and cyclooctyne analogues for use in the present disclosure include:

[0066] These groups can alternatively be called cyclic alkynes.

[0067] Tetrazine and tetrazine derivatives for use in the present disclosure include:

[0068]

[0069] Strained alkenes for use in the present disclosure may have the structure:

[0070] In some embodiments, the click group attached to the urea group may be a dibenzoazacyclooctyne (DIBAC / DBCO) group, a 1-ethylhept-6-enoxy group, or tetramethylthiocycloheptyne sulfoximine (TMTHSI):

[0071] In some embodiments, the click group linked to the urea group is azide.

[0072] In some embodiments, the click group linked to the urea group is tetrazine or a tetrazine derivative.

[0073] In some embodiments, the click group linked to the urea group is an alkyne (-CCH).

[0074] In some embodiments, the click group linked to the urea group is cyclooctyne or a cyclooctyne derivative.

[0075] In some embodiments, the click group linked to the urea group is norbonene or a norbonene derivative.

[0076] In some embodiments, the click group linked to the urea group is methylcyclopropene (1-MCP).

[0077] In some embodiments, the click group linked to the urea group is phenyl-tetrazine. In some embodiments, the click group linked to the urea group is selected from the following groups:

[0078] , or

[0079] In some embodiments, the click group linked to the urea group is:

[0080] In some embodiments, one click group is linked to the urea group.

[0081] In some embodiments, two or more click groups are linked to the urea group. In some of these embodiments, two click groups are linked to the urea group.

[0082] The at least one click group may be linked to the urea group by a second spacer group (B1). In some embodiments, the second spacer group is branched, such that two or more click groups (for example two click groups) are linked to the urea group. In other embodiments, the second spacer group is not branched, such that a single click group is linked to the urea group. In some embodiments where two or more click groups are linked to the urea group, they are the same.

[0083] In other embodiments where two or more click groups are linked to the urea group, they are selected from orthogonal click-group pairs.

[0084] In some embodiments, the second spacer group (B1) is of formula (B1 -1):

[0085] In some embodiments, xl3 is 0 to 2. In some embodiments, xl3 is 0. In some embodiments, xl3 is 1 . In some embodiments, xl3 is 2.

[0086] In some embodiments, xl4 is 0. In some embodiments, xl4 is 1 .

[0087] In some embodiments, xe1 is 2-4. In some embodiments, xe1 is 2. In some embodiments, xe1 is 3. In some embodiments, xe1 is 4.

[0088] In some embodiments, xe2 is 2-4. In some embodiments, xe2 is 2. In some embodiments, xe2 is 3. In some embodiments, xe2 is 4.

[0089] In some of these embodiments the second spacer group (B1) is of formula (B1 -2): where xd is 0 to 3,

[0090] RNB1is -(C2H4O)xei-(CH2)xfi-(NH)xgi-(C(=O)CH2)xhi- where xe1 is 0 or 1 , xf1 is 0 to 2, xg1 is 0 or 1 , and xh1 is 0 or 1 ,

[0091] RNB2is H 0r -(C2H4O)xe2-(CH2)xf2-(NH)xg2-(C(=O)CH2)xh2- where xe2 is 0 or 1 , xf2 is 0 to 2, xg2 is 0 or 1 , and xh2 is 0 or 1 .

[0092] In some embodiments, RNB2is H. In some embodiments, RNB2is -(C2H4O)xe2-(OH2)xi2-(NH)xg2- (C(=O)CH2)xh2-. In other embodiments, RNB2is the same as RNB1. In some embodiments, xd is 0 to 2. In some embodiments, xd is 0 to 1 . In some embodiments, xd is 0. In some embodiments, xd is 1 . In some embodiments, xd is 2. In some embodiments, xd is 3. In some embodiments, xd is 0 or 2.

[0093] In some embodiments, xe1 is 0. In some embodiments, xe1 is 1 .

[0094] In some embodiments, xf1 is 0 to 1 . In some embodiments, xf1 is 0. In some embodiments, xf1 is 1 . In some embodiments, xf1 is 2. In some embodiments, xf1 is 0 or 2.

[0095] In some embodiments, xg1 is 0. In some embodiments, xg1 is 1 .

[0096] In some embodiments, xh1 is 0. In some embodiments, xh1 is 1 .

[0097] In some embodiments, xe2 is 0. In some embodiments, xe2 is 1 .

[0098] In some embodiments, xf2 is 0 to 1 . In some embodiments, xf2 is 0. In some embodiments, xf2 is 1 . In some embodiments, xf2 is 2. In some embodiments, xf2 is 0 or 2.

[0099] In some embodiments, xg2 is 0. In some embodiments, xg2 is 1 .

[0100] In some embodiments, xe1 is 1 , xf1 is 2, xg1 is 0, and xh1 is 0. In some embodiments, xe1 is 0, xf1 is 0, xg1 is 0, and xh1 is 1 . In some embodiments, xe1 is 1 , xf1 is 2, xg1 is 1 , and xh1 is 1.

[0101] In some embodiments, xe2 is 1 , xf2 is 2, xg2 is 0, and xh2 is 0. In some embodiments, xe2 is 0, xf2 is 0, xg2 is 0, and xh2 is 1 . In some embodiments, xe2 is 1 , xf2 is 2, xg2 is 1 , and xh2 is 1 .

[0102] In some embodiments, the second spacer group (B1) is selected from the groups containing:

[0103] In some embodiments, the present disclosure provides a linker moiety which is:

[0104]

[0105] The present disclosure provides a linker comprising:

[0106] (a) an amino group conjugated to an antigen-binding moiety;

[0107] (b) at least one payload comprising moiety clicked to a click group; (c) the urea group: wherein RNis selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

[0108] Examples of the clicked group are shown below:

[0109]

[0110] As shown in the table above, the reaction between the first and second members of the click group pairs can result in two isomeric products, i.e. a mixture. The present disclosure includes both isomeric forms and mixtures thereof in any ratio when only one is shown.

[0111] The amino group may be linked to the urea group by a first spacer group (A1) as defined above.

[0112] The at least one click group may be linked to the urea group by a second spacer group (B1) as defined above. In some embodiments, the linker comprises one of the following groups:

[0113]

[0114]

[0115]

[0116] The present disclosure provides a conjugate comprising:

[0117] (a) at least one payload; (b) an antigen-binding moiety; wherein the linker between the payload and the antibody comprises a moiety derived from a compound of the first aspect.

[0118] Thus, the conjugate comprises: (a) an amino group conjugated to an antigen-binding moiety;

[0119] (b) at least one payload comprising moiety clicked to a click group;

[0120] (c) the urea group: wherein RNis selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group. Payload comprising moieties

[0121] A payload comprising moiety according to the present disclosure may comprise a payload, and a linker moiety between the payload and a click group, which click group is selected from the click group pairs described above. The linker moiety may be a cleavable linker moiety or a non-cleavable moiety.

[0122] Cleavable linkers typically utilise differences between the environment of systemic circulation and that in cancer cells / the tumor microenvironment to release the payload moiety in a targeted manner. Cleavable linkers include chemical cleavage linkers (e.g. acid-cleavable linkers, GSH-cleavable linkers, Fe(ll)- cleavable linkers) and enzyme cleavage linkers (e.g. cathepsin-cleavable linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers, sulfatase-cleavable linkers).

[0123] In some embodiments, a linker moiety according to the present disclosure is a chemical cleavage linker. In some embodiments, a linker moiety according to the present disclosure is an enzyme cleavage linker. In some embodiments, a linker moiety is an acid-cleavable linker, e.g. comprising a hydrazone group (e.g. a 6-maleimidocaproylhydrazone linker or a (4-(4-acetylphenoxy)butanoic acid) hydrazaone linker), a carbonate group or a silyl ether group. In some embodiments, a linker moiety is a GSH-cleavable linker, e.g. comprising a disulfide group. In some embodiments, a linker moiety is a Fe(ll)-cleavable linker, e.g. comprising a 1 ,2,4-trioxolane group. In some embodiments, a linker moiety is a cathepsin-cleavable linker, e.g. comprising a dipeptide (e.g. a valine-citru Hine linker, a phenylalanine-lysine linker or a valinealanine linker), a triglycyl peptide (CX) or a cBu-Cit group. In some embodiments, the linker moiety is GGFG (Glycine-Glycine-Phenylalanine-Glycine). In some embodiments, a linker moiety is a glucuronidase-cleavable linker, e g. comprising a -giucuronide group. In some embodiments, a linker moiety is a glycosidase-cleavable linker, e.g. comprising a p-galactoside group. In some embodiments, a linker moiety is a phosphatase-cleavable linker, e.g. comprising a pyrophosphate group. In some embodiments, a linker moiety is a sulfatase-cleavable linker, e.g. comprising an arylsulfate group. In some embodiments, a linker moiety is a photo-responsive linker, e.g. comprising a heptamethine cyanine fluorophore group, an O-nitrobenzyl group or a PC4AP group. In some embodiments, a linker moiety is a biorthogonal cleavable linker, e.g. comprising a dsProc group.

[0124] Non-cleavable linkers remain inert in common chemical and enzymatic environments in the body, with the payload moiety being released following processing of the ADC by cellular lysosomal proteases. Non- cleavable linkers include linkers comprising thioether or maleimidocaproyl groups.

[0125] In some embodiments, a linker moiety is a thioether linker. In some embodiments, a linker moiety is a maleimidocaproyl linker, e.g. comprising a 2-(maleimidomethyl)-1 ,3-dioxane (MD) group or a Mal-PAB group. In some embodiments, a linker moiety comprises a polyethylene glycol (PEG) group and an alkyne, triazole or piperazine group.

[0126] In some embodiments, a linker moiety further comprises a spacer moiety. Spacer moieties are sometimes required due to the bulky nature of payload moieties. Commonly employed spacer moieties include para- aminobenzyl (PAB), para-aminobenzyl carbamate (PABC), hemiaminal groups, PEG groups, polar acyl sulfamide groups, polar carbamoyl sulfamide groups and HydraSpace (described e.g. in Verkade et al., Antibodies (Basel) (2018) 7(1 ):12 and WO 2016 / 053107 A1 , both of which are hereby incorporated by reference in their entirety). PABC is commonly employed as a spacer moiety in cathepsin-cleavable dipeptide linkers, p-glucuronidase-cleavable linkers, p-galactosidase-cleavable linkers and phosphatase cleavable linkers.

[0127] In some embodiments, there is a functional group to connect the click group. This functional group may be selected from carbonyl (C=O) and oxy (O). Where the click group is DBCO or TMTHSI, the functional group may be carbonyl. Where the click group is TCO, the functional group may be oxy.

[0128] In some embodiments, the link between the payload moiety and the click moiety comprises: payload

[0129] Q is: , where Qxis such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue;

[0130] X is: where a = 0 to 5, b = 0 to 8, c = 0 or 1 , d = 0 to 5. a may be 0, 1 , 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1 . In further embodiments, a is 1 . b may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. In some embodiments, b is 0 to 6. In some of these embodiments, b is 0 to 4, and may be 0, 1 , 2, 3 or 4. In further embodiments, b is 3. c may be 0 or 1 . In some of these embodiments, c is 1 . d may be 0, 1 , 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 0, 1 or 2. In further embodiments, d is 2. In other further embodiments, d is 1 . In other further embodiments, d is 0.

[0131] In some embodiments of X, a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8.

[0132] In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a nonnatural amino acid.

[0133] In one embodiment, Q is selected from: Phe, Lys, Vai, Ala, Cit, Leu, lie, Arg, Ser, and Trp, where Cit is citrulline. In one, Q is a serine derivative (see WO2018 / 234636A1).

[0134] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.

[0135] In one embodiment, Q is selected from: co-Phe-Lys-NH, co-Val-Ala-NH, co-Val-Lys-NH, co-Ala-Lys-NH, co-Val-Cit-NH, co-Phe-Cit-NH, co-Leu-Cit-NH, co-lle-Cit-NH, co-Phe-Arg-NH, co-Gly-Cit-NH, co-Gly-Ala-NH, and co-Trp-Cit-NH; where Cit is citrulline.

[0136] In some of these embodiments, Q is selected from: co-Phe-Lys-NH, co-Val-Ala-NH, co-Val-Lys-NH, co-Ala-Lys-NH, co-Val-Cit-NH.

[0137] In further embodiments, Q is selected fromco-Phe-Lys-NH,co-Val-Cit-NHandco-Val-Ala-NH. In some embodiments, the link between the payload moiety (e g. the DDR inhibitor moiety or the TOP1 inhibitor moiety) and the click group comprises: PABC, a cathepsin-cleavable dipeptide, and a PEG2 to PEG4 (e.g. a PEG3) group. In some of these embodiments, the link between the payload moiety and the click group comprises, or is:

[0138] In some embodiments, the link between the payload moiety comprises GGFG (Glycine-Glycine-

[0139] Phenylalanine-Glycine). This may be directly linked to the payload or linked via a CH2 group. In some of these embodiments, the link between the payload moiety and the click group comprises, or is:

[0140] In some embodiments, the payload comprising moiety comprises more than one payload. Thus, in some embodiments, the payload moiety comprises two or more payloads. In some embodiments, the payload moiety comprises two payloads.

[0141] Where the payload moiety comprises two or more payloads, the payload moiety comprises a branching group. In some embodiments, the branch is at a carbon atom. In some embodiments, the branch is at a nitrogen atom. In some of these embodiments, the branching group is:

[0142] In some embodiments, the link between two payloads and the click group comprises:

[0143] where each of Q1and Q2are independently: , where Qxis such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; where a1 = 0 to 5, b1 = 0 to 8, c1 = 0 or 1 , d1 = 0 to 5. each of X2and X3are independently: where a2 = 0 to 5 and b2 = 0 to 8; a3 = 0 to 5, b3 = 0 to 8, c2 = 0 or 1 , d2 = 0 to 5, and e1 = 0 to 8. a1 may be 0, 1 , 2, 3, 4 or 5. In some embodiments, a1 is 0 to 3. In some of these embodiments, a1 is 0 or 1. In further embodiments, a1 is 1 . b2 may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. In some embodiments, b1 is O to 6. In some of these embodiments, b1 is 0 to 4, and may be 0, 1 , 2, 3 or 4. In further embodiments, b1 is 3. c1 may be 0 or 1. In some of these embodiments, c1 is 1. d1 may be 0, 1 , 2, 3, 4 or 5. In some embodiments, d1 is 0 to 3. In some of these embodiments, d1 is 0, 1 or 2. In further embodiments, d1 is 2. In other further embodiments, d1 is 1 . In other further embodiments, d1 is 0. a2 may be 0, 1 , 2, 3, 4 or 5. In some embodiments, a2 is 0 to 3. In some of these embodiments, a2 is 0 or 1. In further embodiments, a2 is 1 . b2 may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. In some embodiments, b2 is 0 to 6. In some of these embodiments, b2 is 0 to 4, and may be 0, 1 , 2, 3 or 4. In further embodiments, b2 is 3. a3 may be 0 or 1. In some of these embodiments, a3 is 1. b3 may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. In some embodiments, b3 is 0 to 6. In some of these embodiments, b3 is 0 to 4, and may be 0, 1 , 2, 3 or 4. In further embodiments, b3 is 3. In some embodiments, b3 is 8. c2 may be 0 or 1. In some of these embodiments, c2 is 1. d2 may be 0, 1 , 2, 3, 4 or 5. In some embodiments, d2 is 0 to 3. In some of these embodiments, d2 is 0 or 1. In further embodiments, d2 is 1 . e1 may be 0, 1 , 2, 3, 4, 5, 6, 7 or 8. In some embodiments, e1 is 0 to 6. In some of these embodiments, e1 is 0 to 4, and may be 0, 1 , 2, 3 or 4. In further embodiments, e1 is 3.

[0144] Q1and Q2are selected from the same options as Q above.

[0145] In some embodiments, Q2and X3are the same as Q1and X2respectively.

[0146] In some of these embodiments, the link between the payload moieties and the click group comprises, or is: In some of these embodiments, the link between the payload moieties and the click group comprises, or is:

[0147] A payload moiety according to the present disclosure comprises or consist of a cytotoxic agent. Payload moieties are described e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14, Goundry and Parker, Org. Process Res. Dev. (2022) 26, 8, 2121-2123, Fu et al., Signal Transduction and Targeted Therapy (2022) 7:93, Wang et al., Acta Pharmaceutica Sinica B (2023) 13 (10): 4025-4059 and Conilh et al., J. Hematol.

[0148] & Oncol. (2023) 16:3, all of which are hereby incorporated by reference in their entirety.

[0149] In some embodiments, a payload moiety according to the present disclosure may comprise or consist of a microtubule-targeting agent, a DNA-targeting agent, an RNA-targeting agent, an immune system- activating agent, an apoptosis-promoting agent, a metabolism-inhibiting agent and a proteasome inhibiting agent.

[0150] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a microtubule-targeting agent. Microtubules play important roles in maintaining proper cellular morphology, signal transduction, organelle transportation, cell motility and cell division. Microtubules are formed of tubulin, and agents that disrupt the tubulin polymerization dynamics, resulting in cell cycle arrest and apoptosis. Tubulin inhibitors have a stronger toxicity to rapidly-dividing cancerous cells than slower- growing, non-cancerous cells. Microtubule-targeting agents include tubulin polymerization enhancers (e g. auristatins, taxanes), and tubulin polymerization inhibitors (e g. maytansinoids, colchicine). In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a maytansinoid, e.g. maytansine or a derivative thereof, e.g. mytansine, DM1 (mertansine) or DM4 (ravtansine). In some embodiments, a payload moiety comprises, or consists of colchicine or a derivative thereof. In some embodiments, a payload moiety comprises, or consists of, an auristatin, e.g. a dolastatin 10 derivative, e.g. monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PE, auristatin PYE, PF-06380101 , auristatin F-hydroxypropylamide (AF-HPA) or azastatin. In some embodiments, a payload moiety comprises, or consists of, a halichondrin B derivative, e.g. eribulin. In some embodiments, a payload moiety comprises, or consists of, a tubulysin or a derivative thereof, e.g. tubulysin A, D, H, U or V. In some embodiments, a payload moiety comprises, or consists of, a cryptophycin or a derivative thereof, e.g. cryptophycin-1 , cryptophycin-52, cryptophycin-55 or cryptophycin-55gly. In some embodiments, a payload moiety comprises, or consists of, an EG5 (i.e. kinesin / KSP / KIF1 1) inhibitor, e.g. ispinesib (SB715992) or a derivative thereof, or filanesib (ARRY-520) or a derivative thereof. In some embodiments, a payload moiety comprises, or consists of, a taxane, e.g. paclitaxel, docetaxel or cabazitaxel. In some embodiments, a payload moiety comprises, or consists of vinca alkaloid, e.g. vinblastine, vincristine, vindesine, vinorelbine or vinflunine. In some embodiments, a payload moiety comprises, or consists of hemiasterlin or a derivative thereof, e.g. hemiasterlin, hemisterlin A or HTI-286.

[0151] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a DNA-targeting agent. DNA-targeting agents include agents that directly or indirectly destroy DNA through introducing / promoting the formation of single- and / or double-strand breaks (e.g. enediynes, topoisomerase inhibitors), DNA alkylating agents (e g. pyrrolo[2,1 -c][1 ,4] benzodiazepines, indolinobenzodiazpines, duocarmycins), and DNA crosslinking agents (e.g. mitomycin C). In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, an enediyne, e.g. a Cal-like enediyne or an anthraquinone fusion enediyne, e g. calciheamicin yh, calciheamicin Oh or uncialamycin. In some embodiments, a payload moiety comprises, or consists of, a topoisomerase inhibitor, e.g. a TOP1 or TOP2 inhibitor, e.g. camptothecin or a derivative thereof, e.g. SN- 38, exatecan, exatecan mesylate (DX-8951f), / V-glycyl-exatecan or deruxtecan (DXd); e.g. an anthracycline, e.g. doxorubicin, daunorubicin, epirubicin, PNU-159682 or idarubicin. In some embodiments, a payload moiety comprises, or consists of, a pyrrolo[2,1 -c][1 ,4] benzodiazepine (PBD) dimer, or a derivative thereof, e.g. a PDB, KMR-28-39, SJG-136 SGD-1882 or SG3199 dimer. In some embodiments, a payload moiety comprises, or consists of, indolinobenzodiazpine (IGN; monoimine) or a derivative thereof. In some embodiments, a payload moiety comprises, or consists of, a pyridinobenzodiazepine (PDD) dimer, or a derivative thereof, e.g. a PDD or FGX5-67 dimer. In some embodiments, a payload moiety comprises, or consists of, a duocarmycin or a derivative thereof, e.g. duocarmycin A, CC1065, duocarmycin SA, DUBA, seco-DIBA or seco-CBI. In some embodiments, a payload moiety comprises, or consists of, mitomycin C.

[0152] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a RNA-targeting agent. Small molecule inhibitors that target RNA can kill both dividing and dormant tumor cells. RNA-targeting agents include RNA splicing inhibitors (e.g. thailanstatin and derivatives thereof) and RNA polymerase II inhibitors (e.g. amatoxins, RNA polymerase ll-IN-2). In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, thailanstatin or a derivative thereof, e.g. thailanstatin A, thailanstatin B, thailanstatin C or FR901464. In some embodiments, a payload moiety comprises, or consists of, an amatoxin, e.g. a-amanitin or -amanitin. In some embodiments, a payload moiety comprises, or consists of, RNA polymerase ll-IN-2.

[0153] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, an immune system-activating agent. Immune-stimulating antibody conjugates (ISACs) employ small molecule-based engagement of the innate and / or adaptive immune systems. A variety of immune- modulating payloads are in development, including Toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists and glucocorticoid receptor modulators (GRMs). In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a TLR agonist, e.g. an agonist of TLR7, TLR8 or TLR9. In some embodiments, a payload moiety comprises, or consists of, a STING agonist, e g. a cyclic dinucleotide (CDN; e.g. 2,3 cGAMP; 3,3 cGAMP; c-di-GMP or c-di-AMP) or a benzimidazole. In some embodiments, a payload moiety comprises, or consists of, a glucocorticoid receptor modulator, e.g. dexamethasone or a derivative thereof.

[0154] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, an apoptosis-promoting agent. Anti-apoptotic proteins such as Bcl-xL can play important roles in tumorigenesis, metastasis and drug resistance. In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a Bcl-xL inhibitor. In some embodiments, a payload moiety comprises, or consists of, ABT-737.

[0155] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a metabolism-inhibiting agent. Metabolism-inhibiting agents such as niacinamide phosphate ribose transferase (NAMPT) inhibitors control the concentration of NAD+ within cells, inducing energy crisis and cell death, and antifolate antimetabolites such as methotrexate, that inhibit dihydrofolate reductase (DHFR) and thereby DNA synthesis. In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a NAMPT inhibitor, e.g. FK-866 or A-1293201 . In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a DHFR inhibitor, e.g. methotrexate of a derivative thereof.

[0156] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a proteasome-inhibiting agent. Proteasome-inhibiting agents include carmaphycins. In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a carmaphycin or a derivative thereof, e.g. carmaphycin A or carmaphycin B.

[0157] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a DNA damage response (DDR) inhibitor. Hereinbelow, for conciseness, ‘a payload moiety which is a DDR inhibitor’ may be referred to simply as a ‘DDR inhibitor moiety’.

[0158] The DNA Damage Response (DDR) is a complex network of mechanisms for detecting and repair DNA damage, in order to preserve genomic stability. The DDR is reviewed e.g. in Groelly et al., Nature Reviews Cancer (2023) 23:78-94 and Molinaro et al., Cancers (Basel). (2021) 13(15): 3819, both of which are hereby incorporated by reference in their entirety.

[0159] The detection of DNA damage and initiation of repair pathways is mediated by proteins such as ATM (Ataxia-Telangiectasia Mutated) and ATR (Ataxia-Telangiectasia and Rad3-Related). ATM is a protein kinase activated by double-strand breaks in DNA, and which initiates downstream signaling. ATR is activated by DNA damage and replication stress, and in particular responds to single-strand breaks and stalled DNA replication forks. CHK1 and CHK2 (Checkpoint Kinases 1 and 2) are downstream effectors of ATM and ATR, and phosphorylate various target proteins to stop cell cycle progression, and facilitate DNA repair. PARP (Poly ADP-Ribose Polymerase) is involved in repairing single-strand DNA breaks, helping to recruit repair factors and the formation of repair complexes at the sites of DNA damage. DNA- PK (DNA-Dependent Protein Kinase) helps bring broken DNA ends together for non-homologous endjoining (NHEJ), for repairing double-strand breaks. The DDR is facilitated by cell cycle regulation through WEE1 and PLK1 (Polo-Like Kinase 1). WEE1 is a kinase that phosphorylates and inhibits CDKs (Cyclin- Dependent Kinases), thereby delaying cell cycle progression and allowing more time for DNA damage repair prior to cell division. PLK1 regulates the cell cycle checkpoint and promotes repair processes, through phosphorylation of Pole. RAD51 is an ATPase involved in DNA repair. Ubiquitin-specific proteases (USPs) modulate the DDR by influencing the ubiquitination of proteins involved in the DDR. Protein kinase membrane associated tyrosine / threonine 1 (PKMYT1) regulates cell cycle and participates in DDR-related signaling. Aurora-A may contribute to the G2 DNA damage checkpoint through PLK1 and CDC25B activation, and is important in the mitotic DNA damage response.

[0160] Most cancerous cells have a greater dependency on the DDR than non-cancerous cells. DDR inhibitors and their use for the treatment of cancers is described e g. in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et al., Front Immunol. (2022) 13:854730 and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701 , all of which are hereby incorporated by reference in their entirety.

[0161] In some embodiments, a DDR inhibitor moiety according to the present disclosure is, or comprises, a DDR inhibitor selected from:

[0162] (a) a PARP inhibitor (e.g. olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X-121 , AMXI-5001 , JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397, fluzoparib, NMS-03305293, AZD9574); (b) an ATM inhibitor (e.g. CP-466722, KU-55933, KU-60019, KU-59403, AZ31 , AZ32, AZD0156, AZD1390, XRD-0394, M4076, M3541 , WSD-0628, SYH-2051 , IMP-08, SP-1161 , INT-6C4 / 5C4);

[0163] (c) an ATR inhibitor (e.g. M6620 (berzosertib), M4344 (VX-803), AZD6738 (ceralasertib), BAY1895344 (elimusertib) RP3500 (camonsertib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027);

[0164] (d) a WEE1 inhibitor (e.g. adavosertib, Debio 0123, PD0166285, PD0407824, AZD1775, ZN-c3 (azenosertib), IMP7068, SY4835, SCO191 , IMP7068);

[0165] (e) a CHK1 / 2 inhibitor (e g. CBP-501 , prexasertib, MK-8776, GDC-0575, SRA-737, PF-00477736, AZD7762, LY2603618 (rabusertib), LY2880070, XL884, BEBT260, MU380, NU7441 , KU-5778);

[0166] (f) a DNA-PK inhibitor (e.g. CC-115, LY-3023414, AsiDNA, M3814 (nedisertib, peposertib), VX-984 (M9831), BR-101801 , XRD-0394, SL901 , XZP-6877, IMP-11 , ZL-2201 , BR-2006, AZD7648, NU7441);

[0167] (g) a PLK1 inhibitor (e.g. BI-6727 (volasertib), PCM-075 (onvansertib), CYC140 (plogosertib));

[0168] (h) a Pole inhibitor (e.g. ART4215, ART6043, novobiocin, RP-6685, RP-3467);

[0169] (i) a RAD51 inhibitor (e.g. CYT0851);

[0170] (j) an inhibitor of a ubiquitin-specific protease (USP) family enzyme (e.g. an inhibitor of USP11 , USP7, USP4, USP37, USP39, USP45, USP24 and / or USP1 ; e.g. KSQ-4279);

[0171] (k) a PKMYT1 inhibitor (e g. RP6306); and

[0172] (l) an Aurora-A inhibitor (e.g. alisertib, WJ05129 (JS112), JAB-2485).

[0173] In some embodiments, the DDR inhibitor moiety is, or comprises, ceralasertib.

[0174] In some embodiments, the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is an ATR inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, berzosertib:

[0175] In some embodiments, the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is a CHK1 / 2 inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, prexasertib:

[0176]

[0177] In some embodiments, the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is a WEE1 inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, adavosertib, which can be linked as follows, as well as through other positions:

[0178] In some embodiments, the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is an ATM inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, AZD0156, which can be linked as follows, as well as through other positions:

[0179] In some embodiments, the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is a DNA-PK inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, nedisertib:

[0180]

[0181] In some embodiments, the DDR inhibitor moiety is not, or does not comprise, veliparib. In some embodiments, where the DDR inhibitor moiety is or comprises a PARP inhibitor, the PARP inhibitor is not veliparib. In some embodiments, the DDR inhibitor moiety is not, or does not comprise, a PARP inhibitor.

[0182] In some embodiments, a payload moiety according to the present disclosure comprises, or consists of, a TOP1 inhibitor. Hereinbelow, for conciseness, ‘a payload moiety which is a TOP1 inhibitor’ may be referred to simply as a ‘TOP1 inhibitor moiety’.

[0183] During DNA replication and transcription, significant torsional strain is placed on the DNA helix, and this is relieved through the action of DNA topoisomerases I and II (TOP1 and TOP2), which cleave the DNA strand and allow it to untwist, before resealing the breaks (see e g. Delgado et al., Biochem J. (2018) 475(2): 373-398). DNA topoisomerase inhibitors block the resealing step, resulting in DNA fragmentation and cell death. DNA topoisomerase I inhibitors and their use for the treatment of cancers is described e g. in Pommier, Chem Rev. (2009) 109(7): 2894-2902, Li et al., Am J Cancer Res. (2017) 7(12): 2350-2394 and Thomas and Pommier, Clin Cancer Res. (2019) 25(22): 6581 -6589, all of which are hereby incorporated by reference in their entirety.

[0184] In some embodiments, a TOP1 inhibitor moiety according to the present disclosure is, or comprises, a TOP1 inhibitor selected from: camptothecin or a derivative thereof, exatecan, exatecan mesylate (DX- 8951 f), / V-glycyl-exatecan, SN-38, irinotecan, etirinotecan, FL1 18, topotecan, gimatecan, belotecan, deruxtecan, belotecan, rubitecan, lurtotecan, diflomotecan, karenitecan, Silat can, namitecan, elomotecan, DRF-1042, delimotecan, NSC606985, chimmitecan, ZBH-1205, Genz-644282, non-CPT1 , indotecan (LMP-400), indimitecan (LMP-776), AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021 , VIP126, ZBH-01 and LMP-744.

[0185] In some embodiments, the TOP1 inhibitor moiety is not, or does not comprise, PBX-7016 described in WO 2023 / 249473 A1 (the structure of which is shown in Example 3 of WO 2023 / 249473 A1). In some embodiments, where the TOP1 inhibitor moiety is or comprises a camptothecin derivative, the camptothecin derivative is not PBX-7016 described in WO 2023 / 249473 A1. In some embodiments, the TOP1 inhibitor moiety is not, or does not comprise, a camptothecin derivative described in WO 2023 / 249473 A1 .

[0186] In some embodiments, the TOP1 inhibitor moiety is, or comprises, exatecan:

[0187] In some embodiments, the TOP1 inhibitor moiety is, or comprises, belotecan: In some embodiments, the TOP1 inhibitor moiety is, or comprises, SN38:

[0188] In some embodiments, the TOP1 inhibitor moiety is, or comprises, DXd:

[0189]

[0190] In particular, the present disclosure relates to linkers and conjugates which comprise both (a) a payload which is a DNA damage response (DDR) inhibitor, and (b) a payload which is a DNA topoisomerase I (TOP1) inhibitor.

[0191] Such conjugates of the present disclosure are provided with unexpected and advantageous properties relative to known antibody-drug conjugates. In particular, DNA damage repair (DDR) is a key mode of resistance to Topoisomerase I (TOP1) DNA damaging agents, and the combination of both payloads into a single antibody drug conjugate is expected to show improved, possibly synergistic, effects. The dual payload ADC is also expected to possess advantageous properties compared to combining a chemotherapeutic DDR inhibitor with a TOP1 -inhibiting ADC, in reducing the overlapping toxicities of the chemotherapeutic DDR inhibitor and a TOP1 inhibiting ADC, such as neutropenia and thrombocytopenia. The dual DDR inhibitor, TOP1 inhibitor approach is also expected to mitigate against resistance to TOP1 inhibitors, which is sometimes observed on treatment with ADCs comprising a TOP1 inhibitor payload. Such resistance is described in Mosele, et al., Nat Med (2023) 29(8) 2110-2120 (PMID37488289); Zhang, et a!., Br J Cancer (2021) 125(10) 1333-1340 (PMID34294893); Muai, et al., Mol Cell (2018) 69(3) 371 - 384 (PMID29395061).

[0192] DDR inhibitor and TOP1 inhibitor combinations have also been shown precli nica lly and clinically to resensitize tumors to TOP1 inhibitors, restoring responsiveness to validated TOP1 inhibitor therapy. Josse et al., Cancer Res (2014) 74(23): 6968-6979 describes berzosertib (an ATR inhibitor) potentiating the effect of irinotecan in CRC CDX model, COLO205; Coussy et al., Sci Trans Med (2020) 12 (531): eaax2625 describes berzosertib (an ATR inhibitor) increasing sensitivity to irinotecan in SLFN11 - negative, TNMC patient-derived xenograft tumors with BRCAness; Thomas et al., Cancer Cell (2021) 39(4): 566-579. e7 describes berzosertib (an ATR inhibitor) enhancing the efficacy of topotecan in chemotherapy-resistant SCLC patients; Slotkin, et al. (ASCO 2022 Abstract 11503) describes prexasertib (a CHK1 inhibitor) resensitising relapsed or refractory desmoplastic small round cell tumours to irinotecan.

[0193] In some embodiments, the DDR inhibitor and TOP1 inhibitor payloads are linked to the same click group. In some embodiments, the DDR inhibitor and TOP1 inhibitor payload moieties are linked to separate click groups which are then linked to the urea group.

[0194] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload linked to the same click group has the structure:

[0195] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload linked to the same click group has the structure:

[0196] Antigen-binding molecules and antigen-binding moieties

[0197] An ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen. Antigen-binding molecules comprise one or more antigen-binding moieties through which the antigen-binding molecule binds to its target antigen(s). For example, aspects and embodiments of the present disclosure relate to antigen-binding molecules comprising a target antigen-binding moiety.

[0198] Antigen-binding moieties may comprise, or may be derived from, antibodies ( / .e. immunoglobulins (Igs)) and antigen-binding fragments of antibodies. As used herein, ‘antibodies’ include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH, etc ). Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments.

[0199] Antigen-binding moieties also include target antigen-binding aptamers, e.g. a nucleic acid aptamers (reviewed, for example, in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3):181 -202). In some embodiments, an antigen-binding moiety comprises or consists of an antigen-binding peptide / polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (j.e. a single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0200] The skilled person is readily able to produce antigen-binding molecules that bind to a given target antigen, in view of techniques that are well-known in the art. For example, Park and Smolen, Advances in Protein Chemistry (2001) 56: 369-421 describes approaches to the production of monoclonal antibodies suitable for use in humans, including raising xenogeneic antibodies and their subsequent humanisation, identification by human antibody gene-phage display, and production of antibodies in transgenic mice harbouring human antibody genes. Briefly, in the human antibody gene-phage display technique, genes encoding the VH and VL chains are generated by PCR amplification and cloning from naive’ human lymphocytes, and assembled into a library from which they can be expressed either as disulfide-linked Fab fragments or as single-chain Fv (scFv) fragments. The Fab- or scFv-encoding genes are fused to a surface coat protein of filamentous bacteriophage and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with antigen. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab / scFv fragment. In the transgenic mouse technique, mice in which the endogenous murine Ig gene loci have been replaced by homologous recombination with their human homologues are immunised with antigen, and monoclonal antibody is prepared by conventional hybridoma technology, to yield a fully-human monoclonal antibody.

[0201] In some embodiments, an antigen-binding moiety according to the present disclosure comprises, or consists of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody). Antigen-binding moieties may be derived from antibodies. Antibody-derived antigen-binding moieties may comprise, or consist of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody). In some embodiments, an antigen-binding moiety may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody that binds to a given target antigen, or the whole antibody.

[0202] The antigen-binding moieties of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to a given target antigen. Antigen-binding regions of antibodies, such as variable fragment (Fv), Fab and F(ab’)2 fragments may also be used / provided. An ‘antigen-binding region’ is any fragment of an antibody that binds to the target antigen for which the given antibody is specific. Aspects and embodiments of the present disclosure relate to antigen-binding molecules that bind ( / .e. through an antigen-binding moiety) to a given target.

[0203] The target antigen for a target antigen-binding moiety according to the present disclosure may be any molecule. In some embodiments, a target antigen may be a peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A target antigen may be expressed at the cell surface of a cell expressing the target antigen.

[0204] In some embodiments, a target antigen is a disease-associated antigen. A ‘disease-associated antigen’ refers to an antigen whose presence is indicative of a given disease / disease state, or an antigen for which an elevated level of the antigen is positively-correlated with a given disease / disease state. The disease-associated antigen may be an antigen whose expression is associated with the development, progression or severity of symptoms of a given disease. The disease-associated antigen may be associated with the cause or pathology of the disease, or may be expressed abnormally as a consequence of the disease. A disease-associated antigen may be an antigen of an infectious agent or pathogen, a cancer-associated antigen or an autoimmune disease-associated antigen.

[0205] In some embodiments, the disease-associated antigen is an antigen of a pathogen. The pathogen may be prokaryotic (bacteria), eukaryotic (e.g. protozoan, helminth, fungus), virus or prion. In some embodiments, the pathogen is an intracellular pathogen. In some embodiments the pathogen is a virus, e.g. a virus as described hereinabove. In some embodiments the pathogen is a bacterium.

[0206] In some embodiments, the target antigen is a cancer-associated antigen. A cancer-associated antigen is an antigen whose expression or overexpression is associated with cancer. In some embodiments, the cancer-associated antigen is a receptor molecule, e.g. a cell surface receptor. In some embodiments, the cancer-associated antigen is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine. In some embodiments, the cancer-associated antigen is a growth factor or a hormone. In some embodiments, the cancer-associated antigen is a viral antigen. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (i.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by an antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e. is extracellular). The cancer cell antigen may be a cancer-associated antigen. In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and / or protein level) by cells of a cancer, e.g. as compared to the level of expression by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0207] Cancer-associated antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6thedition. Hamilton (ON): BC Decker; 2003. Cancer-associated antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: fibroblast activation protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2 / neu, BING-4, calcium- activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1 , survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1 R, prostate specific antigen; mutated antigens: p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF- pRI I ; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10:2250, which is hereby incorporated by reference in its entirety.

[0208] In some embodiments, the target antigen is an immune cell surface molecule. An immune cell surface molecule is any molecule which is expressed in or at the cell membrane of an immune cell. In some embodiments, the part of the immune cell surface molecule which is bound by the antigen-binding moiety is on the external surface of the immune cell (i.e. is extracellular). The immune cell surface molecule may be expressed at the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be e g. a T cell, B cell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell). The immune cell may express a CD3 polypeptide (e.g. CD3y CD3e CD3£ or CD36), a TCR polypeptide (TCRa or TCRp), CD27, CD28, CD4 or CD8. In some embodiments, the immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)). In some embodiments, the immune cell is a T cell or an NK cell.

[0209] In some embodiments, an immune cell surface molecule may be a CD3-TCR complex polypeptide, e.g. TCRa, TCRp, TCRy, TCR6, TRAC, TRBC1 , TRBC2, TRGC1 , TRGC2, TRDC, CD3e, CD38, CD3y, CD3^ or CD3r). In some embodiments, an immune cell surface molecule is CD3, CD8, CD4 or CD28. In some embodiments, an immune cell surface molecule is a checkpoint molecule {e g. PD-1 , CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA), or a ligand for a checkpoint molecule {e g. PD-L1 , PD-L2, CD80, CD86, MHC class I, MHC Class II, Galectin 9, VSIG3, VSIG8, LRIG1 , PSGL1 , CD155 or HVEM). In some embodiments the immune cell surface molecule is a costimulatory molecule {e.g. CD28, 0X40, 4-1 BB, ICOS or CD27), or a ligand for a costimulatory molecule {e g. CD86, CD80, OX40L 4-1 BBL, ICOSL or CD70).

[0210] In some embodiments, an antigen-binding moiety comprises the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. In some embodiments, the antigen-binding moiety is or comprises the Fv {e g. provided as an scFv) of an antibody. In some embodiments, the antigen-binding moiety is or comprises the Fab region of an antibody. In some embodiments, the antigen-binding moiety is or comprises the whole antibody ( / .e. comprising variable and constant regions).

[0211] An antigen-binding moiety may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex. An antigen-binding moiety may comprise more than one polypeptide which together form an antigen-binding moiety. The polypeptides may associate covalently or non-covalently. In some embodiments, the polypeptides form part of a larger polypeptide comprising the polypeptides {e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).

[0212] In some embodiments, an antigen-binding moiety according to the present disclosure comprises, or consists of, a polypeptide complex formed by protein:protein interaction between constituent peptides / polypeptides of the antigen-binding moiety. An antigen-binding moiety may refer to a non- covalent or covalent complex of more than one polypeptide {e g. 2, 3, 4, 6, or 8 polypeptides), e g. an IgG-like antigen-binding moiety comprising two heavy chain polypeptides and two light chain polypeptides.

[0213] Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0214] The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.

[0215] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5lhEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et a!., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671 -D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In preferred embodiments, the CDRs and FRs of antigenbinding moieties referred to herein are defined according to the IMGT information system.

[0216] In some embodiments, an antigen-binding moiety according to the present disclosure comprises, or consists of, an Fv region that binds to the relevant target antigen. In some embodiments, the VH and VL regions of the Fv are provided as single polypeptide joined by a linker sequence, I.e. a single chain Fv (scFv).

[0217] The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the antigen-binding moiety comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH- CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e g. a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH1 (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).

[0218] In some embodiments, an antigen-binding moiety described herein comprises, or consists of, a whole antibody which binds to the relevant target antigen. As used herein, ‘whole antibody’ refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.

[0219] Immunoglobulins of type G (i.e. IgG) are ~150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chains comprise a VL followed by a CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (K).

[0220] In some embodiments, the antigen-binding moiety comprises, or consists of, an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to the relevant target antigen.

[0221] In some embodiments, an antigen-binding moiety ofthe present disclosure comprises one or more regions (e.g. CH1 , hinge, CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG {e g. IgG 1 , lgG2, lgG3, lgG4), IgA {e.g. lgA1 , lgA2), IgD, IgE or IgM, e.g. a human IgG e.g. hlgG1 , hlgG2, hlgG3, hlgG4), hlgA {e.g. hlgA1 , hlgA2), hlgD, hlgE or hlgM. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human Ig G 1 allotype {e.g. G1 ml , G1 m2, G1 m3 or G1 ml 7).

[0222] In some embodiments, an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2 or 7. In some embodiments, an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4 or 19. In some embodiments, an antigenbinding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:5 or 8. In some embodiments, an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:9, 10, 20 or 21. In some embodiments, an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 , 6, 17 or 18.

[0223] In some embodiments, an antigen-binding moiety of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.

[0224] In some embodiments, an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:11 , 12, 13, 14, 15 or 16.

[0225] In some embodiments described herein, one or more amino acids of an amino acid sequence referred to herein (e.g. an amino acid sequence of an antigen-binding moiety, e.g. an amino acid sequence of a CDR or VH / VL region) are substituted with another amino acid. A substitution comprises substitution of an amino acid residue with a non-identical ‘replacement’ amino acid residue. A replacement amino acid residue of a substitution according to the present disclosure may be a naturally-occurring amino acid residue (j.e. encoded by the genetic code) which is non-identical to the amino acid residue at the relevant position of the equivalent, unsubstituted amino acid sequence, selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie): leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). In some embodiments, a replacement amino acid may be a non-naturally occurring amino acid residue - i.e. an amino acid residue other than those recited in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogues such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301 -336.

[0226] In some embodiments, a substitution may be biochemically conservative. In some embodiments, where an amino acid to be substituted is provided in one of rows 1 to 5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row:

[0227] By way of illustration, in some embodiments wherein substitution is of a Met residue, the replacement amino acid may be selected from Ala, Vai, Leu, lie, Trp, Tyr, Phe and Norleucine.

[0228] In some embodiments, a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, a replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

[0229] That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another, non- identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another, non-identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another, non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another, non-identical negative amino acid.

[0230] In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e g. target binding) of the antigen-binding moiety comprising the substitution as compared to the equivalent unsubstituted molecule.

[0231] Aspects and embodiments of the present disclosure contemplate multispecific antigen-binding molecules. By ‘multispecific’ it is meant that the antigen-binding molecule binds to more than one target antigen (e.g. one of 1 , 2, 3, 4, 5, 6 or more target antigens). In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two, different antigen-binding moieties. In some embodiments, the antigen-binding molecule comprises at least two antigen-binding moieties, wherein each antigen-binding moiety binds to a different target antigen.

[0232] In some embodiments, the antigen-binding molecule comprises: (I) an antigen-binding polypeptide (e.g. a scFv, scFab, polypeptide aptamer or VhH) or and an antigen-binding polypeptide complex (e.g. a Fv, Fab or whole antibody) that binds to a first antigen, and (ii) an antigen-binding polypeptide (e.g. a scFv, scFab, polypeptide aptamer or VhH) or and an antigen-binding polypeptide complex (e.g. a Fv, Fab or whole antibody) that binds to an antigen other than the first antigen. In some embodiments, the antigen-binding molecule comprises: (i) an antigen-binding moiety comprising the VH and VL of an antibody that binds to a first antigen, and (ii) an antigen-binding moiety comprising the VH and VL of an antibody that binds to an antigen other than the first antigen.

[0233] Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety. Multispecific antigenbinding molecule formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. IgGz, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KA-body common HC; CH1 / CL fusion proteins, e.g. scFv2-CH1 / CL, VHH2-CH1 / CL; ‘variable domain only’ bispecific antigen-binding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAb / VHH, tetravalent dAb.VHH; Non-lg fusion proteins, e.g. scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g. scFv-Fc(kih), scFv-Fc(CH3 charge pairs), scFv-Fc (EW-RVT), scFv-fc (HA- TF), scFv-Fc (SEEDbody), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv- Fc(BEAT), Fab-scFv-Fc (SEEDbody), DART-Fc, scFv-CH3(kih), TriFabs; Fc fusions, e.g. Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-lg, scFv2-Fcab; CH3 fusions, e.g. Dia- diabody, scDb-CH3; IgE / IgM CH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g. Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab- VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Faba, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-lgG(kih), Fab-scFab-lgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1 / CL charge pairs, hinge / CH3 charge pairs, SEED-body, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*; appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA- TF-Fv, lgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g. Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv- Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgGs - HC fusions, e.g. IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CctCP) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-lgG(CaCp Fab), Fab-lgG(CR3), Fab-hinge-lgG(CR3); appended IgGs - LC fusions, e.g. IgG-scFv(LC), scFv(LC)-lgG, dAb-IgG; appended IgGs - HC and LC fusions, e.g. DVD-lg, TVD-lg, CODV-lg, scFv4-lgG, Zybody; Fc fusions, e.g. Fab-scFv-Fc, scFv4-lg; F(ab’)2 fusions, e.g. F(ab’)2-scFv2; CH1 / CL fusion proteins e . scFv2-CH1-hinge / CL; modified IgGs, e.g. DAF (two-in one-IgG), DutaMab, Mab2; and non-lg fusions, e.g. DNL-Fa 4-lgG.

[0234] The skilled person is able to design and prepare multispecific antigen-binding polypeptides / polypeptide complexes comprising at least two antigen-binding moieties. Methods for producing bispecific antigenbinding polypeptides / polypeptide complexes include chemically crosslinking antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1 - 2.13.16, which is hereby incorporated by reference in its entirety. For example, / V-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.

[0235] Other methods for producing bispecific antigen-binding polypeptides / polypeptide complexes include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001 . Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1 - 2.13.16. Antigen-binding polypeptides / polypeptide complexes according to the present disclosure may also be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides / polypeptides can be synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.

[0236] Molecular biology techniques suitable for recombinant production of antigen-binding polypeptides / polypeptide complexes are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thEdition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding polypeptides / polypeptide complexes are also described in Frenzel et a / ., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety. The multispecific (e.g. bispecific) antigen-binding polypeptides / polypeptide complexes according to the present disclosure may be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigen-binding polypeptide / polypeptide complex, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber- Schwarz), or French, How to make bispecific antibodies, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference.

[0237] For example, a DNA construct encoding the light and heavy chain variable domains for the two antigenbinding moieties, and including sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques. A recombinant bispecific antigen-binding polypeptide / polypeptide complex can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e g. a mammalian host cell), and expressed recombinant bispecific polypeptide / polypeptide complex can then optionally be purified.

[0238] Fc regions

[0239] In some embodiments, an antigen-binding molecule of the present disclosure (e g. an antigen-binding moiety thereof) comprises an Fc region. As used herein, an Fc region’ refers to a polypeptide complex formed by interaction between two polypeptides, each polypeptide comprising the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

[0240] Herein, a ‘CH2 domain’ refers to an amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). The CH2 domain is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85. A ‘CH3 domain’ refers to an amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). The CH3 domain is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85. A ‘CH2-CH3 region' refers to an amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.

[0241] In some embodiments, a CH2 domain, CH3 domain and / or a CH2-CH3 region according to the present disclosure corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e g. lgA1 , lgA2), IgD, IgE or IgM. In some embodiments, the CH2 domain, CH3 domain and / or a CH2-CH3 region corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of a human IgG (e.g. h IgG 1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. h Ig A1 , hlgA2), hlgD, h Ig E or hlgM. In some embodiments, the CH2 domain, CH3 domain and / or a CH2-CH3 region corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of a human IgG 1 allotype (e.g. G1 ml , G1 m2, G1 m3 or G1 ml 7). In some embodiments, the CH2 domain, CH3 domain and / or a CH2-CH3 region corresponds to the CH2 domain / CH3 domain / CH2-CH3 region of human lgG1 allotype G1 m3.

[0242] Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects. Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about through Fc- mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells) through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0243] In some embodiments, an antigen-binding molecule according to the present disclosure comprises one or more (e g. two) polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:9 or 10. In some embodiments, the antigen-binding molecule comprises an Fc region comprising one or more e.g. two) polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:9 or 10.

[0244] Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et a!., Protein Cell (2018) 9(1):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification to increase or reduce an Fc-mediated function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region. Where an Fc region / CH2 / CH3 is described as comprising modification(s) ‘corresponding to’ reference substitution(s), equivalent substitution(s) in the homologous Fc / CH2 / CH3 are contemplated.

[0245] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and / or CH3 regions.

[0246] In some embodiments, the Fc region comprises modification to reduce / prevent an Fc-mediated function (e.g. ADCC, ADCP, CDC). In some embodiments, the Fc region comprises modification to reduce / prevent ADCC. In some embodiments, the Fc region comprises modification to reduce / prevent CDC. In some embodiments, the Fc region comprises modification to reduce / prevent binding to an Fc receptor. In some embodiments, the Fc region comprises modification to reduce / prevent binding to an Fey receptor. In some embodiments, the Fc region comprises modification to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0247] In some embodiments, the Fc region comprises modification at the amino acid residue corresponding to N297. In some embodiments, the Fc region comprises modification corresponding to N297A or N297Q or N297G as described in Leabman et al., Mabs. (2013) 5:896-903. Substitution of ‘N297’ with ‘A’, ‘G’ or ‘Q’ is known to eliminate glycosylation, and thereby reduce Fc binding to C1 q and Fey receptors, and thus also reducing CDC and ADCC. In some embodiments, the Fc region comprises modification corresponding to N297A. In some embodiments, an antigen-binding molecule according to the present disclosure comprises one or more (e.g. two) polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20 or 21 . In some embodiments, the antigen-binding molecule comprises an Fc region comprising one or more (e g. two) polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20 or 21.

[0248] Functional properties of the antigen-binding molecules

[0249] The antigen-binding molecules described herein may be characterised by reference to certain functional properties. In some embodiments, an antigen-binding molecule described herein may possess one or more of the following properties: binds to cells expressing the target antigen for the antigen-binding molecule; inhibits proliferation of cells expressing the target antigen for the antigen-binding molecule; increases killing of cells expressing the target antigen for the antigen-binding molecule; inhibits proliferation and / or increases killing of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; inhibits tumor growth and / or reduces tumor size / volume (e.g. of a cancer expressing the target antigen for the antigen-binding molecule); increases survival of subjects having a cancer (e g. a cancer expressing the target antigen for the antigen-binding molecule).

[0250] It will be appreciated that a given antigen-binding molecule may display more than one of the properties recited in the preceding paragraph. A given antigen-binding molecule may be evaluated for the properties recited in the preceding paragraph using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in non-human animals. In some embodiments, the assays may be e g. ex vivo assays, i.e. performed using cells / tissue / an organ obtained from a subject.

[0251] Where assays are cell-based assays, they may comprise treating cells with an antigen-binding molecule in order to determine whether the antigen-binding molecule displays one or more of the recited properties. Assays may employ species labelled with detectable entities in order to facilitate their detection. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities / concentrations of a given antigen-binding molecule (e.g. a dilution series).

[0252] Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained. The concentration of a given agent at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the agent in relation to the relevant activity, which may also be referred to as the ‘ECso’. Depending on the property, the EC50 may also be referred to as the ‘half-maximal inhibitory concentration’ or ‘IC50’, this being the concentration of the agent at which 50% of the maximal level of inhibition of a given property is observed.

[0253] In some embodiments, the antigen-binding molecule of the present disclosure binds to its target antigen in a region which is accessible to an antigen-binding molecule ( / .e., an extracellular antigen-binding molecule) when the relevant antigen is expressed at the cell surface (f.e. in or at the cell membrane). In some embodiments, the antigen-binding molecule binds to its target antigen when it is expressed at the cell surface. In some embodiments, the antigen-binding molecule binds to cells expressing its target antigen.

[0254] The ability of an antigen-binding molecule to bind to a given cell type can be analyzed by contacting cells with the antigen-binding molecule, and detecting antigen-binding molecule bound to the cells, e.g. after a washing step to remove unbound antigen-binding molecule. The ability of an antigen-binding molecule to bind to cells expressing a given target antigen can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0255] In some embodiments, the antigen-binding molecule inhibits proliferation of cells expressing the target antigen for the antigen-binding molecule. The ability of an antigen-binding molecule to inhibit proliferation of a given cell type can be analyzed by contacting cells with the antigen-binding molecule, and subsequently evaluating proliferation of the cells ( / .e. after a period of time sufficient for an effect on cell proliferation to be observed). Cell proliferation can be evaluated e.g. by detecting changes in number of cells over time, or by in vitro analysis of incorporation of3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.

[0256] In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting proliferation of cells expressing the target antigen for the antigen-binding molecule to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of proliferation of the same cells observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to influence proliferation of cells expressing the relevant target antigen), in a given assay.

[0257] In some embodiments, the antigen-binding molecule described herein inhibits proliferation of cells expressing the target antigen for the antigen-binding molecule with an ICso of 100 nM or less, preferably one of <50 nM, <40 nM, <30 nM, <20 nM, <10 nM, <5 nM, <4 nM, <3 nM, <2 nM, <1 nM, <900 pM, <800 pM, <700 pM, <600 pM or <500 pM. In some embodiments, the antigen-binding molecule inhibits proliferation of cells {e g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule.

[0258] As used herein, ‘in proximity to’ a cell expressing the target antigen for the antigen-binding molecule, refers to the region / area within 100 pm of the cell expressing the target antigen. Cells in proximity to a cell expressing the target antigen for the antigen-binding molecule, may also be referred to as surrounding cells or bystander cells. Such cells may or may not express the target antigen for the antigen-binding molecule themselves. In some embodiments, cells in proximity to a cell expressing the target antigen for the antigen-binding molecule may be within 100 pm (e.g. within 90 pm, 80 pm, 70 pm, 60 pm, 50 pm, 40 pm, 30 pm, 20 pm, or 10 pm) of the cell expressing the target antigen.

[0259] In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting proliferation of cells {e g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of proliferation of the same cells observed in the absence of the antigenbinding molecule (or in the presence of an appropriate control antigen-binding molecule known not to influence proliferation of cells in proximity to a cell expressing the target antigen for the antigen-binding molecule).

[0260] In some embodiments, the antigen-binding molecule does not inhibit proliferation of cells {e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigenbinding molecule.

[0261] In some embodiments, the antigen-binding molecule according to the present disclosure potentiates {i.e. upregulates, enhances) cell killing of cells comprising / expressing the target antigen for the antigenbinding molecule.

[0262] In some embodiments, an antigen-binding molecule according to the present disclosure may inhibit growth or reduce metastasis of a cancer comprising cells comprising / expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule may potentiate {i.e. upregulate, enhance) cell killing of cells comprising / expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule may potentiate {i.e. upregulate, enhance) cell killing of cells {e.g. cells that do not comprise / express the target antigen) in proximity to a cell comprising / expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule does not potentiate {i.e. upregulate, enhance) cell killing of cells {e.g. cells that do not comprise / express the target antigen) in proximity to a cell comprising / expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule may inhibit growth of cells of a cancer, or may inhibit growth of a tumor, comprising cells comprising / expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule may inhibit metastasis of a cancer / tumor comprising cells comprising / expressing the target antigen for the antigenbinding molecule.

[0263] In some embodiments, an antigen-binding molecule according to the present disclosure may inhibit growth or reduce metastasis of a cancer comprising (i) cells comprising / expressing the target antigen for the antigen-binding molecule, and (II) cells not comprising / expressing the target antigen for the antigenbinding molecule. In some embodiments, an antigen-binding molecule may inhibit growth of cells of a cancer, or may inhibit growth of a tumor, comprising (i) cells comprising / expressing the target antigen for the antigen-binding molecule, and (ii) cells not comprising / expressing the target antigen for the antigenbinding molecule. In some embodiments, an antigen-binding molecule may inhibit metastasis of a cancer / tumor comprising (i) cells comprising / expressing the target antigen for the antigen-binding molecule and (ii) cells not comprising / expressing the target antigen for the antigen-binding molecule. Cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (201 1), 9(6):601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity / cell killing assays include release assays such as the51Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) release assay, ATP release assay using Cell Titre Gio, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells.

[0264] In some embodiments an antigen-binding molecule according to the present disclosure is capable of reducing the number / proportion of cells expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule according to the present disclosure is capable of depleting / enhancing depletion of such cells.

[0265] In some embodiments, an antigen-binding molecule of the present disclosure displays anticancer activity. In some embodiments, the antigen-binding molecule increases killing of cancer cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g. as compared to an appropriate control condition. The cancer may be a cancer as described herein, e.g. a cancer expressing / overexpressing the target antigen for the antigen-binding molecule.

[0266] In some embodiments, an antigen-binding molecule according to the present disclosure reduces / inhibits growth of a cancer and / or of a tumor of a cancer. In some embodiments, an antigen-binding molecule reduces tissue invasion by cells of a cancer. In some embodiments, an antigen-binding molecule reduces metastasis of a cancer. In some embodiments, an antigen-binding molecule displays anticancer activity. In some embodiments, an antigen-binding molecule reduces the growth / proliferation of cancer cells. In some embodiments, an antigen-binding molecule reduces the survival of cancer cells. In some embodiments, an antigen-binding molecule increases the killing of cancer cells. In some embodiments, an antigen-binding molecule of the present disclosure causes a reduction in the number of cancer cells e.g. in vivo. The cancer may be a cancer comprising cells expressing the target antigen for the antigenbinding molecule. The cancer may be a cancer comprising cells expressing the target antigen for the antigen-binding molecule and cells that do not express the target antigen for the antigen-binding molecule.

[0267] An antigen-binding molecule of the present disclosure may be analyzed for the properties described in the preceding paragraph in appropriate assays. Such assays include e.g. in vivo models.

[0268] In some embodiments, administration of an antigen-binding molecule according to the present disclosure may cause one or more of: inhibition of the development / progression of the cancer, a delay to / prevention of onset of the cancer, a reduction in / delay to / prevention of tumor growth, a reduction in / delay to / prevention of tissue invasion, a reduction in / delay to / prevention of metastasis, a reduction in the severity of one or more symptoms of the cancer, a reduction in the number of cancer cells, a reduction in the cancer burden, a reduction in tumor size / volume, and / or an increase in survival of subjects having the cancer (e.g. progression free survival or overall survival), e.g. as determined in an appropriate model.

[0269] It will be appreciated that the properties recited in the preceding paragraph are evaluated after a period of time sufficient for an effect associated with treatment using the antigen-binding molecule to be observed. Tumor growth may be monitored by investigating tumor volume over time. Tumor growth may be evaluated by measuring tumor volume (e g. in mm3) overtime.

[0270] In some embodiments, an antigen-binding molecule of the present disclosure is capable of reducing tumor size / volume (e.g. the mean tumor size / volume for the treatment group in an in vivo model, e g. of a cancer expressing the target antigen for the antigen-binding molecule) to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the tumor size / volume observed at the same time point in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence tumor growth), in a given assay. In some embodiments, evaluation of tumor size / volume for the purposes of such comparison is performed after more than 5 days, e.g. one of >10 days, >15 days, >20 days, >25 days, >30 days, >35 days, >40 days, >35 days, >50 days, >55 days, >60 days, >65 days, >70 days, >75 days, >80 days, >85 days, >90 days, >95 days or >100 days following administration of the first dose of the antigen-binding molecule, in the relevant model.

[0271] In some embodiments, an antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition (e.g. expressed as % tumor growth inhibition, e.g. calculated relative to tumor growth observed on treatment with an appropriate control antigen-binding molecule) which is greater than 1 times, e g. one of >1.01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1.05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of tumor growth inhibition observed at the same time point in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence tumor growth), in a given assay. In some embodiments, evaluation of tumor growth inhibition for the purposes of such comparison is performed after more than 5 days, e.g. one of >10 days, >15 days, >20 days, >25 days, >30 days, >35 days, >40 days, >35 days, >50 days, >55 days, >60 days, >65 days, >70 days, >75 days, >80 days, >85 days, >90 days, >95 days or >100 days following administration of the first dose of the antigen-binding molecule, in the relevant model.

[0272] In some embodiments, an antigen-binding molecule of the present disclosure is capable of increasing median survival of subjects having a cancer (e g. in an in vivo model, e.g. of a cancer expressing the target antigen for the antigen-binding molecule) to greater than 1 times, e.g. one of >1.01 times, >1.02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the median survival observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence survival of subjects having the cancer), in a given assay. Median survival may be expressed in days from the start of the experiment, for subjects in the relevant treatment groups. It will be appreciated that a cancer expressing the target antigen for the antigen-binding molecule may comprise cells comprising / expressing the target antigen and may further comprise cells which do not comprise / express the target antigen.

[0273] In some embodiments, an antigen-binding molecule according to the present disclosure possesses one or more novel, similar or improved functional properties as compared to: (i) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only a TOP1 inhibitor moiety (i.e. not also comprising a DDR inhibitor moiety), and / or (ii) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only a DDR inhibitor moiety (i.e. not also comprising a TOP1 inhibitor moiety).

[0274] In some embodiments, an antigen-binding molecule according to the present disclosure possesses one or more novel, similar or improved functional properties as compared to: (i) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same TOP1 inhibitor moiety as the antigen-binding molecule (i.e. not comprising the DDR inhibitor moiety of the antigen-binding molecule), and / or (ii) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same DDR inhibitor moiety as the antigen-binding molecule (i.e. not comprising the TOP1 inhibitor moiety of the antigen-binding molecule). For conciseness, in the following paragraphs, ‘an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same TOP1 inhibitor moiety as the antigen-binding molecule (i.e. not comprising the DDR inhibitor moiety of the antigen-binding molecule)’ is referred to simply as ‘an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety’, and ‘an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same DDR inhibitor moiety as the antigen-binding molecule (i.e. not comprising the TOP1 inhibitor moiety of the antigen-binding molecule)’ is referred to simply as ‘an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety’. In some embodiments, an antigen-binding molecule according to the present disclosure may display one or more of the following: similar binding to cells expressing the relevant target antigen for the antigen-binding molecule, as compared to an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety; increased killing of cells expressing the relevant target antigen for the antigen-binding molecule, as compared to killing of such cells displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety; similar killing of cells (e g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; increased inhibition of tumor growth and / or a greater reduction of tumor size / volume (e.g. of a cancer expressing the relevant target antigen for the antigen-binding molecule), as compared to tumor growth inhibition / reduction in tumor size / volume displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety; similar internalization into cells expressing the relevant target antigen for the antigen-binding molecule, as compared to internalization displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety; similar toxicological properties, as compared to those of an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety.

[0275] In accordance with the preceding paragraph, a level of a given property / outcome which is ‘similar to’ a reference level may be >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1.1 times the reference level. In some embodiments, a level of a given property / outcome which is ‘increased’ relative to a reference level may be greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1.03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times or >5 times the reference level. In some embodiments, a level of a given property / outcome which is ‘reduced’ relative to a reference level may be less than 1 times, e g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the reference level.

[0276] It will be appreciated that for the purposes of such evaluations, equivalent amounts of the antigen-binding molecule and the comparator antigen-binding molecule only comprising the TOP1 / DDR inhibitor moiety may be compared. In some embodiments, amounts of the different antigen-binding molecules providing equivalent amounts of the respective payloads are employed. In some embodiments, the antigen-binding molecule of the present disclosure binds to cells expressing the relevant target antigen for the antigen-binding molecule with an ECso which is similar to the ECso with which an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety binds to the relevant cells, in a given assay. In some embodiments, the ECso for binding of the antigen-binding molecule to cells expressing the relevant target antigen is >0.5 times and <2 times, e.g. one of >0.55 times and <1.9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the ECso for binding of an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety to cells of the same type, as determined in the same assay.

[0277] In some embodiments, the antigen-binding molecule of the present disclosure increases the killing of cells expressing the relevant target antigen for the antigen-binding molecule to a level that is greater than the level of killing of the same cells displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, in a given assay. In some embodiments, the antigen-binding molecule increases the killing of cells expressing the relevant target antigen for the antigen-binding molecule (e.g. cancer cells expressing the relevant target antigen for the antigen-binding molecule) to a level that is greater than 1 times, e g. one of >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1.05 times, >1.1 times, >1.2 times, >1.3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of cell killing of the relevant cells displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0278] In some embodiments, the antigen-binding molecule of the present disclosure kills cells expressing the relevant target antigen for the antigen-binding molecule with an ECso which is less than the ECso with which an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety kills the relevant cells, in a given assay. In some embodiments, the ECso of the antigen-binding molecule of the present disclosure for killing cells expressing the relevant target antigen is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the ECso for killing cells of the same type for an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0279] In some embodiments, the antigen-binding molecule of the present disclosure kills cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule with an ECso which is similar to the ECso with which an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety kills the relevant cells, in a given assay. In some embodiments, the ECSQ of the antigen-binding molecule of the present disclosure for killing cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule is >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1.8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1.3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the EC$o for killing cells of the same type for an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0280] In some embodiments, the antigen-binding molecule of the present disclosure reduces tumor size / volume to a level that is greater than the level of reduction in tumor size / volume observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, in a given assay. In some embodiments, the antigen-binding molecule is capable of reducing tumor size / volume (e.g. the mean tumor size / volume for the treatment group in an in vivo model, e g. of a cancer expressing the relevant target antigen for the antigen-binding molecule) to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the tumor size / volume observed at the same time point following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay. In some embodiments, evaluation of tumor size / volume for the purposes of such comparison is performed after more than 5 days, e.g. one of >10 days, >15 days, >20 days, >25 days, >30 days, >35 days, >40 days, >35 days, >50 days, >55 days, >60 days, >65 days, >70 days, >75 days, >80 days, >85 days, >90 days, >95 days or >100 days following administration of the first dose of the relevant antigen-binding molecule, in the relevant model.

[0281] In some embodiments, the antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition that is greater than the tumor growth inhibition observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, in a given assay. In some embodiments, the antigen-binding molecule achieves a level of tumor growth inhibition (e.g. expressed as % tumor growth inhibition, e.g. calculated relative to tumor growth observed on treatment with an appropriate control antigen-binding molecule) which is greater than 1 times, e.g. one of >1.01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of tumor growth inhibition observed at the same time point following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay. In some embodiments, evaluation of tumor size / volume for the purposes of such comparison is performed after more than 5 days, e.g. one of >10 days, >15 days, >20 days, >25 days, >30 days, >35 days, >40 days, >35 days, >50 days, >55 days, >60 days, >65 days, >70 days, >75 days, >80 days, >85 days, >90 days, >95 days or >100 days following administration of the first dose of the relevant antigen-binding molecule, in the relevant model.

[0282] In some embodiments, the antigen-binding molecule of the present disclosure achieves a synergistic level of killing of cells expressing the relevant target antigen for the antigen-binding molecule, as compared to the level of cell killing achieved individually by equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety. That is, in some embodiments, the antigen-binding molecule of the present disclosure achieves a level of cell killing that is synergistic ( / .e. super-additive), relative to what is observed when equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety are used alone.

[0283] In some embodiments, the antigen-binding molecule of the present disclosure achieves a synergistic level of tumor growth inhibition and / or a synergistic reduction in tumor size / volume, as compared to the level of achieved individually by equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety. That is, in some embodiments, the antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition and / or a reduction of tumor size / volume that is synergistic (i.e. super-additive), relative to what is observed when equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety are used alone.

[0284] As used herein, a ‘synergistic’ or ‘super-additive’ level of a relevant effect (e g. cell killing, inhibition of tumor growth, reduction in tumor size / volume) for a given antigen-binding molecule refers to a level of the effect which is greater than the sum of the effects observed for the individual comparator molecules (i.e. an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety, and an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety), when used alone.

[0285] Quantitative methods for assessing synergism are described e.g. in Tallarida, Genes Cancer. (2011) 2(11):1003— 1008 and Chou, Cancer Res (2010) 70:440-446, both of which are hereby incorporated by reference in their entirety. Additive, synergistic and antagonistic effects may be evaluated in experiments in which a range of different doses of an antigen-binding molecule of the disclosure and equivalent antigen-binding molecules comprising only the TOP1 or DDR inhibitor moiety are evaluated for the relevant effect. Dose-response curves may be plotted, and evaluated in order to determine whether the antigen-binding molecule of the disclosure achieves a synergistic level of the relevant effect relative to the equivalent antigen-binding molecules comprising only the TOP1 or DDR inhibitor moiety. In some embodiments, synergy may be evaluated using combination / composition index (Cl) values calculated using the Chou-Talalay method described in Chou, Cancer Res (2010) 70:440-446. According to the Chou-Talalay method, for a given antigen-binding molecule of the disclosure Cl = 1 indicates an additive effect, Cl <1 indicates synergism, and Cl >1 indicates antagonism. In some embodiments, the antigen-binding molecule of the present disclosure displays similar internalization into cells expressing the relevant target antigen for the antigen-binding molecule, as compared to internalization into cells of the same type displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety. In some embodiments, the antigen-binding molecule is internalized into cells expressing the relevant target antigen for the antigen-binding molecule to a level that is >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the level of internalization of an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety into the relevant cells, as determined in the same assay. In some embodiments, evaluation of internalization for the purposes of such comparison is performed after incubation of the relevant antigen-binding molecule with the relevant cells for more than 5 min, e.g. one of >30 min, >1 h, >1 .5 h, >2 h or >2.5 h.

[0286] In some embodiments, the antigen-binding molecule of the present disclosure displays similar toxicity to subjects administered the antigen-binding molecule as compared to subjects administered an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety. In some embodiments, the antigen-binding molecule has a similar toxicological profile as compared to an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety.

[0287] In some embodiments, the number / proportion of red blood cells / white blood cells / lymphocytes / monocytes / neutrophils / platelets in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and <2 times, e.g. one of >0.55 times and <1.9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1.2 times or >0.95 times and <1 .1 times the number / proportion of such cells observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0288] In some embodiments, the hemoglobin concentration / hematocrit percentage / mean corpuscular volume, mean corpuscular hemoglobin / mean corpuscular hemoglobin concentration in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the level observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0289] In some embodiments, the level of a correlate of hepatic function (e.g. alkaline phosphatase, alanine aminotransferase, albumin, total protein) in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the level observed in the peripheral blood of a subject following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0290] In some embodiments, the level of a correlate of renal function (e.g. blood urea nitrogen and / or creatinine) in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and <2 times, e.g. one of >0.55 times and <1.9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the level observed in the peripheral blood of a subject following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0291] In some embodiments, the level of a correlate of pancreatic function (e.g. glucose and / or amylase) in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1.1 times the level observed in the peripheral blood of a subject following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.

[0292] In some embodiments, the level of sodium / potassium / phosphate / calcium in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and <2 times, e.g. one of >0.55 times and <1 .9 times, >0.6 times and <1 .8 times, >0.65 times and <1 .7 times, >0.7 times and <1 .6 times, >0.75 times and <1 .5 times, >0.8 times and <1 .4 times, >0.85 times and <1 .3 times, >0.9 times and <1 .2 times or >0.95 times and <1 .1 times the level observed in the peripheral blood of a subject following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and / or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay. Additional sequences

[0293] The antigen-binding molecules of the present disclosure and their constituent polypeptides may additionally comprise further amino acids or sequences of amino acids.

[0294] The polypeptides of the present disclosure may comprise one or more linker sequences between sequences of amino acids. Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and / or serine residues.

[0295] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e g. 1 , 2, 3, 4, 5 or 6) copies (e g. in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)s. In some embodiments, the linker sequence has a length of 1 -2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

[0296] The antigen-binding molecules of the present disclosure and their constituent polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule / polypeptide. For example, antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.

[0297] The antigen-binding molecules of the present disclosure and their constituent polypeptides may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

[0298] The signal peptide may be present at the N-terminus of the polypeptide, and may be present in the newly synthesised polypeptide. The signal peptide provides for efficient trafficking of the polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature polypeptide. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 201 1 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).

[0299] Labels and conjugates

[0300] In some embodiments, the antigen-binding molecules of the present disclosure and their constituent polypeptides comprise a detectable moiety.

[0301] In some embodiments, a detectable moiety is a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. An antigen-binding molecule or a constituent polypeptide thereof may be covalently or non-covalently labelled with the detectable moiety.

[0302] Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Hydrogen3, Sulfur35, Carbon14, Phosphorus32, Iodine123, Iodine125, Iodine126, Iodine131, Iodine133, Bromine77, Technetium99m, Indium111, lndium113m, Gallium67, Gallium68, Ruthenium95, Ruthenium97, Ruthenium103, Ruthenium105, Mercury207, Mercury203, Rhenium99111, Rhenium101, Rhenium105, Scandium47, Tellurium121"1, Tellurium122"1, Tellurium125"1, Thulium165, Thulium167, Thulium168, Copper67, Fluorine18, Yttrium90, Palladium100, Bismuth217and Antimony211. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides / polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers.

[0303] In some embodiments, an antigen-binding molecule or a constituent polypeptide thereof comprises an epitope tag, e g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the antigen-binding molecule / polypeptide.

[0304] In some embodiments, an antigen-binding molecule or a constituent polypeptide thereof polypeptide comprises a moiety having a detectable activity, e.g. an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e g. beta-galactosidase), glucoronidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases. Producing the antigen-binding molecules

[0305] Antigen-binding polypeptides / polypeptide complexes according to the present disclosure may be conjugated to linker-payload moieties according to the present disclosure for the production of antigenbinding molecules according to the present disclosure by any suitable technigues, which are well known to the skilled person and routinely employed in the art.

[0306] General methods for the conjugation of antigen-binding polypeptides / polypeptide complexes to linkerpayload moieties are described e.g. in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah et al., Molecules. (2021) 26(10): 2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are hereby incorporated by reference in their entirety. Conjugation of antigen-binding moieties and linkerpayload moieties and purification of antigen-binding molecules produced by such conjugation is described e.g. in Beck et al., (2017) Nat Rev Drug Discov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351 ; Jackson, Org Process Res Dev (2016) 20: 852-866; and Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer (2016) Wiley.

[0307] Antigen-binding moieties according to the present disclosure may be conjugated to linker-payload moieties according to the present disclosure by any suitable techniques, which are well known to the skilled person and routinely employed in the art. Such methods are described e.g. in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah et al., Molecules. (2021) 26(10): 2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all ofwhich are hereby incorporated by reference in their entirety.

[0308] Conjugation of antigen-binding moieties and linker-payload moieties and the purification of antigenbinding molecules produced by such conjugation can be performed e.g. as described in in Beck et al., (2017) Nat Rev Drug Discov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351 ; Jackson, Org Process Res Dev (2016) 20: 852-866; or Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer, (2016) Wiley, all of which are hereby incorporated by reference in their entirety. Other relevant disclosures relating to conjugation and linkers are: Tsuchikama and An, Protein Cell. (2018) 9(1): 33-46, Khongorzul et al., Mol Cancer Res (2020) 18 (1): 3-19 and Drago et al., Nature Reviews Clinical Oncology (2021) 18: 327-344, all of which are hereby incorporated by reference in their entirety.

[0309] Lysine amide coupling Lysine-based conjugation is one of the most widely used non-specific conjugation strategies. Such conjugation occurs on reactive amine side chains of lysine residues due to their good nucleophilicity. Immunoglobulin scaffolds contains over 80 lysine residues, most ofwhich are exposed on the surface of the molecule. Among the surface lysine residues, more than 20 have been shown as highly solvent- accessible and can serve as potential ADC conjugation sites. Lysine conjugation follows two main strategies that result in the formation of a stable amide or amidine bond between the protein and the drug-linker complex. Generally speaking, activated esters on the drug-linker complexes, often O- succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters, react with the antibody lysine residues and achieve the conjugation via amide bonds. On the other hand, stable amidine bonds can be generated on an antibody by the reaction of imido ester compounds, such as Traut’s reagent, with antibody lysine residues.

[0310] A one-step conjugation of a drug-linker moiety containing an amine-reactive group to the antibody via amide bonds is known, as well as two-step conjugation, where in the first step, a small bi-functional reagent containing both an amine- and a thiol-reactive functional groups is reacted with the available lysine s-amino groups to serve as a chemical adaptor, leaving free thiol-reactive groups on the antibody. In the second step, the payload drugs or drug-linker complexes are attached to the thiol-reactive groups introduced previously to form the ADC. The two-step approach is often used when the drug / drug-linker complex contains a thiol-reactive module or as an alternative route when introducing an amine-reactive module into the drug or drug-linker complex is proven to be difficult. Four small adaptors commonly used in the two-step conjugation: SPDB disulfide, MCC (maleimidomethyl cyclohexane-1 -carboxylate), sulfo- SPDB, and Hydrazine.

[0311] Cysteine coupling

[0312] Cysteine modification occurs most commonly by 1 ,4-conjugate addition to / V-substituted maleimides. Maleimides are particularly attractive reagents due to their synthetic accessibility and rapid reaction rates with cysteine under mild conditions. The resulting thiosuccinimide conjugates are inherently unstable, due to their propensity towards retro-Michael addition. This instability can be mitigated by forcing postconjugation hydrolysis of the thiosuccinimide, creating a stable chemical linkage. Accordingly, a number of “self-hydrolysing” maleimides have now been developed, with ring-opening catalysed by adjacent functional groups such as primary amine, polyethylene glycol (PEG) and / V-aryl amongst the most promising. Other reagents including a-halocarbonyls, palladium oxidative-addition complexes, ethynylphosphonamidates, vinylphosphonites and ethynylbenziodoxolones.

[0313] Some non-maleimide cysteine conjugations are summarised in Kang, et al., Chem Sci (2021) 12, 13613- 13647 (doi: 10.1039 / D1 SC02973H), and include the use of:

[0314] (i) alkynyl carboxylic acid derivatives;

[0315] (ii) 5-methylene pyrrolone (5MP);

[0316] (iii) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB);

[0317] (iv) phenyloxadiazole sulfone (PODS);

[0318] (v) aza-dibenzocyclooctyne (DBCO);

[0319] (vi) phosphonamidite;

[0320] (vii) 3-arylpropionitrile (APN);

[0321] (viii) perfluoroarene;

[0322] (ix) ethynylbenziodoxolone (EBX);

[0323] (x) bicyclo[1 .1 .Ojbutane (BOB) carboxylic amide; and

[0324] (xi) allenamide. Another possible approach is described in Cheng, et al., Front. Oncol. 12:951589 (doi: 10.3389 / fonc.2022.951589) where 2-methylsulfonyl pyrimidine is used instead of a maleimide.

[0325] Genetic modification of the number of accessible cysteine residues on an antibody surface is a method to achieve site-selective and homogeneous modification. For example, in THIOMABs, the engineered cysteine is installed on an anti-MUC16 antibody by mutation of heavy chain alanine 114 (HC-A114).

[0326] Other approaches have engineered antibodies to contain cysteine mutations at D265C, S239C, E269C, K326C or A327C, or to insert additional cysteines before and after positions HC-S239, HC-A114, and LC- V205.

[0327] Non-natural amino acid incorporation by genetic engineering

[0328] Site-specific incorporation of non-canonical amino acids (ncAAs) into antibodies results in an efficient approach to the site-specific modification of antibodies, and therefore homogeneous ADCs.

[0329] NcAAs bearing unique functionalities, such as ketones, azides, cyclopropenes or diene functional groups, have been developed and incorporated into antibodies. Such ncAAs include p-acetylphenylalanine (pAcF), which has a ketone side chain which can participate in oxime ligation reactions; Ne-(1- methylcycloprop-2-enecarboxamido)-lysine (CpK), which has a cyclopropene side chain which can participate in EDDA reactions; para-azidomethyl phenylalanine (pAMF), which has a an azide side chain which can undergo click reactions; spiro[2.4]hepta-4,6-diene-lysine (SCpHK), which has a spiro[2.4]hepta-4,6-diene side chain which can participate in Diels-Alder reactions; and N6-(2- azidoethoxy)-carbonyl-L-lysine (AzK), which has a an azide side chain which can undergo click reactions.

[0330] Azide-containing ncAAs can undergo rapid CuAAC or SPAAC reactions under physiological conditions, para-azidophenylalanine (pAzF) can undergo reactions with, for example, cyclooctyne-functionalised linkers and dibenzylcyclooctyne (DBCO)-functionalised linkers. A cyclopropene derivative of lysine (N e- [((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l-lysine; CypK) can undergo a rapid and efficient inverse-electron demand Diels-Alder (EDDA) reaction with a tetrazine-functionalised linker.

[0331] Cyclopentadiene-containing ncAAs, spiro[2.4]hepta-4,6-diene-lysine (SCpHK) and cyclopentadiene-lysine (CpHK), can undergo irreversible Diels-Alder cycloadditions with maleimide-modified drugs.

[0332] Enzymatic conjugation

[0333] Enzymes can be used to achieve site-selective antibody modification due to their high specificity and mild reaction conditions. Enzymes can either directly attach a payload to a specific amino acid sequence or introduce a reactive functionality on the antibody that can be further functionalised with the desired payload.

[0334] Transpeptidation using sortase

[0335] Sortase-mediated antibody conjugation (SMAC) technology is an additional enzymatic ligation approach. SMAC-technology uses S. aureus sortase A, which is a transpeptidase that cleaves the amide bond between threonine and glycine residues in the LPXTG (X = any amino acid) pentapeptide motif, and subsequently catalyses the attachment of glycine-functionalised payloads to the newly generated C- terminus. The sortase recognition motif and a Strep II tag, which is used to aid removal of unreacted antibody, were fused to the light and heavy chain C-terminus of different antibodies. Sortase-mediated conjugation can then be used to attach a series of penta-glycine tagged payloads.

[0336] Transpeptidation using microbial transglutaminase

[0337] The use of bacterial transglutaminases is a powerful approach for site-specific incorporation of the payload into the antibody. A transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR arising from the conjugation of 2 linker-payloads (one conjugation site per heavy chain). An N297Q mutation prior to this conjugation provides two more reaction sites (resulting in the conjugation of 4-1 in ker- payloads). An alternative version using a peptide sequence-specific transglutaminase. This enzyme recognizes and utilizes LLQG motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation. Another advantage of this LLQG-specific bacterial transglutaminase is that conjugation sites can be flexibly laid by inserting this short peptide motif within the antibody structure. Further alternative approaches allow for the use of transglutaminase without deglycosylation.

[0338] The DAR will depend on the number of payloads per linker-payload moieties conjugated.

[0339] N-Glycan engineering

[0340] Asn297 (N297) within the Fc domain and the N-glycan on this residue are conserved in all IgG classes, making these components attractive reaction sites for broadly applicable ADC conjugation. Incorporation of an aldehyde group on the N-glycan terminus using p-1 ,4-galactosyltransferase (GalT) and a-2,6- sialyltransferase (SialT) introduce a sialic acid on each N-glycan terminus, which is subsequently converted into an aldehyde group using NalO4 under mild oxidation conditions. The aldehyde groups generated can then be used to conjugate aminooxyfunctionalized payloads.

[0341] Another approach is to incorporate non-natural saccharides possessing orthogonal reaction handles into the antibody. A technology based on this strategy is the GlycoConnect in which the glycan chain at Asn297 is trimmed using the endoglycosidase Endo S2 and then azide groups are introduced using a mutant galactosyl transferase GalT(Y289L) and N-azidoacetylgalactosamine (GalNAz). The azide handles can be used for a strain-promoted click reaction with payloads.

[0342] In some embodiments, the linker-payload terminates in an amino group that may be conjugated to the antigen-binding molecule using transglutaminase. It is to be understood that alternative conjugation methods (e.g., lysine amide coupling or cysteine coupling) may be employed to produce the antigenbinding molecules described herein. It is also to be understood that any of the linker moieties described herein may be further modified to contain one or more groups suitable for conjugation to an antigenbinding moiety via alternative conjugations methods known by the skilled person or described herein). For example, the amino group of the linker moiety for conjugation to an antigen-binding moiety may be further modified to contain one or more groups suitable for conjugation to an antigen-binding moiety via alternative conjugations methods known by the skilled person or described herein.

[0343] In some embodiments, the method further comprises purifying / isolating the antigen-binding molecule ( / .e. from unreacted precursors and / or by-products). In some embodiments, the antigen-binding molecule may be purified / isolated by chromatography, e g. size-exclusion chromatography.

[0344] The present disclosure also provides an antigen-binding molecule obtained or obtainable by the methods of the present disclosure.

[0345] Antigen-binding moieties according to the present disclosure may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides / polypeptides can be synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.

[0346] Alternatively, antigen-binding moieties according to the present disclosure may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thEdition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding polypeptides are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety.

[0347] In some cases, the antigen-binding moieties of the present disclosure are comprised of more than one polypeptide chain. In such cases, production of the antigen-binding moiety may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding moiety.

[0348] For recombinant production according to the present disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments, the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments, the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e g. a cell described hereinabove. In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media. In some embodiments polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according to a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431 , which is hereby incorporated by reference in its entirety.

[0349] Production of antigen-binding moieties may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thEdition; incorporated by reference herein above). Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0350] Following culturing the cells that express the polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e g. by Bonification, rapid freeze-thaw or osmotic lysis.

[0351] It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins. Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be performed subsequently to precipitation.

[0352] Once the polypeptide(s) of interest have been isolated from culture it may be desired or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation. Conjugation of linker to antigen-binding molecule

[0353] The linker moieties, including linker-payload molecules, of the present disclosure may be conjugated to the antigen-binding molecules by enzymatic conjugation. The use of bacterial transglutaminases is a powerful approach for site-specific incorporation of the payload into the antibody. A transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR arising from the conjugation of 2 linkerpayloads (one conjugation site per heavy chain). An N297Q mutation prior to this conjugation provides two more reaction sites (resulting in the conjugation of 4-linker-payloads). An alternative version using a peptide sequence-specific transglutaminase. This enzyme recognizes and utilizes LLQG motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation. Another advantage of this LLQG-specific bacterial transglutaminase is that conjugation sites can be flexibly laid by inserting this short peptide motif within the antibody structure. Further alternative approaches allow for the use of transglutaminase without deglycosylation.

[0354] In some embodiments, the linker moiety is conjugated to the antigen-binding moiety via a glutamine residue of the antigen-binding moiety. For example, the conjugation is between the amine group of the linker moiety and the glutamine at Q295 (EU numbering) of the antigen-binding moiety. In some embodiments, the antigen-binding moiety is an antibody or an antigen-binding fragment thereof.

[0355] The DAR will depend on the number of payloads per linker-payload moieties conjugated. In some embodiments, the ratio between the linker moiety to the antigen-binding moiety is from about 1 :1 to about 2:1 . In some embodiments, the ratio between the linker moiety to the antigen-binding moiety is about 2:1 . In some embodiments, the ratio between the linker-payload moiety and the antigen-binding moiety is 1 :1. In some embodiments, the ratio between the linker-payload moiety and the antigen-binding moiety is 2:1 . In some embodiments, the linker-payload moiety comprises a first payload (e.g. a TOP1 inhibitor) and a second payload (e.g., a DDR inhibitor), wherein the ratio between the first payload and the second payload is about 1 :1. In some embodiments, the antigen-binding molecule describe herein has a DAR for the first payload of from about 3 to about 4. In some embodiments, the antigen-binding molecule describe herein has a DAR for the second payload of from about 3 to about 4. In some embodiments, the antigen-binding molecule describe herein has a DAR for the first payload of about 4 and a DAR for the second payload of about 4.

[0356] In some embodiments, the method further comprises purifying / isolating the antigen-binding molecule ( / .e. from unreacted precursors and / or by-products). In some embodiments, the antigen-binding molecule may be purified / isolated by chromatography, e g. size-exclusion chromatography.

[0357] The present disclosure also provides an antigen-binding molecule obtained or obtainable by the methods of the present disclosure. Compositions

[0358] The present disclosure provides a composition comprising an antigen-binding molecule according to the present disclosure.

[0359] The antigen-binding molecules described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. Thus, the present disclosure provides a pharmaceutical composition / medicament comprising an antigen-binding molecule described herein.

[0360] The pharmaceutical compositions / medicaments of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0361] The term pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rdEdition (2020), Academic Press.

[0362] Pharmaceutical compositions and medicaments of the present disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0363] Suitable formulations may comprise the antigen-binding molecule provided in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0364] In some embodiments, the pharmaceutical compositions / medicament is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or a tumor.

[0365] The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing an antigen-binding molecule described herein; isolating / purifying an antigen-binding molecule described herein; and / or mixing an antigen-binding molecule described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.

[0366] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g. a disease / condition described herein), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.

[0367] Therapeutic and prophylactic applications

[0368] The antigen-binding molecules and compositions described herein find use in therapeutic and prophylactic intervention for disease, e.g. cancers.

[0369] It will be appreciated that the antigen-binding molecules and compositions of the present disclosure may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level of expression or activity of the target antigen for the antigen-binding molecule, or a reduction in the number or activity of cells comprising / expressing the target antigen for the antigen-binding molecule.

[0370] For example, the disease / condition may be a disease / condition in which the target antigen for the antigen-binding molecule, or cells expressing / overexpressing the target antigen for the antigen-binding molecule are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of the target antigen for the antigen-binding molecule, or an increase in the number / proportion of cells comprising / expressing the target antigen for the antigen-binding molecule is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of the target antigen for the antigen-binding molecule, or an increase in the number / proportion of cells comprising / expressing the target antigen for the antigen-binding molecule may be a risk factor for the onset, development or progression of the disease / condition. The present disclosure provides an antigen-binding molecule or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is an antigen-binding molecule or composition described herein for use in a method of treating or preventing a cancer (e.g. a cancer described herein). Also provided is the use of an antigen-binding molecule or composition described herein in the manufacture of a medicament for treating or preventing a cancer (e g. a cancer described herein). Also provided is a method of treating or preventing a cancer (e.g. a cancer described herein) in a subject, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigenbinding molecule or composition described herein.

[0371] The methods may be effective to reduce the development or progression of a cancer, alleviation of the symptoms of a cancer or reduction in the pathology of a cancer. The methods may be effective to prevent progression of the cancer, e.g. to prevent worsening of, or to slow the rate of development of, the cancer. In some embodiments, the methods may lead to an improvement in the cancer, e.g. a reduction in the symptoms of the cancer or reduction in some other correlate of the severity / activity of the cancer. In some embodiments, the methods may prevent development of the cancer to a later stage (e.g. a chronic stage or metastasis).

[0372] As used herein, a ‘cancer’ may be or comprise any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant. The cancer may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues / cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, biliary tract, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.

[0373] Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumors may originate in any other non-nervous tissue; examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, endometrial cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma. In some embodiments, the cancer is breast cancer, lung cancer (e g. small cell lung cancer), gastric cancer or endometrial cancer. In some embodiments, the cancer is breast ductal carcinoma, small cell lung carcinoma, gastric carcinoma or endometrial carcinoma.

[0374] In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is a myeloid hematologic cancer, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, AIDS-related lymphoma, cutaneous T cell lymphoma, mycosis fungicides, primary central nervous system lymphoma, Sezary syndrome, Waldenstrom macroglobulinemia, leukemia, T cell leukemia, B cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, acute promyelocytic leukemia, chronic promyelocytic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, myeloma, multiple myeloma, myelodysplastic syndrome, or a myeloproliferative disorder.

[0375] In some embodiments, the cancer to be treated / prevented comprises cells expressing the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented is a cancer which is positive for the target antigen for the antigen-binding molecule. In some embodiments, the cancer comprises cells that overexpress the target antigen for the antigen-binding molecule.

[0376] Overexpression can be determined by detection of a level of expression which is greater than the level of expression by equivalent non-cancerous cells / non-tumor tissue.

[0377] In some embodiments, the cancer to be treated / prevented is a cancer which is heterogenous for expression of the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented comprises a population of cells heterogenous for expression of the target antigen for the antigen-binding molecule.

[0378] In some embodiments, the cancer to be treated / prevented comprises cells that do not express the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented comprises cells that are negative for the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented comprises cells expressing the target antigen for the antigen-binding molecule and cells that do not express the target antigen for the antigen-binding molecule. In some embodiments the cancer to be treated / prevented comprises cells which are positive for the target antigen for the antigen-binding molecule and cells which are negative for the target antigen for the antigen-binding molecule.

[0379] Expression may be determined by any suitable means. Expression may be gene expression or protein expression. Gene expression can be determined e.g. by detection of mRNA encoding the target antigen for the antigen-binding molecule, for example by quantitative real-time PCR (qRT-PCR). Protein expression can be determined e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA. In some embodiments the cancer is a cancer in which the target antigen for the antigen-binding molecule is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of the target antigen for the antigen-binding molecule, a cancer for which expression of the target antigen for the antigen-binding molecule is a risk factor and / or a cancer for which expression of the target antigen for the antigen-binding molecule is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by expression of the target antigen for the antigen-binding molecule, e.g. the cancer may comprise cells (e.g. cells of tumor tissue) expressing the target antigen for the antigen-binding molecule. Such cancers may be referred to as being positive for the target antigen for the antigen-binding molecule. A cancer which is ‘positive’ for the target antigen for the antigen-binding molecule may be a cancer comprising cells expressing the target antigen for the antigen-binding molecule (e.g. at the cell surface). A cancer which is ‘positive’ for the target antigen for the antigen-binding molecule may overexpress the target antigen for the antigen-binding molecule.

[0380] In some embodiments, the cancer to be treated / prevented comprises cells harboring a genetic variant (e.g. a mutation) which causes increased (gene and / or protein) expression and / or activity of the target antigen for the antigen-binding molecule, relative to comparable cells harboring a reference allele not comprising the genetic variant (e.g. a non-mutated, or ‘wildtype’ allele). The genetic variant may be or comprise insertion, deletion, substitution to, or larger-scale translocation / rearrangement of, the nucleotide sequence relative to the reference allele.

[0381] A mutation resulting in’ increased expression of the target antigen for the antigen-binding molecule may be known or predicted to cause, or may be associated with, increased gene / protein expression of the target antigen for the antigen-binding molecule. Mutations resulting in increased expression and / or activity of the target antigen for the antigen-binding molecule may be referred to as ‘activating’ mutations.

[0382] A mutation which causes increased expression of the target antigen for the antigen-binding molecule may result in gene or protein expression of the target antigen for the antigen-binding molecule which is not expressed by, and / or not encoded by genomic nucleic acid of, an equivalent cell not harboring the mutation. That is, the expression of the target antigen for the antigen-binding molecule may be a result of the mutation, and thus ‘increased expression’ may be from no expression.

[0383] A mutation which causes increased expression of the target antigen for the antigen-binding molecule may result in increased gene or protein expression of the target antigen for the antigen-binding molecule which is expressed by, and / or which is encoded by genomic nucleic acid of, an equivalent cell not comprising the mutation. By way of illustration, a cell may comprise mutation(s) resulting in an increase in the level of transcription of nucleic acid encoding the target antigen for the antigen-binding molecule relative to the level of transcription of the relevant nucleic acid(s) by an equivalent cell not comprising the mutation(s).

[0384] In some embodiments, a mutation which causes increased expression of the target antigen for the antigen-binding molecule may cause an increase in gene expression of the gene encoding the relevant antigen relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of the target antigen for the antigen-binding molecule may cause an increase in protein expression of the relevant protein relative to an equivalent cell not comprising the mutation.

[0385] In some embodiments, a mutation which causes increased expression of the target antigen for the antigen-binding molecule may cause an increase in the level of the target antigen for the antigen-binding molecule on or at the cell surface of a cell comprising the mutation, relative to an equivalent cell not comprising the mutation.

[0386] Cells having increased expression of the target antigen for the antigen-binding molecule relative to the level of expression of the target antigen for the antigen-binding molecule by a reference cell (e.g. as a result of mutation) may be described as ‘overexpressing’ the target antigen for the antigen-binding molecule, or having ‘upregulated expression’ of the target antigen for the antigen-binding molecule. For example, a cancer comprising cells harboring a mutation resulting in increased expression of the target antigen for the antigen-binding molecule relative to equivalent cells lacking the mutation may be described as a cancer comprising cells displaying overexpression / upregulated expression of the target antigen for the antigen-binding molecule. In some embodiments, the reference cell lacking the mutation may be a non-cancerous cell (e.g. of equivalent cell type) or a cancerous cell (e.g. of equivalent cancer type).

[0387] A mutation which causes increased activity of the target antigen for the antigen-binding molecule may result in an increase in the target antigen for the antigen-binding molecule-mediated activity relative to the level of the target antigen for the antigen-binding molecule-mediated activity by an equivalent cell not comprising the mutation.

[0388] In some embodiments, a cancer to be treated / prevented in accordance with the present disclosure may be characterised by an increase in the expression and / or activity of the target antigen for the antigenbinding molecule ( / .e. gene and / or protein expression) in an organ / tissue / subject affected by the disease / condition e.g. as compared to normal organ / tissue / subject ( / .e. in the absence of the disease / condition). In some embodiments, cells and / or a tumor of a cancer to be treated / prevented may be characterised by an increase in the expression and / or activity of the target antigen for the antigenbinding molecule, e g. as compared to the level of expression and / or activity observed in equivalent non- cancerous cells / non-tumor tissue.

[0389] In some embodiments, the cancer may be a relapsed cancer. As used herein, a ‘relapsed’ cancer refers to a cancer which responded to a treatment (e.g. a first line therapy for the cancer), but which has subsequently re-emerged / progressed, e.g. after a period of remission. For example, a relapsed cancer may be a cancer whose growth / progression was inhibited by a treatment (e.g. a first line therapy for the cancer), and which has subsequently grown / progressed. A cancer that is relapsed with respect to given treatment may be described as having acquired resistance to such treatment. In some embodiments, the cancer may be a refractory cancer. As used herein, a ‘refractory’ cancer refers to a cancer which has not responded to a treatment (e.g. a first line therapy for the cancer). For example, a refractory cancer may be a cancer whose growth / progression was not inhibited by a treatment (e.g. a first line therapy for the cancer). In some embodiments a refractory cancer may be a cancer for which a subject receiving treatment for the cancer did not display a partial or complete response to the treatment. A cancer that is refractory with respect to given treatment may be described as having intrinsic resistance to such treatment.

[0390] In some embodiments, the cancer is a cancer that is relapsed or refractory with respect to treatment with a DNA damage response (DDR) inhibitor. In some embodiments, the cancer is refractory with respect to treatment with a DDR inhibitor. In some embodiments, the cancer is relapsed with respect to treatment with a DDR inhibitor. In some embodiments, the cancer has intrinsic resistance to treatment with a DDR inhibitor. In some embodiments, the cancer has acquired resistance to treatment with a DDR inhibitor. In accordance with such embodiments, the DDR inhibitor may have been administered in the form of an antigen-binding molecule comprising a payload moiety comprising or consisting of the DDR inhibitor, or may have been administered in unconjugated form.

[0391] Herein, where a cancer is described as being relapsed / refractory / resistant, etc. with respect to a given intervention, it may be simply described as being ‘relapsed / refractory / resistant to’ the relevant intervention.

[0392] DDR inhibitors and their use for the treatment of cancers is described e.g. in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et a!., Front Immunol. (2022) 13:854730 and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701 , all of which are hereby incorporated by reference in their entirety. In some embodiments, a DDR inhibitor according to the present disclosure is selected from: a PARP inhibitor (e.g. olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X- 121 , AMXI-5001 , JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397, fluzoparib, NMS-03305293, AZD9574), an ATM inhibitor (e.g. CP-466722, KU-55933, KU-60019, KU- 59403, AZ31 , AZ32, AZD0156, AZD1390), an ATR inhibitor (e g. M6620 (berzosertib), M4344 (VX-803), AZD6738 (ceralasertib), BAY1895344 (elimusertib), RP3500 (camonsertib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027), a WEE1 inhibitor (e.g. adavosertib, Debio 0123, PD0166285, PD0407824, AZD1775 ZN-c3 (azenosertib), IMP7068, SY4835, SCO191 , IMP7068), a CHK1 / 2 inhibitor (e g. CBP-501 , prexasertib, MK-8776, GDC-0575, SRA-737, PF-00477736, AZD7762, LY2603618 (rabusertib), LY2880070, XL884, BEBT260, CC-115, MU380, NU7441 , KU-5778), a DNA-PK inhibitor (e.g. CC-115, LY-3023414, AsiDNA, M3814 (nedisertib, peposertib), M9831 (VX-984), BR-101801 , XRD-0394, SL901 , XZP-6877, IMP-11 , ZL-2201 , BR-2006, AZD7648, NU7441), a PLK1 inhibitor (e.g. BI-6727 (volasertib), PCM-075 (onvansertib), CYC140 (plogosertib)), a PolO inhibitor (e.g. ART4215, ART6043, novobiocin, RP-6685, RP-3467), a RAD51 inhibitor (e.g. CYT0851), a USP inhibitor (e g. an inhibitor of USP11 , USP7, USP4, USP37, USP39, USP45, USP24 and / or USP1 ; e.g. KSQ-4279), a PKMYT1 inhibitor (e.g. RP6306), or an Aurora-A inhibitor (e.g. alisertib, WJ05129 (JS112), JAB-2485). In some embodiments, the cancer is a cancer that is relapsed or refractory with respect to treatment with a DNA topoisomerase I (TOP1) inhibitor. In some embodiments, the cancer is refractory with respect to treatment with a TOP1 inhibitor. In some embodiments, the cancer is relapsed with respect to treatment with a TOP1 inhibitor. In some embodiments, the cancer has intrinsic resistance to treatment with a TOP1 inhibitor. In some embodiments, the cancer has acquired resistance to treatment with a TOP1 inhibitor. In accordance with such embodiments, the TOP1 inhibitor may have been administered in the form of an antigen-binding molecule comprising a payload moiety comprising or consisting of the TOP1 inhibitor, or may have been administered in unconjugated form.

[0393] DNA topoisomerase I inhibitors and their use for the treatment of cancers is described e g. in Pommier, Chem Rev. (2009) 109(7): 2894-2902, Li et al., Am J Cancer Res. (2017) 7(12): 2350-2394 and Thomas and Pommier, Clin Cancer Res. (2019) 25(22): 6581 -6589, all of which are hereby incorporated by reference in their entirety. In some embodiments, a TOP1 inhibitor according to the present disclosure is selected from: camptothecin, irinotecan, etirinotecan, SN-38, DX-8951f (extatecan mesylate), exatecan, FL118, topotecan, gimatecan, belotecan, deruxtecan, belotecan, rubitecan, lurtotecan, diflomotecan, karenitecan, silatecan, namitecan, elomotecan, DRF-1042, delimotecan, NSC606985, chimmitecan, ZBH- 1205, Genz-644282, non-CPT1 , indotecan (LMP-400), indimitecan (LMP-776), LMP744, AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021 , VIP126 and ZBH-01.

[0394] In some embodiments, the cancer to be treated / prevented in accordance with the present disclosure is a cancer that is: relapsed or refractory with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and relapsed or refractory with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein). In some embodiments, the cancer is: refractory with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and refractory with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein). In some embodiments, the cancer is: relapsed with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and relapsed with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein). In some embodiments, the cancer is: refractory with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and relapsed with respect to treatment with a TOP1 inhibitor (e g. a TOP1 inhibitor as described herein). In some embodiments, the cancer is: relapsed with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and refractory with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein). In accordance with such embodiments, the TOP1 inhibitor and / or DDR inhibitor may have been administered in the form of an antigen-binding molecule comprising a payload moiety comprising or consisting of the TOP1 inhibitor / DDR inhibitor, or may have been administered in unconjugated form.

[0395] Treatment of a cancer in accordance with the methods of the present disclosure achieves one or more of the following treatment effects: reduces the number of cancer cells in the subject, reduces the size of a cancerous tumor / lesion in the subject, inhibits (e.g. prevents or slows) growth of cancer cells in the subject, inhibits (e g. prevents or slows) growth of a cancerous tumor / lesion in the subject, inhibits (e g. prevents or slows) the development / progression of a cancer (e g. to a later stage, or metastasis), reduces the severity of symptoms of a cancer in the subject, increases survival of the subject (e.g. progression free survival or overall survival), reduces a correlate of the number or activity of cancer cells in the subject, and / or reduces cancer burden in the subject.

[0396] Subjects may be evaluated in accordance with the Revised Criteria for Response Assessment: The Lugano Classification (described e.g. in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated by reference hereinabove) in order to determine their response to treatment. In some embodiments, treatment of a subject in accordance with the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.

[0397] Prevention may refer to prevention of development of a cancer, and / or prevention of worsening of a cancer, e.g. prevention of progression of a cancer, e.g. to a later stage (e.g. metastasis).

[0398] In some embodiments, administration of an antigen-binding molecule / composition according to the present disclosure may be associated with one or more of: inhibition of the development / progression of the cancer, a delay to / prevention of onset of the cancer, a reduction in / delay to / prevention of tumor growth, a reduction in / delay to / prevention of tissue invasion, a reduction in / delay to / prevention of metastasis, a reduction in the severity of one or more symptoms of the cancer, a reduction in the number of cancer cells, a reduction in the cancer burden, a reduction in tumor size / volume, and / or an increase in survival of subjects having the cancer (e g. progression free survival or overall survival).

[0399] In accordance with various aspects of the present disclosure, a method of treating and / or preventing a cancer according to the present disclosure may comprise inhibiting the growth of a tumor, reducing the size / volume of a tumor and / or increasing the survival of a subject having the cancer.

[0400] In accordance with various aspects of the present disclosure, methods are provided which are for, or which comprise (e.g. in the context of treatment / prevention of a cancer, e.g. a cancer described herein), one or more of the following: binding to cells expressing the target antigen for the antigen-binding molecule; inhibiting the proliferation of cells expressing the target antigen for the antigen-binding molecule; killing cells expressing the target antigen for the antigen-binding molecule; inhibiting proliferation and / or increasing killing of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; inhibiting tumor growth and / or reducing tumor size / volume, e.g. of a cancer expressing the target antigen for the antigen-binding molecule; and / or increasing the survival of subjects having a cancer, e.g. a cancer expressing the target antigen for the antigen-binding molecule.

[0401] Also provided are antigen-binding molecules and compositions according to the present disclosure for use in such methods, and the use of antigen-binding molecules and compositions according to the present disclosure in manufacture of compositions (e.g. medicaments) for use in such methods. It will be appreciated that the methods typically comprise administering an antigen-binding molecule according to the present disclosure to a subject.

[0402] Similarly, one or more of the following may be observed in a subject following therapeutic or prophylactic intervention in accordance with the present disclosure (e.g. compared to the level / number / proportion etc. prior to intervention): inhibition of proliferation of cells expressing the target antigen for the antigen-binding molecule; killing of cells expressing the target antigen for the antigen-binding molecule; inhibition of proliferation and / or increased killing of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; inhibition of tumor growth and / or reduction of tumor size / volume, e.g. of a cancer expressing the target antigen for the antigen-binding molecule; and / or increased survival of a subject having a cancer, e.g. a cancer expressing the target antigen for the antigen-binding molecule.

[0403] In some embodiments, therapeutic / prophylactic intervention in accordance with the present disclosure may be described as being ‘associated with’ one or more of the effects described in the preceding paragraph. The skilled person is readily able to evaluate such properties using techniques that are routinely practiced in the art.

[0404] Administration of the antigen-binding molecules and compositions of the present disclosure is preferably in a therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and timecourse of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rdEdition (2020), Academic Press.

[0405] Administration of the antigen-binding molecules and compositions of the present disclosure may be e.g. parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral or transdermal. Administration may be by injection, infusion or ingestion.

[0406] In some aspects and embodiments, articles of the present disclosure may be administered to a tissue / organ of interest (e.g. a tissue / organ affected by the disease / condition affected by the condition (e.g. a tissue / organ in which symptoms of the disease / condition manifest). In some aspects and embodiments, articles of the present disclosure may be administered to the blood ( / .e. intravenous / intra- arterial administration) by injection or infusion (e.g. via cannula), or may be administered subcutaneously or orally. In some aspects and embodiments, articles of the present disclosure may be administered to a tumor.

[0407] In some embodiments, therapeutic or prophylactic intervention according to the present disclosure may further comprise administering another agent for the treatment / prevention of the relevant disease / condition. Administration of antigen-binding molecules and compositions described herein may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Simultaneous administration refers to administration with another therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other (e g. within 1 , 4, 6, 8 or 12 hours) and optionally via the same route of administration (e.g. to the same tissue, artery, vein or other blood vessel). Sequential administration refers to administration of one agent followed after a given time interval by separate administration of another agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.

[0408] Multiple doses of the antigen-binding molecules and compositions may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

[0409] Subjects

[0410] The subject in accordance with aspects described herein may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g. a cancer, e.g. a cancer described herein), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0411] In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the present disclosure herein is a subject having, or at risk of developing, a cancer, e.g. a cancer described herein. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease / condition.

[0412] In some embodiments, a patient may be selected for treatment described herein based on the detection of a cancer expressing / overexpressing the target antigen for the antigen-binding molecule, e.g. in a sample obtained from the subject (e.g. a biopsy, e.g. of a tumor). Kits

[0413] The present disclosure also provides kits of parts. A kit according to the present disclosure may comprise components for performing a method described herein, in whole or in part.

[0414] 5 The kit may have at least one container having a predetermined quantity of an antigen-binding molecule or composition described herein.

[0415] In some aspects of the present disclosure a kit of parts is provided. In some embodiments, the kit may comprise an antigen-binding molecule or composition described herein, and which may be provided in a 10 predetermined quantity.

[0416] The kit may provide an antigen-binding molecule or composition described herein together with instructions for administration to a patient in order to treat a specified disease / condition (e.g. a disease / condition described herein, e.g. a cancer).

[0417] 15

[0418] The kit may provide an antigen-binding moiety according to the disclosure, and a linker-payload moiety according to the present disclosure. The kit may further comprise reagents for conjugating the antigenbinding moiety and the linker-payload moiety.

[0419] 20 The kit may further comprise reagents, buffers and / or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0420] 25 Sequence identity

[0421] As used herein, ‘sequence identity' refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining

[0422] 30 percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Sbding, J. 2005, Bioinformatics 21 , 951 -960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When

[0423] 35 using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0424] Sequences

[0425] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0426] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0427] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0428] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0429] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a’, ‘an’, and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about’, it will be understood that the particular value forms another embodiment. Where a nucleic acid sequence is disclosed or referred to herein, the reverse complement thereof is also expressly contemplated.

[0430] Methods described herein may preferably be performed in vitro. The term ‘in vitro' is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo' is intended to encompass procedures with / on intact multi-cellular organisms. Values may be expressed herein as ‘about’ a particular value. Similarly, ranges may be expressed herein as from ‘about’ a particular value, and / or to ‘about’ another particular value. The term ‘about’ in relation to a numerical value is optional, and means for example + / - 10 %. By way of illustration, reference e.g. to ‘about 10 %’ is to be construed as 9 % to 11 %. In instances herein where ‘about’ is recited, the value it precedes is also specifically contemplated. By way of illustration, reference e.g. to ‘about 10 %’ also specifically contemplates 10 %.

[0431] Brief Description of the Figures

[0432] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0433] Figure 1 shows general synthetic schemes for LM3, LM5, LM6, and LM7.

[0434] Figure 2A and 2B show general synthetic schemes for LP-2, LP-4, LP-5, and LP-6.

[0435] Figure 3 shows a process flowchart for ADC production. Figure 4 shows the efficacy of ADC1 against cells expressing the antigen of the antibody in the ADC.

[0436] General methods

[0437] All chemicals, raw materials and solvents were purchased from commercial sources, unless indicated otherwise. All chemical reactions were run under ambient conditions, unless otherwise indicated. Flash column chromatography was performed with CombiFlash® NEXTGEN 100, and the column was purchased from Agela Technologies. Prep-HPLC purifications were carried out using AUNO LC-2000, and the column is of Phenomenex Luna C18, 250 x 100 mm, 10pm, 10nm.1H NMR spectra were recorded on a Bruker spectrometer (400 MHz).1H NMR chemical shifts are expressed in parts per million (6) downfield from tetramethylsilane (with the CDCIs peak at 7.26 ppm used as a standard). Mass Spectrometric data were recorded on SHIMADZU LCMS-2020 (ESI-MS) and Agilent 1260\G6125B (ESIMS), and the column is of Kinetex® EVO C18 4.6x50mm, 5pm, Kinetex® EVO C18 2.1*30mm, 5pm, Shim-pack Scepter C18-120 3.0x33mm 3um and Poroshell 120 EC C18 2.7pm 3.0*30mm.

[0438] Synthesis of intermediate 17 a) Oxybis(ethane-2, 1-diyl)dimethanesulfonate (12)

[0439] To the solution of 2,2'-oxybis(ethan-1-ol) 11 (20 g, 188.46 mmol, 1.0 eq.) in 400 mL of CH2CI2 at 0 °C, triethylamine (1 18.8 mL, 848.07 mmol, 4.5 eq.) was added followed by the dropwise addition of methanesulfonyl chloride (32 mL, 414.62 mmol, 2.2 eq.) under nitrogen atmosphere. The reaction mixture was stirred at rt for 16 h. The reaction mixture was quenched with sulfate buffer solution and extracted with CH2CI2. Combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get compound I2 as a pale yellow solid (49 g, Crude). b) 2-(2-azidoethoxy)ethyl methanesulfonate (13)

[0440] To the solution of oxybis(ethane-2,1-diyl) dimethanesulfonate 12 (20 g, 76.33 mmol, 1 .0 eq.) in 500 mL of Acetonitrile at rt was added TBAB (2.46 g, 7.63 mmol, 0.1 eq.) followed by portion wise addition of NaNs (4.96 g, 76.33 mmol, 1 .0 eq.) under nitrogen atmosphere. The reaction mixture was stirred at 70 °C for 40 h. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to get crude compound. The crude product was purified by MPLC Flash Column chromatography using EtOAc in Hexane (20:80) eluent to afford the title compound 13 as pale-yellow liquid (7.8 g, 48%). c) Tert-butyl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate (15)

[0441] To a stirred solution of tert-butyl (2-aminoethyl)carbamate 14 (3.0 g, 18.73 mmol, 1.0 eq.) and 2-(2- azidoethoxy)ethyl methane sulfonate I3 (7.83 g, 37.47 mmol, 2.0 eq .) in 100 mL of acetonitrile were added K2CO3 (7.76 g, 56.19 mmol, 3.0 eq.) and KI (310 mg, 1 .87 mmol, 0.1 eq,) at rt under nitrogen atmosphere. The reaction was stirred at 100° C for 16 h. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to get crude compound. The crude product was purified by MPLC Flash Column chromatography using EtOAc in Hexane (25:75) eluent to afford the title compound I5 as pale-yellow liquid (4.0 g, 55%). ES / APCI: (M+1): 387.3 d) N1,N1-bis(2-(2-azidoethoxy)ethyl)ethane-1,2-diamine dihydrochloride (16)

[0442] To the stirred solution of tert-butyl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate 15 ((0.65 g, 1 .6819 mmol,1 .0 eq.) in 10 mL CH2CI2 at 0°C 4M HCI in 1 ,4-dioxane (5 mL) was added and stirred at rt for 2 h. The reaction mixture was evaporated under reduced pressure to get crude product I6 (0.7 g) as a pale brown liquid. HRMS: (M+1): 287.2390. e) 2,5-dioxopyrrolidin-1-yl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate (17)

[0443] To the solution of N1 ,N1 -bis(2-(2-azidoethoxy)ethyl)ethane-1 ,2-diamine dihydrochloride I6 (0.7 g, 1.9484 mmol, 1.0 eq.) and bis(2,5-dioxopyrrolidin-1-yl) carbonate (0.6 g, 2.3381 mmol, 1.2 eq.) in 10 mL of THF, DIPEA (1.01 mL, 5.8452 mmol, 3.0 eq.) was added and refluxed for 1 h. The reaction mixture was evaporated under reduced pressure to get crude product. The crude product was diluted with EtOAc and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get 0.81 g of crude pale brown liquid 17.

[0444]

[0445] LM1 a) tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3-oxa-6,9, 11-triazahexadecan-16-yl)carbamate

[0446] (19)

[0447] To the solution of 2,5-dioxopyrrolidin- 1 -yl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate I7 (0.5 g, 1.1698 mmol, 1 .0 eq.) in 10 mL of CH2Cl2 at RT, DIPEA (0.61 mL, 3.5094 mmol, 3.0 eq.) and catalytic DMAP (2 mg) were added followed by the addition of tert-butyl (5-aminopentyl)carbamate 18 (0.28 g, 1.4037 mmol, 1.2 eq.) and stirred at RT for 16 h. The reaction mixture was evaporated and diluted with Ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude product. The crude product was purified by MPLC Flash Column chromatography using MeOH in CH2CI2 (2:98) eluent to afford the title compound 19 as pale yellow liquid (0.18 g, 30%). HRMS:(M+1): 515.2715. b) 1 -(5-aminopentyl)-3-(2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)urea (LM1)

[0448] To the solution of tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3-oxa-6,9,11-triazahexadecan-16- yl)carbamate I9 (0.18 g, 0.3497 mmol, 1.0 eq.) in 5.0 mL CH2CI2 at O°C, 4M HCI in 1 ,4-dioxane (1 mL) was added and stirred at RT for 3 h. The reaction mixture was evaporated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude material. The crude material was washed with ether and the residue was dissolved in CH2CI2. The organic layer was washed with saturated NaHCOs solution then aqueous layer was extracted with 10% MeOH in CH2CI2. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford LM1 (0.07 g, 48%) as pale brown liquid.1H NMR (DMSO-d6) 400 MHz) 6 ppm 5.95 (t, J=5.2 Hz, 1 H), 5.59 (t, J=5.6 Hz, 1 H), 3.57-3.56 (m, 4H), 3.48-3.47 (m, 4H), 3.39-3.38 (m, 4H), 3.02-3.0 (m, 2H), 2.95-2.93 (m, 2H), 2.55-2.53 (m, 4H), 1 .36- 1.33 (m, 4H), 1.24-1.23 (m, 2H). HRMS: (M+1): 415.2792. ELSD HPLC purity: tRet: 7.207 min (purity: 98.34%). Example 2 - Synthesis of LM2

[0449] N, Kl

[0450] LM2 a) tert-butyl (16-azido- 11 -(2-(2-azidoethoxy)ethyl)-7-oxo-3, 14-dioxa-6, 8, 11 -triazahexadecyl)carbamate (111)

[0451] To the solution of 2,5-dioxopyrrolidin-1-yl(2-(bis(2-(2-azidoethoxy)ethyl)amino) ethylcarbamate 17 (0.5 g, 1.1698 mmol, 1.0) in 10 mL of CH2Cl2 at RT DIPEA (0.61 mL, 3.5094 mmol, 3.0 eq.) and cat. DMAP (0.2 g) were added followed by the addition of tert-butyl (2-(2-aminoethoxy)ethyl)carbamate 110 (0.28 g, 1 .4037 mmol, 1 .2 eq.) and stirred at RT for 16 h. The reaction mixture was evaporated and diluted with Ethyl acetate and washed with water. The organic layer was dried over anhydrous Na2SC>4 and concentrated under reduced pressure to get crude product. The crude product was purified by MPLC Flash Column chromatography using MeOH in CH2CI2 (2:98) eluent to afford the title compound 111 as pale yellow liquid (0.21 g, 34.74%). HRMS: (M+1): 515.2571. b) 1-(2-(2-aminoethoxy)ethyl)-3-(2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)urea (LM2) To the solution of tert-butyl (16-azido-11-(2-(2-azidoethoxy)ethyl)-7-oxo-3,14-dioxa-6,8,11- triazahexadecyl)carbamate, 111 (0.21 g, 0.4065 mmol, 1.0 eq.) in 5 mL of CH2CI2 at O °C, 2 mL of 4M HCI in 1 ,4-dioxane was added and stirred at RT for 4h. The reaction mixture was evaporated and dried over anhydrous Na2SO4 and concentrated under reduced pressure to get crude. The crude product was washed with ether and the residue was dissolved in CH2CI2. The organic layer was washed with saturated NaHCOs solution then aqueous layer was extracted with 10% MeOH in CH2CI2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford LM2 (0.12 g, 71 %) as pale brown liquid.1H NMR (DMSO-d6) 400 MHz) 5 ppm 6.04 (t, J=5.6 Hz, 1 H), 5.78 (t, J=5.6 Hz, 1 H), 3.57 (t, J=6 Hz, 4H), 3.49 (t, J=6 Hz, 4H), 3.39-3.33 (m, 9H), 3.15-3.11 (m, 2H), 3.03-3.01 (m, 2H), 2.68- 2.65 (m, 5H), 2.55-2.50 (m, 2H). HRMS: (M+1): 417.2622. ELSD HPLC purity: tRel: 7.32 min (99.03% purity). a) tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3,14, 17,20-tetraoxa-6,9, 11-triazadocosan-22- yl)carbamate (113)

[0452] To the solution of 2,5-dioxopyrrolidin-1 -yl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate 17 (0.5 g, 0. mmol) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate 112 (0.410 g, 1.403 mmol) in DCM was added, DIPEA (0.64 mL, 3.509 mmol) and was added and stirred the reaction mixture at rt for 16h . The reaction mixture was diluted with DCM and washed with water. The organic layer was dried over anhydrous Na2SC>4 and concentrated under reduced pressure to get crude product. The crude product was purified by MPLC Flash Column chromatography using MeOH in CH2CI2 (7:93) eluent to afford the title compound 113 as a colorless liquid (0.3 g, 42%). Confirmed by1H NMR (DMSO- 6, 400 MHz) & ESMS.: (M+1): 605.4. b) 1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)urea (LM3)

[0453] To the solution of tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3,14,17,20-tetraoxa-6,9,11- triazadocosan-22-yl)carbamate 113 (0.15 g, 0.248 mmol) in CH2Cl2 was added 4M HCI in 1 ,4-dioxane (1.0 mL) at 0 °C, stirred at RT for 1 h. The reaction mixture was evaporated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude material. The crude product was washed with ether and the residue was dissolved in CH2CI2. The organic layer was washed with saturated NaHCOs solution then aqueous layer was extracted with 2% MeOH in CH2CI2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford LM3 (0.1 g, 80%) as pale yellow liquid.1H NMR (DMSO-d6) 400 MHz) 5 ppm 6.04 (t, J=5.6 Hz, 1 H), 5.78 (t, J=5.6 Hz, 1 H), 3.58-3.56 (m, 4H), 3.52-3.39 (m, 12H), 3.37-3.33 (m, 10H), 3.14-3.10 (m, 2H), 3.04-3.01 (m, 2H), 2.68-2.62 (m, 6H). HRMS: (M+1): 505.2753. ELSD HPLC purity: 6.615 min (99.42% purity).

[0454] Example 4 - Synthesis of linker LM4 a) tert-butyl 3-(2-bromoethoxy)propanoate (115)

[0455] To the solution of tert-butyl 3-(2-hydroxyethoxy)propanoate 114 (1 g, 5.2598 mmol,1 .0 eq.) in 10 mL CH2CI2 at 0°C, CBri (2.61 g, 7.8897 mmol, 1 .5 eq.) was added followed by the portion wise addition of PPhs (2.06 g, 7.8897 mmol,1 .5 eq.). The reaction mixture was stirred at RT for 4h. The reaction mixture was diluted with ethylacetate and washed with water. The organic layer was dried over anhydrous Na2SC and concentrated under reduced pressure to get crude. The crude product was purified by MPLC Flash Column chromatography using EtOAc in Hexane (10:90) eluent to afford the title compound 115 as Pale-yellow liquid (1.18 g, 88.62%). Confirmed by1H NMR (DMSO-d6, 400 MHz). b) tert-butyl 2,2-dimethyl-4-oxo-3,8, 11, 14,20-pentaoxa-5, 17-diazatricosan-23-oate (117)

[0456] To the solution of tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate 116 (0.64 g, 2.1889 mmol,1 .0 eq.) and tert-butyl 3-(2-bromoethoxy)propanoate 115 (0.66 g, 2.6267 mmol, 1 .2 eq.) in CH3CN at RT, DIPEA (1.14 mL, 6.6 mmol, 3.0 eq.) was added and heated to reflux for 16h in sealed tube. The reaction mixture was diluted with ethylacetate and washed with water. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to get crude. The crude product was purified by MPLC Flash Column chromatography using MeOH in DCM (7:93) eluent to afford the title compound 117 as Paleyellow liquid (0.4 g, 39%). Confirmed by1H NMR (DMSO-d6, 400 MHz) & HRMS: (M+1): 465.3497. c) tert-butyl 17-((2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamoyl)-2,2-dimethyl-4-oxo-3,8, 11, 14,20- pentaoxa-5, 17-diazatricosan-23-oate (118)

[0457] To the solution of 2,5-dioxopyrrolidin-1-yl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate I7 (0.81 g, 1.8950 mmol, 1.2 eq.) in CH2CI2 at RT DIPEA (0.82 mL, 4.7376 mmol, 2.5eq.) and cat. DMAP (10 mg) were added followed by the addition of tert-butyl 2,2-dimethyl-4-oxo-3,8,11 ,14,20-pentaoxa-5,17- diazatricosan-23-oate 117 (0.73 g, 1.6 mmol, 1.0 eq.) and stirred at RT for 16 h. The reaction mixture was evaporated and diluted with Ethyl acetate and washed with water. The organic layer was dried over anhydrous Na2SC>4 and concentrated under reduced pressure to get crude product. The crude product was purified by MPLC Flash Column chromatography using MeOH in DCM (2.5:97.5) eluent to afford the title compound as pale brown liquid 118 (0.47 g, 38.32%). Confirmed by1H NMR (DMSO-d6, 400 MHz) & HRMS.:(M+1): 777.5654. d) 1 -amino- 12-((2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamoyl)-3, 6, 9, 15-tetraoxa-12- azaoctadecan-18-oic acid (LM4)

[0458] To the solution of tert-butyl 17-((2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamoyl)-2,2-dimethyl-4-oxo- 3,8,11 ,14,20-pentaoxa-5,17-diazatricosan-23-oate 118 (0.11 g, 0.1416 mmol, 1 .0 eq.) in 5.0 mL CH2CI2 at 0°C, 4M HCI in 1 ,4-dioxane (2.0 mL) was added and stirred at RT for 2h. The reaction mixture was evaporated and dried over anhydrous NazSC and concentrated under reduced pressure to get crude. The crude product was washed with ether and the residue was dissolved in water. 5 equivalents of NaHCOs were added and stirred for 30 min then 0.1 N HCI solution was added until the reaction mixture just turned to acidic. The aqueous solution was saturated with NaCI and extracted with 5%MeOH in DCM. The organic layer was dried over anhydrous Na2SC>4 and concentrated under reduced pressure to afford LM4 (0.06 g, 68.26%) as pale brown liquid.1H NMR (DMSO-d6) 400 MHz) 5 ppm 6.083 (t, J=5.2 Hz, 1 H), 3.57-3.37 (m, 30H), 3.06-3.01 (m, 2H), 2.81-2.80 (m, 2H), 2.68-2.67 (m, 4H), 2.55-2.50 (m, 2H), 2.23 (t, J=6 Hz, 2H). HRMS: (M+1): 621.3833. ELSD HPLC purity: tRet: 6.877 min (98% purity).

[0459] Example 4A - Synthesis of LM3X

[0460] Protocol for the synthesis of LM3X from common intermediate 113

[0461] Synthesis of 2,5-dioxopyrrolidin-1-yl 2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)acetate (Compound 9a):

[0462] To a solution of compound 9 (1.5 g, 6.51 mmol, 1 .0 equiv.) in CH2CI2 (15 mL), DIG (863 mg, 6.84 mmol, 1 .05 equiv.) and HOSu (787 mg, 6.84 mmol, 1 .05 equiv.) were added. The mixture was stirred at 25 °C for 30 min. LC-MS analysis showed compound 9 was consumed completely, and the desired product mass was detected. The reaction mixture was concentrated under reduced pressure to get the crude product. The residue was further purified by column chromatography (SiC>2, Petroleum ether / Ethyl acetate = 1 / 0 to 0 / 1) to afford compound 9a. MS (ESI): [M+H]+:328.2.

[0463] 3-7 3B-1

[0464] A 100 mL round-bottom flask was purged with N2 gas and Pd / C (300 mg) was carefully added into it. THF (10 mL) was then added to infiltrate the Pd / C completely, followed by the addition of compound 3-7 (1 .0 g, 1.65 mmol, 1.0 eq.) in THF (10.0 mL) slowly. The reaction mixture was purged with H2 gas and stirred at 28 °C for 6 h under H2 atmosphere (15 psi). The reaction was monitored by LC-MS analysis. Upon completion of the reaction, the reaction mixture was filtered carefully under N2 atmosphere. The filtrate was concentrated under reduced pressure to give compound 3B-1 that was used directly for the next step without further purification. MS (ESI): [M+H]+:553.2.

[0465] NMM (512 mg, 5.06 mmol, 556 pL, 4.0 eq.) was added to a solution of 3B-1 (0.70 g, 1 .26 mmol, 1.0 eq.) and Cpd_9a (870 mg, 2.66 mmol, 2.1 eq.) in DMF (7.00 mL). The mixture was stirred at 25 °C for 30 min. LC-MS analysis showed compound 3B-1 was consumed, and desired product mass was detected. After completion, isopropyl ether (70 mL), was added to the reaction mixture and the precipitated material was centrifuged and filtered to afford 3B-1 (> 800 mg, crude) that was used directly in the next step without further purification MS (ESI): [M+H]+:978.6.

[0466] Compound 3B-2 (1.0 g, 1.01 mmol, 1.0 equiv.) was added to a 2N HCI / dioxane (10 mL) solution. The mixture was stirred at 25 °C for 30 min. LC-MS analysis showed the complete consumption of compound 3B-2. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (HCI condition) to obtain LM3X as a solid (HCI salt form). The solid was then dissolved in sat. NaHCOs solution and stirred for 10 minutes. The resultant To a solution of triphosgene (50.7 g, 171 mmol, 26.7 mL, 2.0 eq.) in CH2Cl2 (250 mL) was added amine 1 (25.0 g, 85.5 mmol, 1.0 eq.) in CH2CI2 (250 mL) followed by compound 2 (25 g, 85.5 mmol, 1.0 eq.) in CH2CI2 (250 mL). The mixture was stirred at 0 °C for 2 h. Further, triethylamine (28.4 mL, 256 mmol, 3.0 eq.) was added followed by the addition of the second batch of compound 2 (25.0 g, 85.5 mmol, 1.0 eq.) at 0 °C. The reaction mixture was stirred at 0 °C for 3 h. LC-MS analysis showed compound 1 was consumed completely, and desired product mass was detected. Upon completion, the reaction mixture was concentrated under reduced pressure to give the crude product. The crude residue was purified by prep- HPLC (TFA condition, ELSD) and compound 4 (24.0 g, 67.6 mmol, 75% yield, 95% purity) was obtained as a colourless oil. LCMS purity: 95% (ELSD); MS (ESI): [M+H]+: 380.4. ’H NMR (400 MHz, DMSO-d6): 6.75 (s, 1 H), 5.97 (s, 2 H), 3.50 (d, J = 1 .25 Hz, 8 H), 3.34 - 3.39 (m, 6 H), 3.13 (t, J = 5.63 Hz, 2 H), 3.02 - 3.08 (m, 4 H), 1.37 (s, 9 H).

[0467] To a mixture of DMSO (9.67 g, 123 mmol, 8.78 mL, 2.70 eq.) and CH2CI2 (121 mL) was slowly added oxalyl dichloride (12.8 g, 100 mmol, 8.83 mL, 2.2 eq.) in CH2CI2 (87mL) at -78 °C for over 30 minutes. Further compound 4 (17.4 g, 45.8 mmol, 1 .00 eq.) in CH2CI2 (104 mL) was charged into the solution and stirred for another 1 h at -78 °C. Triethylamine (27.8 g, 275 mmol, 38.2 mL, 6.0 eq.) was then added at -78 °C and the reaction temperature was increased slowly from -78 °C to -20 °C. LC-MS analysis showed compound 4 was consumed completely and the desired product peak was detected. The reaction mixture was concentrated, and the crude mass (compound 5) was used directly for the next reaction.

[0468] Synthesis of tert-butyl N-[2-[2-[2-[2-(2-oxoethylcarbamoylamino)ethoxy]ethoxy]ethoxy]ethyl] carbamate (compound 7):

[0469] To a solution of compound 5 (0.5 g, 1 .33 mmol, 1.0 eq.) and compound 6 (389 mg, 927 pmol, 0.7 eq) in CH2CI2 (50 mL) was added NaBHsCN (250 mg, 4 mmol, 3.0 eq.) and AcOH (318 mg, 5.3 mmol, 303 pL, 4.0 eq.). The mixture was stirred at 20 °C for 30 min. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent to furnish the crude product that was further purified by prep-HPLC (0.1 % TFA condition, ELSD) to afford compound 7 (450 mg, 575 pmol, 43% yield for two steps, 99.8% purity by LCMS) as a colorless oil. MS (ESI): [M+H]+:781 .3. A solution of compound 7 (200 mg, 256 pmol, 1.0 eq.) in HCI / 1 ,4-dioxane (2 mL) was stirred at 20 °C for 30 min. LC-MS analysis showed that compound 7 was consumed completely, and the desired mass peak was detected. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent, followed by the addition of saturated NaHCCh solution. The product was extracted from the

[0470] To a solution of compound 8A (1 g, 6.3 mmol, 1 eq.) and compound 6C (2.3 g, 9.42 mmol, 1.5 eq.) in CH2CI2 (10 mL) was added NaBH3CN (1.18 g, 18.85 mmol, 3.0 eq.) and AcOH (1.51 g, 25.13 mmol, 1.44 mL, 4.0 eq.). The mixture was stirred at 0 °C for 24 h. LCMS analysis showed compound 8A was consumed completely, and desired product mass was detected. Upon completion, the reaction mixture was concentrated under reduced pressure to give crude product. The crude residue was purified by prep-HPLC (FA condition, ELSD) to afford compound 8B (890 mg, 1 .750 mmol, 27.862% yield, 76% purity) as a colourless oil. LCMS Purity: 76% (ELSD); MS (ESI): [M+H]+: 387.4. A solution of compound 8B (390 mg, 1.01 mmol, 1.0 eq.) in 2N HCI / 1 ,4-dioxane (4 mL) was stirred at 25°C for 1 h. LCMS analysis showed compound 8B was consumed completely, and desired mass peak was detected. Upon completion, the reaction mixture was concentrated under reduced pressure to obtain the crude product, compound 8C (290 mg, crude) as a colourless oil. The crude material was used directly for the next step without any further purification. MS (ESI): [M+H]+: 287.2.

[0471] Synthesis of tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3, 14, 17, 20, 23, 26, 29, 32,35- nonaoxa-6,9, 11-triazaheptatriacontan-37-yl)carbamate (Compound 11):

[0472] Compound 8 (200 mg, 390 pmol, 1 .0 eq.) in CH2CI2 (2 mL) and compound 8C (223 mg, 780 pmol, 2.0 eq.) in CH2CI2 (1 mL) were added to a solution of triphosgene (116 mg, 390 pmol, 61 pL, 1.0 eq.) in CH2CI2 (1 mL). The mixture was stirred at 0 °C for 2 h. Further, EtjN (197mg, 1.95 mmol, 270 pL, 5.0 eq.) was added at 0 °C to keep the reaction medium basic and stirred for 3 h at 0 °C. LCMS analysis showed compound 8 was consumed completely, and the desired mass peak was detected. Upon, completion, the reaction mixture was concentrated under reduced pressure to get the crude product. The crude residue was purified by prep-HPLC (FA condition, ELSD) to obtain compound 11 (125 mg, 148 pmol, 38% yield, 96.3% purity) as a colorless oil. LCMS Purity: 96.3% (ELSD); MS (ESI): [M+H]+: 825.6

[0473] A solution of compound 11 (75 mg, 91 pmol, 1.0 eq.) in 2N HCI / 1 ,4-dioxane (1 mL) was stirred at 25 °C for 1 h. LCMS analysis showed compound 11 was consumed completely, and the desired mass was detected. Upon completion, saturated NaHCOs (30 mL) solution was added to the reaction mixture and stirred for 10 min, followed by extraction with dichloromethane. The combined organic layers were dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give a residue. The crude residue was purified by prep-HPLC (ELSD) to afford LM6 (50 mg, 67 pmol, 74% yield, 97.3% purity) as a colourless oil. Purity by HPLC: 97.3%; MS (ESI): [M+H]+:725.4.1H NMR (400 MHz, DMSO-cfs): 5 ppm 6.00 (t, J = 5.7 Hz, 1 H), 5.76 (t, J = 5.6 Hz, 1 H), 3.58 (s, 4H), 3.51 - 3.50 (m, 30H), 3.49 (br s, 4H), 3.47 (br s, 2H), 3.37 (br d, J = 5.1 Hz, 6H), 3.14 - 3.10 (m, 2H), 3.02 (q, J = 6.3 Hz, 2H), 2.69 - 2.67 (m, 2H), 2.65 (d, J = 2.3 Hz, 2H), 2.64 - 2.61 (m, 2H), 1 .24 (br s, 2H).

[0474] Example 4D - Synthesis of LM7

[0475] NaBHsCN (592 mg, 9.4mmol, 3.0 eq.) and AcOH (377 mg, 6.3mmol, 359 pL, 2.0 eq.) were added to a solution of compound 8A (500 mg, 3.14 mmol, 1 .0 eq.) and compound C (1.32 g, 3.14 mmol, 1.0 eq.) in CH2CI2 (5 mL). The mixture was stirred at 35 °C for 48 h. LCMS analysis showed compound 8A was consumed completely, and desired mass peak was detected. Upon completion, the reaction mixture was concentrated under reduced pressure to get the crude product. The crude residue was purified by prep-HPLC (FA condition, ELSD) to obtain Compound 8B-1 (250 mg, 416 pmol, 13% yield, 93.7% purity) as a colourless oil. LCMS Purity: 93.7 % (ELSD); MS (ESI): [M+H]+: 563.3. A solution of Compound 8B-1 (250 mg, 444 pmol, 1 .0 eq.) in 2N HCI / 1 ,4-dioxane (2.5 mL) was stirred at 25 °C for 1 h. LCMS analysis showed compound 8B-1 was consumed completely, and desired mass peak was detected. Upon completion, the reaction mixture was concentrated under reduced to obtain crude Compound 8C-1 (200 mg, crude) as a colourless oil. The crude material was as such for the next step without any further purification. MS (ESI): [M+H]+: 463.4. Synthesis of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[bis[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl] amino]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]car bamate (Compound 12):

[0476] Compound 8C-1 (100 mg, 216 pmol, 1 .0 eq.) in CH2CI2 (1 mL) and compound 8 (223 mg, 432 pmol, 2.0 eq.) in CH2CI2 (0.5 mL) were added to a solution of triphosgene (64 mg, 216 pmol, 33.8 pL, 1.0 eq.) in CH2CI2 (0.5 mL). The mixture was stirred at 0 °C for 2 h. EtsN (109 mg, 1.1 mmol, 145 pL, 5.0 eq.) was then added at 0 °C to maintain the medium basic and stirred for another 3 h at 0 °C. LCMS analysis showed compound 8C-1 was consumed completely, and the desired mass peak was detected. The reaction mixture was concentrated under reduced pressure to give crude product. The crude was purified by prep- HPLC (FA condition, ELSD). Compound 12 (80 mg, 78 pmol, 36% yield, 97.5% purity) was obtained as a colourless oil. LCMS Purity: 97.5% (ELSD); MS (ESI): [M+H]+: 1001.5.

[0477] Synthesis of 1-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethyl]-3-[2-[bis[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]amino]ethyl]urea (LM7):

[0478] A solution of compound 12 (80 mg, 78 pmol, 1.0 eq.) in 2N HCI / 1 ,4-dioxane (1 mL) was stirred at 25 °C for 1 h. LCMS analysis showed compound 12 was consumed completely, and the desired mass peak was detected. Upon completion of the reaction, sat. NaHCOs solution was added to the reaction mixture and stirred for 10 min followed by extraction with dichloromethane. The combined organic layers were dried over anhydrous NazSC , filtered, and concentrated under reduced pressure to get the LM7 (50 mg, 55.3 pmol, 71% yield, 99.7% purity) as a colourless oil. Purity by HPLC: 99.7%; MS (ESI): [M+H]+:901 .5.1H NMR (400 MHz, DMSO-cfe): 6 ppm 6.00 (br t, J = 5.4 Hz, 1 H), 5.78 - 5.73 (m, 1 H), 3.61 - 3.59 (m, 4H), 3.55 - 3.54 (m, 6H), 3.52 - 3.49 (m, 30H), 3.44 - 3.43 (m, 1 H), 3.44 (br t, J = 6.2 Hz, 6H), 3.40 - 3.37 (m, 6H), 3.36 - 3.31 (m, 9H), 3.14 - 3.11 (m, 2H), 3.03 - 2.99 (m, 2H), 2.63 (dt, J = 2.3, 6.0 Hz, 6H), 1 .23 (br s, 2H).

[0479] Example 5 - Synthesis of Linker-Payload molecule-1 (LP-1)

[0480] i) (1-(9H-fluoren-9-yl)-3-oxo-2,7, 10, 13-tetraoxa-4-azapentadecan-15-oyl)-L-valyl-L-alanine (compound 34)

[0481] Compound 34 was synthesized using standard solid-phase Fmoc chemistry. a) Resin loading: CH2CI2 (200 mL) was added to 2-chlorotrityl chloride resin (6.0 mmol, 1 .00 equiv.), followed by the addition of Fmoc-Ala-OH (1 .0 equiv.) and DIPEA (6.0 equiv.), the mixture was agitated under N2 atmosphere at 25 °C for 2 h. Thereafter methanol (9.5 mL) was added to the resin and the agitation continued for 30 min. The resin was then filtered and washed with DMF (300 mL x 3). b) Deprotection: 20% piperidine in DMF (200 mL) was added to the resin and agitated under N2 atmosphere at 25 °C for 30 min. The resin was washed with DMF (200 mL x 5) and filtered to get the resin with reactive amine group. c) Coupling: A solution of HBTU (2.85 equiv.), and Fmoc-Val-OH (3.0 equiv.) in DMF (200 mL) was added to the resin followed by the addition of DIEPA (6.0 equiv.). The mixture was agitated under N2 atmosphere at 25 °C for 30 min. The resin was then washed with DMF (200 mL x 3). d) Repeat step b to deprotect Fmoc group. Treat the resulting resin with Fmoc-N-amido-PEG3-acid (2.0 equiv.), HATU (1 .9 equiv.) and DIPEA (4.0 equiv.) in DMF. The mixture was agitated under N2 atmosphere at 25 °C for 30 min. The resulting resin was washed with DMF (200 mL x 3). e) Peptide cleavage and purification: The resin was washed with methanol (200 mL x 3) and dried under vacuum. The dried resin was treated with the cleavage buffer consisting of 20% HFIP in CH2CI2 and stirred for 30 min and filtered. Concentration of the filtrate under reduced pressure furnished the crude compound 34 (1 .71 g) which was taken forward without further purification. ii) ( 9H-fluoren-9-yl)methyl ((2S,5S)-1-(( 4-(hydroxymethyl)phenyl)amino) -5-isopropyl-2-methyl-1,4, 7-trioxo- 9, 12, 15-trioxa-3, 6-diazaheptadecan-17-yl)carbamate (Compound 36)

[0482] FmocHN

[0483] 36

[0484] To a solution of compound 34 (1 .60 g, 2.67 mmol, 1 .0 equiv.) in CH2CI2 (16.0 mL) was added compound 35 (657 mg, 5.34 mmol, 2.0 equiv ), and EEDQ (1.32 g, 5.34 mmol, 2.0 equiv ). The reaction mixture was stirred at 25 °C for 12 h. LCMS analysis showed the starting material 34 was consumed completely, and desired product mass was detected. The reaction mixture was added to 160 mL isopropyl ether, then centrifuged to afford the crude compound 36 (2.00 g) as a yellow oil. The crude product was used for the subsequent reaction without further purification. MS (ESI): [M+Na]+: 727.4.

[0485] To a solution of compound 36 (2.00 g, 2.6 mmol, 1.0 equiv ), and compound 37 (1 .58 g, 5.2 mmol, 2.0 equiv.) in DMF (20 mL) was added DIEPA (671 mg, 5.20 mmol, 905 pL, 2.0 equiv.). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed 31 % of desired product formation and 2% of the starting alcohol 36. The mixture was purified directly by purified by prep-HPLC (TFA condition) and the solvent was removed to furnish compound 38 (680 mg, 765 pmol, 29.4% yield, 97.9% purity) as a yellow solid.

[0486] MS (ESI): [M+Na]+: 870.4.

[0487] To a solution of compound 38 (200 mg, 225 pmol, 1 .0 equiv.), and Exatecan (119 mg, 225 pmol, 1 .00 equiv.) in DMF (3.40 mL) was added HOBt (33.4 mg, 247 pmol, 1.1 equiv.) and DIPEA (58 mg, 450 pmol, 78.4 pL, 2.0 equiv ). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed compound 38 was completely consumed and the desired product mass was identified. The mixture was purified directly by prep-HPLC (TFA condition) to afford compound 39 (180 mg, 148 pmol, 66% yield, 96.3% purity) as a yellow solid. MS (ESI): [M+H]+: 1167.7

[0488] To a solution of compound 39 (180 mg, 148 pmol, 1 .0 equiv.) in DMF (1 .26 mL) was added triethylamine (392 mg, 3.88 mmol, 0.54 mL, 26 equiv.). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed compound 39 was consumed completely, and desired product mass was detected. Compound 40 (140 mg, crude) was obtained as a brown liquid and used directly for the next step. MS (ESI): [M+H]+: 945.4

[0489] LP-1

[0490] To a solution of compound 40 (140 mg, 148 pmol, 1 .0 equiv.) in DMF (1.26 mL) was added triethylamine (392 mg, 3.9 mmol, 0.54 mL, 26.1 equiv.), and DBCO-OSu (59.6 mg, 148 pmol, 1.0 equiv ). The mixture was stirred at 25 °C for 1 h. LC-MS showed compound 40 was consumed completely, and desired mass was detected. The reaction mixture was added to 18.0 mL of isopropyl ether, and the crude product was slowly precipitated out. Centrifuged to get the crude product and discarded the liquid supernatant. The residue was purified by prep-HPLC (TFA condition) to obtain LP-1 (61 .0 mg, 47.5 pmol, 32.5% yield, 97.5% purity) as a yellow solid. MS (ESI): [M+Na]+: 1253.6;1H NMR (400 MHz, DMSO-da) 5 ppm 9.98 (s, 1 H), 8.36 (d, J = 6.80 Hz, 1 H), 8.04 - 8.06 (m, 1 H), 7.71 - 7.78 (m, 2 H), 7.64 - 7.66 (m, 1 H), 7.58 (br d, J =8.8 Hz, 3 H), 7.41 - 7.47 (m, 4 H), 7.25 - 7.37 (m, 6 H), 5.44 (s, 2 H), 5.28 (br s, 2 H), 5.07 (s, 2 H), 5.00 (br d, J =13.88 Hz, 1 H), 4.38 (br t, J =6.94 Hz, 1 H), 4.28 (dd, J =9.13, 6.63 Hz, 1 H), 3.93 (s, 2 H), 3.43 - 3.61 (m, 12 H), 3.27 (brt, J =6.00 Hz, 4 H), 3.03 - 3.11 (m, 2 H), 2.37 (s, 3 H), 2.13 - 2.26 (m, 3 H), 1 .93 - 2.03 (m, 2 H), 1.81 - 1 .92 (m, 2 H), 1 .70 - 1 .80 (m, 1 H), 1 .30 (d, J =7.00 Hz, 3 H), 0.84 - 0.91 (m, 6 H), 0.81 (br d, J =6.75 Hz, 3 H). Example 6 -Synthesis of LP-2

[0491] To a solution of oxalyl chloride (5.9 mL, 136 mmol, 4.0 equiv.) in CH2CI2 (90.0 mL), DMSO (9.6 mL, 68.1 mmol, 2.0 equiv.) was added at -78°C followed by the addition of tert-butyl 2-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)acetate 119 (9.0 g, 34.0 mmol, 1.0 equiv.) and the reaction mixture was stirred at the same temperature for 1 h. Thereaftertriethylamine (28.4 mL, 204 mmol, 6.0 equiv.) was added at -78°C and stirred for further 1 h and allowed to warm to room temperature. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give compound I20 (12.0 g, crude) as a colorless oil, which was used without any further purification.1H NMR (400 MHz, DMSO-cfs): 6 ppm 9.54 (s, 1 H), 4.14 (s, 2 H), 3.55 - 3.60 (m, 2 H), 3.44 - 3.55 (m, 8 H), 1.38 (s, 9 H). ii) Tert-butyl 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxo-ethoxy)ethoxy]ethoxy]ethylamino] ethoxy]ethoxy]ethoxy]acetate (122)

[0492] 122

[0493] To a solution of compound 120 (12.0 g, 1.0 equiv.) and tert-butyl 2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)acetate 121 (17.9 g, 681 pmol, 1.5 equiv.) in MeOH (120 mL), NaBHsCN (2.85 g, 454 mmol, 1.0 equiv.) was added. The mixture was stirred at 20-25°C for 1 h. Upon completion, the reaction mixture was extracted with CH2CI2 (200 mL) and H2O (100 mL). The combined organic layers were dried over Na2SC>4, filtered and concentrated under reduced pressure to give compound 122 (18.0 g, crude) as a yellow oil. MS (ESI): [M+H]+:510.6.

[0494]

[0495] To a solution of compound 122 (18.0 g, 1 .0 equiv.), FmocOSu (17.9 g, 681 pmol, 1 .5 equiv.) in THF (90 mL) and H2O (90 mL), NaHCCh (5.9 g, 70.6 mmol, 2.0 equiv.) was added. The mixture was stirred at 20-25°C for 1 h. Upon completion, the reaction LCMS showed compound I22 was consumed completely. The reaction was extracted with DCM (200 mL) and H2O (100 mL). The combined organics were dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiC>2, Petroleum ether / Ethyl acetate=100 / 1 to 5 / 1) to give compound I23 (2.1 g, 2.87 mmol, 98.1 % purity, and 2.2 g, 3.01 mmol, 92.4% purity. 16.6% yield) as a white solid. Purity by HPLC (220 nm): 98.1 %; MS (ESI): [M+H]+:732.3;1H NMR (400 MHz, DMSO-cfe): 6 ppm 7.88 (d, J = 7.4 Hz, 2 H), 7.63 (d, J = 7.4 Hz, 2 H), 7.37 - 7.44 (m, 2 H), 7.30 - 7.36 (m, 2 H), 4.45 (d, J = 5.6 Hz, 2 H), 4.27 (br t, J = 5.4 Hz, 1 H), 3.95 - 3.99 (m, 4 H), 3.46 - 3.56 (m, 16 H), 3.38 - 3.44 (m, 4 H), 3.13 (br s, 4 H), 1.40 - 1.42 (m, 18 H).

[0496] 124

[0497] Compound 123 (2.1 g, 2.87 mmol, 1.0 equiv.) was dissolved in 50% FA / DCM (21.0 mL) and stirred at 20- 25 °C for 16 hrs. LCMS analysis showed compound I23 was consumed completely. The reaction mixture was quenched by adding NaHCOs (100 mL) at 0-5°C. The resultant mixture was purified directly by prep- HPLC (TFA condition) to give compound I24 (700 mg, 1.03 mmol, 35.9% yield) as a colourless oil. Purity by HPLC (220 nm): 98.2%; MS (ESI): [M+H]+:676.3;1H NMR (400 MHz, DMSO-d8): 5 ppm 7.88 (d, J = 7.5 Hz, 2 H), 7.64 (d, J = 7.4 Hz, 2 H), 7.38 - 7.43 (m, 2 H), 7.30 - 7.36 (m, 2 H), 4.45 (d, J = 5.6 Hz, 2 H), 4.23 - 4.32 (m, 1 H), 3.96 (s, 2 H), 3.92 (s, 2 H), 3.44 - 3.58 (m, 20 H), 3.12 (br s, 4 H), 1.40 (s, 9 H)

[0498] To a solution of compound 124 (200 mg, 295 pmol, 1 .0 equiv.) and compound I25 (Val-Ala-PABC-Exatecan, CAS: 2845164-91-0)(308 mg, 377 pmol, 1 .3 equiv.) in DMF (2.0 mL) DIPEA (58.6 uL, 354 pmol, 1 .2 equiv ), HOBt (47.9 mg, 354 pmol, 1.2 equiv.) and EDCI (67.9 mg, 354 pmol, 1.2 equiv.) were added respectively. The mixture was stirred at 20-25 °C for 3 hrs. LCMS analysis showed compound I24 was consumed completely. The resultant mixture was purified directly by prep-HPLC (TFA condition) to give compound 126 (900 mg, 637 pmol, 68.3% yield) as a white solid. Purity by HPLC (220 nm): 97.3%; MS (ESI): [M+H]+:1413.5. The reactions were performed in parallel to obtain a total of 900 mg of 126.

[0499]

[0500] Compound 126 (900 mg, 637 pmol, 1.0 equiv.) was dissolved in 50% FA / DCM (9.0 mL) and stirred at 20- 25 °C for 72 hrs. LCMS analysis showed compound I26 was consumed completely. The resultant was triturated with isopropyl ether (10 mL) at 0-5 °C to give compound I27 (500 mg, crude) as a colourless oil. MS (ESI): [M+H]+:1356.8. The residue was purified by prep-HPLC (TFA condition) to give compound 130 (1.6 g, 1.81 mmol, 84.0% yield) as a yellow solid. Purity by HPLC (220 nm): 95.8%; MS (ESI): [M+Na]+: 882.8.

[0501] Compound 130 (1.5 g, 1.69 mmol, 1.0 equiv.) was first dissolved in 50% formic acid in CH2CI2 (15.0 mL) and stirred at 20-25 °C for 24 h. LC-MS analysis showed compound I30 was consumed and detected the desired mass. The residue was purified by prep-HPLC (AcOH condition) to give compound 131 (750 mg, 95.9 pmol, 84.0% yield) as a yellow solid. Purity by HPLC (220 nm): 92.5%; MS (ESI): [M+Na]+:782.8.

[0502]

[0503] I3-2

[0504] To a solution of compound I27 (500 mg, 368 pmol, 1.0 equiv.) and compound 131 (392 mg, 479 pmol, 1.3 equiv.) in DMF (5.0 mL), DIPEA (152 uL, 921 pmol, 2.5 equiv ), HOBt (74.7 mg, 552 pmol, 1.5 equiv.) and EDCI (105 mg, 552 pmol, 1 .5 equiv.) were added successively. The mixture was stirred at 20-25 °C for 2 h. LC-MS analysis showed compound 127 was consumed, and the desired mass was identified. The reaction mixture 132 was directly used to the next step without further purifications. MS (ESI): [(M+2H) / 2]2+:1062.5.

[0505] 133

[0506] To the above solution of compound 132, triethylamine (1 .0 mL) was added. The reaction mixture was stirred at 20-25 °C for 4 h. LC-MS showed compound 132 was consumed completely, and the desired mass was detected. The resultant mixture was directly purified by prep-HPLC (TFA condition) to give compound 133

[0507] (200 mg, 105 pmol, 28.5% purity) as a white solid. Purity by HPLC (220 nm): 92.9%; MS (ESI): [(M+2H) / 2]2+:950.5.

[0508] To a solution of compound 133 (85 mg, 42.2 pmol, 1.0 equiv.) and 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13- tetraoxa-4-azapentadecan-15-oic acid I34 (36.4 pL, 82.4 pmol, 2.0 equiv.) in DMF (1 .0 mL) DIEA (17.4 pL, 105 pmol, 2.5 equiv.), HOBt (11.4 mg, 84.4 pmol, 2.0 equiv.) and EDCI (16.1 mg, 84.4 pmol, 2.0 equiv.) were added successively. The mixture was stirred at 20-25 °C for 1 hr. LC-MS showed compound I33 was consumed completely and detected the desired mass. The reaction mixture 135 was directly used for the next step. MS (ESI): [(M+2H) / 2]2+:1155.9.

[0509]

[0510] To the solution of compound I35, in DMF (0.8 mL), triethylamine (0.2 mL) was added. The mixture was stirred at 20-25 °C for 1 hr. LCMS showed compound I35 was consumed completely, and detected the desired mass. The resultant reaction mixture was purified directly by prep-HPLC (TFA condition) to give compound 136 (55 mg, 26.3 pmol, 62% yield) as a white solid. Purity by HPLC (220 nm): 98.5%; MS (ESI): [(M+2H) / 2]2+: 1045.1.

[0511]

[0512] LP 2

[0513] To a solution of compound I36 (40 mg, 19.1 pmol, 1.0 equiv.) and DBCO-OSu (15.4 mg, 38.3 pmol, 2.0 equiv.) in DMF (400 pL) was added NMM (3.8 mg, 38.3 pmol, 4.2 pL, 2.0 equiv.). The mixture was stirred at 20-25 °C for 1 h and monitored by LC-MS analysis. Upon completion, the reaction mixture was concentrated and the the residue was purified by prep-HPLC (NH4HCO3 condition) to give LP-2 (15 mg) as a white solid. Purity by HPLC (220 nm): 95.6%; MS (ESI): [(M+2H) / 2]2+:1188.7.1H NMR (400 MHz, DMSO- cfe) 6 ppm 9.94 - 10.04 (m, 2 H), 8.93 (s, 1 H), 8.37 (d, J = 8.3 Hz, 4 H), 8.04 (s, 1 H), 7.91 - 8.01 (m, 4 H), 7.71 - 7.78 (m, 3 H), 7.64 - 7.69 (m, 1 H), 7.57 - 7.62 (m, 4 H), 7.23 - 7.51 (m, 16 H), 7.18 - 7.19 (m, 1 H), 6.51 (s, 1 H), 5.44 (s, 1 H), 5.27 (br s, 2 H), 5.08 (br s, 3 H), 4.52 (s, 1 H), 4.27 - 4.42 (m, 3 H), 4.15 (s, 2 H), 3.93 (br s, 3 H), 3.41 - 3.60 (m, 36 H), 3.03 - 3.14 (m, 4 H), 2.87 (s, 3 H), 2.57 - 2.62 (m, 8 H), 2.35 (s, 3 H), 2.17 - 2.26 (m, 3 H), 1.94 - 2.03 (m, 4 H), 1.30 (br d, J = 7.1 Hz, 6 H), 1.24 (s, 3 H), 1.19 (d, J = 6.8 Hz, 6 H), 0.85 - 0.90 (m, 9 H), 0.82 (br d, J = 6.6 Hz, 6 H). Example 6 -Synthesis of LP-3

[0514] To a solution of compound I36 (40 mg, 19.1 pmol, 1 .0 equiv.) in DMF, TMTHSI-OSu (8 mg, 24 pmol, 1.2 equiv.), triethylamine (10.6 pL, 76.6 pmol, 4.0 equiv.) and DMAP (4.68 mg, 38.3 pmol, 2.0 equiv.) were added.. The mixture was stirred at 20-25 °C for 2 h and monitored by LC-MS analysis. Upon completion of the reaction, the reaction mixture was concentrated, and the residue was purified by prep-HPLC (HCOOH condition) to give LP-3 (20 mg, 8.64 pmol, 45.1 % yield) as a white solid. Purity by HPLC (220 nm): 97.7%; MS (ESI): [(M+2H) / 2]2+:1157.8;1H NMR (400 MHz, DMSO-cfc): 6 ppm 10.0 (s, 2 H), 8.93 (s, 1 H), 8.35 - 8.41 (m, 4 H), 8.05 (br d, J = 8.4 Hz, 1 H), 7.98 (br d, J = 7.4 Hz, 2 H), 7.93 (d, J = 8.5 Hz, 2 H), 7.73 - 7.78 (m, 2 H), 7.55 - 7.63 (m, 4 H), 7.31 - 7.47 (m, 8 H), 7.30 (s, 1 H), 7.18 (br s, 2 H), 6.64 (br t, J = 5.2 Hz, 1 H), 6.52 (s, 1 H), 5.44 (s, 2 H), 5.27 (br d, J = 4.8 Hz, 3 H), 5.07 (br s, 4 H), 4.52 (s, 2 H), 4.39 (br t, J = 6.7 Hz, 2 H), 4.25 - 4.33 (m, 2 H), 4.16 (s, 2 H), 3.93 (s, 4 H), 3.87 (s, 1 H), 3.84 (s, 1 H), 3.36 - 3.66 (m, 42 H), 3.06 - 3.13 (m, 3 H), 2.87 (s, 3 H), 2.35 (s, 3 H), 2.33 (br s, 1 H), 1 .96 - 2.02 (m, 2 H), 1 .83 - 1 .92 (m, 2 H), 1.28 - 1.34 (m, 12 H), 1.17 - 1.21 (m, 12 H), 0.85 - 0.90 (m, 9 H), 0.82 (br d, J = 6.6 Hz, 6 H)

[0515] Example 6A -Synthesis of common linker Intermediates Synthesis of common linkers 6-5 and Int C

[0516] To a solution of oxalyl dichloride (8.64 g, 68.1 mmol, 5.96 mL, 3.0 eq.) in dichloromethane (60 mL) was added DMSO (8.86 g, 113 mmol, 8.05 mL, 5.0 eq.) at -78 °C followed by the addition of compound 6-1 (6.0 g, 22.7 mmol, 1 .0 eq.). The mixture was stirred at -78 °C for 1 h, then triethylamine (11 .4 g, 113 mmol, 15.7 mL, 5.0 eq.) was added and stirred at -78 °C for 1 h. 'H NMR analysis of the crude reaction mixture showed compound 6-1 was consumed >50%. The reaction mixture was filtered and concentrated under reduced pressure to give compound 6-2 (5.6 g, crude) as a yellow oil. The crude material was used as such for the subsequent reaction.1H NMR (400 MHz, CDCI3): 6 ppm 9.73 - 9.76 (m, 1 H), 4.17 (d, J =0.75 Hz, 1 H), 4.14 - 4.20 (m, 1 H), 4.02 (s, 2 H), 3.71 - 3.74 (m, 8 H), 1 .48 (s, 9 H).

[0517] NaBHsCN (1.0 g, 16.0 mmol, 1 .5 equiv.) was added to the solution of compound 6-2 (5.60 g, 10.6 mmol, 1.0 equiv.) and compound Int 22 (CAS: 478168-42-2; 5.55 g, 21 .3 mmol, 2.0 equiv.) in MeOH (40 mL). The mixture was stirred at 25 °C for 1 h. LCMS analysis showed compound 6-2 was consumed completely. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition) to afford compound 6-3 (5.50 g, 8.23 mmol, 70% purity) as a yellow oil. The crude material (70% purity) was used as such for the next step. MS (ESI): [M+H]+:468.3.

[0518] To a solution of compound 6-3 (5.4 g, 1.0 equiv.) and Fmoc-OSu (1.9 g, 5.65 mmol, 0.70 equiv.) in dichloromethane (50.0 mL) was added DIPEA (2.1 g, 16.1 mmol, 2.67 mL, 2.0 equiv.). The mixture was stirred at 25 °C for 1 h. LCMS analysis showed compound 6-3 was consumed completely. Upon completion, the reaction mixture was partitioned between dichloromethane and H2O. The organic phase was separated, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition) to afford compound 6-4 (4.5 g, 6.44 mmol, 80% yield, 98.8% purity) as a yellow oil. Purity by HPLC: 98.8%; MS (ESI): [M+Na]+:712.2;1H NMR (400 MHz, CDCh): 6 ppm 7.76 (d, J =7.28 Hz, 2 H), 7.59 (d, J=7.28 Hz, 2 H), 7.40 (t, J =7.40 Hz, 2 H), 7.30 - 7.35 (m, 2 H), 4.49 (d, J =6.02 Hz, 2 H), 4.23 (t, J =6.02 Hz, 1 H), 4.16 (s, 2 H), 4.01 (s, 2 H), 3.73 - 3.77 (m, 3 H), 3.65 - 3.73 (m, 8 H), 3.55 - 3.64 (m, 8 H), 3.44 - 3.50 (m, 4 H), 3.27 - 3.37 (m, 4 H), 1.48 (s, 9 H).

[0519] A solution of compound 6-4 (4.5 g, 6.44 mmol, 1 .0 equiv.) in formic acid (100%, 20 mL) was stirred at 25 °C for 3 h. LCMS analysis showed compound 6-4 was consumed completely. Upon completion, the reaction mixture was concentrated under reduced pressure to remove the solvent and lyophilized to remove the residual formic acid to afford compound 6-5 (4.23 g, 5.98 mmol, 96% yield, 96.1 % purity) as a colourless oil. Purity by HPLC: 96.1 %; MS (ESI): [M+H]+:634.4;1H NMR (400 MHz, CDCh): 6 ppm 8.01 (s, 1 H), 7.75 - 7.79 (m, 1 H), 7.77 (d, J =7.51 Hz, 1 H), 7.59 (d, J =7.51 Hz, 2 H), 7.40 (t, J =7.45 Hz, 2 H), 7.30 - 7.35 (m, 2 H), 4.50 - 4.54 (m, 1 H), 4.51 (br s, 1 H), 4.24 (t, J =5.90 Hz, 1 H), 4.15 - 4.19 (m, 4 H), 3.75 (s, 3 H), 3.65 - 3.74 (m, 8 H), 3.55 - 3.65 (m, 8 H), 3.48 (br dd, J =11 .09, 5.25 Hz, 4 H), 3.27 - 3.36 (m, 4 H).

[0520] To a solution of DBCO-OSu (9.9 g, 24.6mmol, 1 .0 eq.) in DMF (90 mL) was added NMM (5.4 mL, 49 mmol, 2.0 eq.) and Int A (5.35 g, 26 mmol, 1.05 eq.). The mixture was stirred at 25 °C for 4 h. LCMS analysis showed DBCO-NHS was consumed, and the desired product mass was detected. Upon completion, the solvent was removed and the residue was directly purified by prep-HPLC (TFA condition) to afford Int B (12 g, 24.3 mmol, 98% yield) as a yellow sticky oil after lyophilization. MS (ESI): [M+H]+:495.4.

[0521] To a solution of Int B (12 g, 24.3 mmol, 1 .0 eq.) in CH2CI2 (120 mL) was added EDCI (6.51 g, 33.97 mmol, 1.4 eq.) and HOSu (3.35 g, 29.2 mmol, 1 .2 eq.). The mixture was stirred at 25 °C for 16 h. LCMS analysis showed Int B was consumed, and the desired mass peak was detected. Upon completion, the reaction mixture was washed twice with water and brine. The combined organic layers were dried over anhydrous Na2SC>4, filtered, and the solvent was evaporated under reduced pressure to furnish crude Int C (13.6 g, 20.46 mmol, 84.2% yield, 89% purity) as a yellow sticky gel, that was used directly for the next step without further purification. MS (ESI): [M+H]+:592.4.

[0522] Example 6B -Synthesis of linker payload LP-4

[0523] To a solution of compound 6-5 (100 mg, 158 pmol, 1 eq.) in DMF (1 mL) was added compound 40 (164 mg, 174 pmol, 1.1 eq.), DIPEA (41 mg, 316 pmol, 52 pL, 2 eq.) and TBTU (61 mg, 190 pmol, 1.2 eq.). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed compound 6-5 was consumed completely, and desired mass peak was detected. The reaction mixture was added to isopropyl ether, and the crude product was precipitated out slowly. Further it was centrifuged to afford the crude product. The supernatant was discarded to furnish compound 6-6 (246 mg, crude) as brown oil. HPLC Purity: 85%; MS (ESI): [M+H]+: 1560.2.

[0524] To a solution of compound 6-6 (246 mg, 134 pmol, 1 eq) in DMF (1 .96 mL) was added EtaN (492 pL). The mixture was stirred at 25 °C for 16 h. LCMS analysis showed compound 6-6 was consumed completely and the desired mass peak was detected. Upon completion, the reaction mixture was added to isopropyl ether, and the crude product was precipitated out slowly. Furter it was centrifuged to afford the crude product. The supernatant was discarded to afford compound 6-7 (179 mg, crude) as a brown solid. HPLC

[0525] Purity: 76.8%; MS (ESI): [M+H]+: 1338.0.

[0526] To a solution of compound 6-7 (179 mg, 103 pmol, 1 eq.) in MeOH (750 L) was added saturated aqueous solution of Na2CC>3 (750 pL). The mixture was stirred at 25 °C for 1 h. LCMS analysis showed compound 6-7 was consumed completely, and desired mass peak was detected. Upon completion, the solvent was removed and the residue was directly purified by prep-HPLC (TFA condition) to afford Compound 6-8 (139 mg, 96 pmol, 93% yield, 91.1 % purity) as a yellow solid. HPLC Purity: 91.177%; MS (ESI): [M+H]+: 1323.5.

[0527] Synthesis of 2-[2-[2-[2-[[2-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12),4,6,8,13,15-hexaen- 10-yn-2-yl)-4-oxo-butanoyl]amino]ethoxy]ethoxy]ethoxy]acetyl]-[2-[2-[2-[2-[2-[2-[2-[2-[[(1S)-1-[[(1S)- 2-[4-[[(10S,23R)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02, 14.04, 13.06, 11.020, 24]tetracosa-1 , 6(11), 12, 14, 16(24), 17, 19-heptaen-23- yl]carbamoyloxymethyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo- ethoxy]ethoxy]ethoxy]ethyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (Compound 6-9):

[0528]

[0529] To a solution of compound 6-8 (109 mg, 75 pmol, 1 eq.) in DMF (1 .1 mL) was added Int C (58mg, 98 pmol, 1.3 eq.) and DIPEA (21 mg, 165 pmol, 27 pL, 2.2 eq.). The mixture was stirred at 25 °C for 16 h. LCMS analysis indicated that >90% of compound 6-8 was used up and the desired compound was formed. Upon, completion, the reaction mixture was concentrated and the residue was purified by prep-HPLC to afford

[0530] Compound 6-9 (58 mg, 27 pmol, 36% yield, 84.5% purity) as a yellow solid. HPLC Purity: 84.6%; MS (ESI): [M / 2+H]+: 900.8.

[0531] Synthesis of [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-[2-[[2-[2-[2-[2-[[2-[2-[2-[2-[[4-(2- azatricyclo[10.4.0.04,9]hexadeca-1(12),4,6,8,13,15-hexaen-10-yn-2-yl)-4-oxo- butanoyi]amino]ethoxy]ethoxy]ethoxy]acetyi]-[2-[2-[2-[2-[2-[2-[2-[2-[[(1S)-1-[[(1S)-2-[4-[[(10S,23R)-

[0532] 10-ethyl-18-fiuoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1, 6(11), 12, 14, 16(24), 17,19-heptaen-23- yl]carbamoyloxymethyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo ethoxy]ethoxy]ethoxy]ethylamino]-2-oxo- ethoxy]ethoxy]ethoxy]ethyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ethoxy]acet yl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[4-[5-[3-amino-6-(4- isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-methyl-carbamate (LP-4):

[0533]

[0534] To a solution of compound 6-9 (29 mg, 30 pmol, 1.2 eq.) in DMF (500 pL) was added TBTU (9.6 mg, 29.8 pmol, 1 .2 eq.) and DIPEA (6.4 mg, 50 pmol, 8.3 pL, 2 eq.). The mixture was stirred at 25 °C for 1 h. LCMS analysis showed >95% of the starting material 6-9 was consumed and the desired compound was formed. Upon completion, the reaction mixture was concentrated, and the residue was directly purified by prep- HPLC (AcOH condition) to give LP-4 (33 mg, 12 pmol, 47% yield, 98.6% purity) as a white solid. HPLC Purity: 98.6%; MS (ESI): [M / 2+H]+: 1377.9.1H NMR (400 MHz, DMSO-cfe): 6 ppm 9.99 (s, 2 H), 8.93 (s, 1 H), 8.37 (br d, J = 8.63 Hz, 4 H), 8.05 (br d, J = 8.25 Hz, 1 H), 7.98 (br d, J = 7.75 Hz, 2 H), 7.93 (d, J = 8.63 Hz, 2 H), 7.71 - 7.79 (m, 3 H), 7.54 - 7.68 (m, 8 H), 7.26 - 7.50 (m, 15 H), 7.16 - 7.23 (m, 2 H), 6.51 (s, 1 H), 5.44 (s, 2 H), 5.21 - 5.33 (m, 3 H), 5.07 (br s, 4 H), 5.01 (d, J = 14.01 Hz, 1 H), 4.52 (s, 2 H), 4.39 (br t, J = 6.94 Hz, 2 H), 4.24 - 4.32 (m, 2 H), 4.17 (s, 2 H), 3.94 (s, 4 H), 3.85 (d, J = 1 .38 Hz, 4 H), 3.38 - 3.62 (m, 56 H), 3.21 - 3.28 (m, 6 H), 3.03 - 3.13 (m, 3 H), 2.87 (s, 3 H), 2.36 (s, 3 H), 2.14 - 2.27 (m, 3 H), 1.94 - 2.03 (m, 3 H), 1.81 - 1.92 (m, 2 H), 1.70 - 1.81 (m, 1 H), 1.30 (br d, J =7.13 Hz, 6 H), 1.19 (d, J = 6.75 Hz, 6 H), 0.84 - 0.90 (m, 9 H), 0.82 (br d, J =6.63 Hz, 6 H). Example 6C -Synthesis of linker payload LP-5

[0535]

[0536] To a solution of 131 (120 mg, 153 pmol, 1.0 eq.) and Int 12 (109 mg, 169 pmol, 1.1 eq.) in DMF (1.2 mL) was added DIPEA (40 mg, 307 pmol, 50.794 pL, 2.0 eq.), HOBt (41 mg, 307 pmol, 2.0 eq.) and EDCI (59 mg, 307 pmol, 2.0 eq.). The mixture was stirred at 20 °C for 2 h. LCMS analysis indicated the complete consumption of Int 5, and desired mass peak was detected. Upon completion, the reaction mixture was triturated with cold isopropyl ether and centrifuged to get the residue to give the crude Int 13 (420 mg, crude) that was used for the next step without further purification. MS (ESI): [M+2H]2+: 1415.5

[0537] To a solution of Int 13 (490 mg, crude) in DMF (3.5 mL) was added EhN (1.1 g, 1 1 mmol, 1.5 mL, 36 eq.). The mixture was stirred at 20 °C for 4.5 h. LCMS analysis showed compound Int 13 was consumed completely, and the desired mass peak was detected. After completion, the reaction mixture was added to the isopropyl ether, and the crude product was precipitated slowly. The crude product was purified by prep- HPLC (0.1% TFA condition) to give Int 14 (95 mg, 75 pmol, 23% yield for two steps, 93.6% purity) as a white solid. Purity by HPLC: 93.6%; MS (ESI): [M+H]+:1192.2.

[0538] To a solution of compound 6-5 (7.81 g, 12.3 mmol, 1.0 eq.) in DMF (85 mL) was added I25 (12.1 g, 16 mmol, 1 .3 eq.), EDCI (7.1 g, 37 mmol, 3.0 eq.), HOBt (5.33 g, 39.4 mmol, 3.2 eq.) and DIPEA (8.15 mL, 49.3 mmol, 4.0 eq.). The reaction mixture was stirred at 25 °C for 4 h. LCMS analysis showed compound 6-5 and int 7 were completely consumed and the desired mass peak was detected. Upon completion, the reaction mixture was concentrated and then triturated by isopropyl ether and centrifuged to obtain the crude compound 6-6 (20.0 g, crude) as a dark residue and it was directly used for the next step without further purification. MS (ESI): [M+H]+: 1371.1.

[0539] To a solution of crude compound 6-6 (20 g, crude) in DMF (160 mL) was added triethylamine (40 mL, 287 mmol). The mixture was stirred at 25 °C for 18 h. LCMS analysis showed compound 6-6 was consumed, and the desired mass peak was detected. Upon completion, the resultant was triturated with isopropyl ether twice, and centrifuged to obtain the crude compound 6-7 (20 g, crude) as a dark residue that was directly used in the next step without further purification. MS (ESI): [M+H]+:1149.0.

[0540] Compound 6-7 (20 g, crude) was dissolved in MeOH / Saturated Na2COs (1 :1) (v / v, 110 mL in total) and the solution was stirred at 25 °C for 2 h. LCMS analysis indicated compound 6-7 was consumed completely and the desired mass peak was detected along with ~3% isomer. The reaction mixture was evaporated and purified directly by prep-HPLC (TFA condition) to afford Compound 6-8 (7.22 g, 6.29 mmol, 51 % yield over 3 steps, 98.8% purity) as a grey solid after lyophilization. LCMS Purity: 98.8%; MS (ESI): [M+H]+:1135.0, [M+2H]2+:568.3.

[0541] To a solution of compound 6-8 (7.22 g, 6.4 mmol, 1 .0 eq.) in DMF (75 mL) was added DIPEA (2.32 mL, 14. mmol, 2.2 eq.) and Int C (4.89 g, 8.28 mmol, 1.3 eq.). The mixture was stirred at 25 °C for 30 h. LCMS analysis showed ~3% of compound 6-8 remained, and the desired product peak was detected. At this timepoint, the reaction mixture was concentrated and directly purified by prep-HPLC (AcOH condition) to afford compound 6-9 (3.07 g, 1 .71 mmol, 27% yield, 93.7% purity) as a grey solid after lyophilization. LCMS Purity: 93.7%; MS (ESI): [M+H]+:1610.9, [M+2H]2+:806.3.

[0542] [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-[2-[2-[2-[[2-[2-[2-[2-[[4-(2- azatricyclo[10.4.0.04,9]hexadeca-1(12),4,6,8,13,15-hexaen-10-yn-2-yl)-4-oxo- butanoyl]amino]ethoxy]ethoxy]ethoxy]acetyl]-[2-[2-[2-[2-[[(1S)-1-[[(1S)-2-[4-[[(10S.23R)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02, 14.04, 13.06, 11.020,24]tetracosa-1, 6(11),12,14,16(24),17,19-heptaen-23- yl]carbamoyloxymethyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[4-[5-[3-amino-6-(4- isopropylsulfonylphenyl)pyrazin-2-yl]isoxazol-3-yl]phenyl]methyl]-N-methyl-carbamate (LP-5):

[0543] To a solution of Int 14 (25.9 mg, 21.7 pmol, 1.4 eq.) and compound 6-9 (55 mg, 15.52 pmol, 1.0 eq.) in DMF (300 pL) were added DIPEA (9 mg, 70 pmol, 11.6 pL, 4.5 eq.), HOBt (6.7 mg, 49.67 pmol, 3.2 eq.) and EDCI (8.0 mg, 42 pmol, 2.7 eq.) successively. The mixture was stirred at 20 °C for 2 h. LCMS analysis showed compound 6-9 was consumed completely, and the desired mass was detected. Upon completion, the reaction mixture was added to isopropyl ether, and the crude product was precipitated out slowly. The crude product was filtered and purified by prep-HPLC (0.5% AcOH condition) to afford LP-5 (33 mg, 11.85 pmol, 55% yield, 97% purity) as a white solid after lyophilization. Purity by HPLC: 97%; MS (ESI): [M+2H]2+:1393.5; [M+3H]3+:929.3;1H NMR (400 MHz, DMSO-cf6): 6 ppm 10.00 (br s, 2 H), 8.93 (br s, 1 H), 8.37 (br d, J = 5.75 Hz, 4 H), 7.90 - 8.08 (m, 5 H), 7.75 (br s, 3 H), 7.51 - 7.69 (m, 8 H), 7.24 - 7.50 (m, 16 H), 7.13 - 7.22 (m, 2 H), 6.52 (br s, 1 H), 5.44 (br s, 2 H), 5.27 (br s, 3 H), 5.07 (br s, 4 H), 4.53 (br s, 2 H), 4.35 - 4.44 (m, 2 H), 4.25 - 4.33 (m, 2 H), 4.16 (br d, J = 1.13 Hz, 2 H), 3.93 (br s, 4 H), 3.85 (br s, 2 H), 3.48 (br s, 66 H), 3.02 - 3.12 (m, 3 H), 2.87 (br s, 3 H), 2.67 (br s, 1 H), 2.32 - 2.38 (m, 4 H), 2.13 - 2.26 (m, 3 H), 1.71 - 2.04 (m, 7 H), 1.30 (br s, 6 H), 1.19 (br d, J = 2.88 Hz, 6 H), 0.79 - 0.91 (m, 15 H).

[0544] Example 6D -Synthesis of linker payload LP-6

[0545]

[0546] To a solution of Fmoc-VC-PAB-PNP (CAS: 863971-53-3, 248 mg, 324 pmol, 1.5 eq) and Berzosertib (0.1 g, 216 pmol, 1.0 eq.) in DMF (1 mL), DIPEA (56 mg, 431 pmol, 71.5 pL, 2.0 eq.) and HOBt (58 mg, 431 pmol, 2.0 eq.) were added. The reaction mixture was stirred at 20 °C for 2 h. LCMS analysis showed Berzosertib was consumed completely, and the desired mass peak was detected. Upon completion, the reaction mixture was triturated with isopropyl ether and centrifuged to obtain Fmoc-Val-Cit-PAB-Berzosertib (0.4 g, crude) as a yellow residue, the crude material was used as such for the next step without any further purification. MS (ESI): [M+H]+:1092.8.

[0547] To a solution of VC1 (0.4 g, crude) in DMF (2.8 mL) was added EtsN (1.2 mL). The mixture was stirred at 20 °C for 16 h, then isopropyl ether was added to the reaction mixture to induce precipitation. The precipitated material was collected and purified by prep-HPLC (0.1 % TFA condition) to furnish VC2 (80 mg, 80 pmol, 22% yield, 87% purity) as a pale-yellow solid. HPLC Purity: 87.4%; MS (ESI): [M+H]+:869.5.

[0548] To a solution of compound 6-5 (0.1 g, 158 pmol, 1.0 eq.) and NHz-Val-Cit-PABC-Exa (139 mg, 166 pmol, 1 .05 eq., CAS: 2227350-99-2) in DMF (1 mL) was added DIPEA (82 mg, 631 pmol, 105 pL, 4.0 eq.), HOBt (68 mg, 505 pmol, 3.2 eq.) and EDCI (91 mg, 473 pmol, 3.0 eq.) were added successively. The mixture was stirred at 20 °C for2 h. LCMS analysis indicated compound 6-5 was consumed completely, and desired mass peak was detected. Upon completion, the reaction mixture was triturated twice with isopropyl ether, and centrifuged to obtain the crude compound 039VC-1 (0.3 g, crude) as a dark residue and was directly used for the next step without further purification. MS (ESI): [M+H]+: 1457.6, [M+2H]2+:729.6. To a solution of crude compound 039VC-1 (0.3 g, crude) in DMF (2.4 mL) was added EtsN (0.6 mL, 287 mmol). The mixture was stirred at 20 °C for 16 h. LCMS analysis indicated compound 039VC-1 was consumed completely, and the desired product mass peak was detected. Upon completion, the reaction mixture was triturated twice with isopropyl ether and centrifuged to obtain the crude compound 039VC-2 (0.3 g, crude) as a dark residue that was directly used for the next step without further purification. MS (ESI): [M+H]+:1235.3, [M+2H]2+:618.4.

[0549] Synthesis of 2-[2-[2-[2-[2-[2-[2-[2-[[(1S)-1-[[(1S)-1-[[4-[[(10S,23R)-10-ethyl-18-fluoro-10-hydroxy-19- methyl-5, 9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02, 14.04, 13.06, 11.020, 24]tetracosa- 1,6(11), 12, 14,16(24), 17, 19-heptaen-23-yl]carbamoyloxymethyl]phenyl]carbamoyl]-4-ureido- butyi]carbamoyl]-2-methyl-propyi]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethylamino]ethoxy]ethoxy]ethoxy]acetic acid (Compound 039VC-3):

[0550] Compound 039VC-2 (0.3 g, crude) was dissolved in MeOH / saturated Na2CC>3 (1 :1) (v / v, 3 mL in total). The solution was stirred at 20 °C for 2 h. LCMS analysis showed compound 039VC-2 was consumed completely, and the desired mass peak was detected. Upon completion the solvent was removed and directly purified by prep-HPLC (0.1 % TFA condition) to furnish compound 039VC-3 (100 mg, 72 pmol, 30% yield forth ree steps, 87.7% purity) as a white solid after lyophilization. The 87.7% purity product was directly for the subsequent reaction without any additional purification. HPLC Purity: 87.7%; MS (ESI):

[0551] To a solution of compound 039VC-3 (100 mg, 66 pmol, 1 .0 eq.) and Int C (50 mg, 85 pmol, 1 .3 eq) in DMF (0.5 mL) was added DIPEA (19 mg, 144 pmol, 24 pL, 2.2 eq.). The mixture was stirred at 20 °C for 20 h. LCMS analysis showed >93% of 039VC-3 was consumed and the desired mass peak was observed. At this timepoint, the solvent was removed, and the resulting residue was directly purified by prep-HPLC (0.5% AcOH condition) to afford Compound 039VC-4 (50 mg, 25 pmol, 36% yield, 85.0% purity) as a white solid after lyophilization. HPLC Purity: 85.0%; MS (ESI): [M+2H]2+:849.4, [M+3H]3+:565.6.

[0552] To a solution of compound 039VC-4 (50 mg, 29.5 pmol, 1.0 eq.) and VC2 (36 mg, 41 pmol, 1 .4 eq.) in DMF (0.4 mL) was added HOBt (13 mg, 94 pmol, 3.2 eq.), EDCI (15 mg, 80 pmol, 2.7 eq.) and DIPEA (17 mg, 133 pmol, 22 pL, 4.5 eq.) were added The mixture was stirred at 20 °C for 2 h. The reaction was monitored by LCMS analysis. Upon completion, the solvent was removed under vacuum, and the resulting residue was directly purified by prep-HPLC (0.5% AcOH condition) to afford LP-6 (34 mg, 13 pmol, 45% yield, 98.3% purity) as a white solid after lyophilization. HPLC Purity: 98.3%; MS (ESI): [M+2H]2+:1274.8, [M+3H]3+:850.21H NMR (400 MHz, DMSO-cfe): 8 ppm 9.99 - 10.14 (m, 2 H), 8.93 (s, 1 H), 8.30 - 8.39 (m, 4 H), 7.96 - 8.01 (m, 2 H), 7.93 (br d, J = 7.00 Hz, 2 H), 7.75 (br s, 3 H), 7.59 (br s, 6 H), 7.25 - 7.50 (m, 16 H), 7.19 (br d, J = 1 .50 Hz, 2 H), 6.51 (s, 1 H), 5.93 - 6.05 (m, 2 H), 5.43 (br d, J = 7.13 Hz, 6 H), 5.20 - 5.31 (m, 3 H), 5.07 (br s, 4 H), 4.52 (br s, 2 H), 4.27 - 4.44 (m, 4 H), 4.15 (s, 2 H), 3.93 (br s, 3 H), 3.55 (br d, J = 14.13 Hz, 10 H), 3.48 (br s, 28 H), 2.98 - 3.13 (m, 6 H), 2.86 (s, 3 H), 2.31 - 2.38 (m, 5 H), 1.92 - 2.05 (m, 4 H), 1.52 - 1.76 (m, 7 H), 1.30 - 1 .49 (m, 5 H), 1.23 (br d, J = 1.50 Hz, 1 H), 1.18 (br d, J = 7.00 Hz, 6 H), 0.74 - 0.92 (m, 17 H).

[0553] Example 7 - ADC synthesis, monomeric purity, potency and DAR determination

[0554] A. Synthesis

[0555] The ADCs in table C were prepared according to the general method outlined below.

[0556] Antibodies were made in-house from transient expression in CHO cells based on known sequences for an antibody to a tumour associated antigen (Ab-TAA).

[0557] The ADC synthesis consists of two steps, the enzymatic addition of a linker moiety, followed by the conjugation of the payload comprising moiety. These events take place at the site of glutamine-295 (Q295), in the CH2 domain on modified antibody containing a mutation of the asparagine 297 into alanine (N297A). The first step of the process is the microbial transglutaminase (MTG or MTGase)-mediated conjugation of the linker moiety onto Q295 of the modified antibody. Having attached the click group, payload comprising moieties are introduced via metal-free click chemistry. Materials

[0558] The following materials were used in the synthesis:

[0559] MTGase Enzyme: Activa® Tl transglutaminase (Ajinomoto), unit activity 98.56 U / g

[0560] PBS pH 7.45 (ThermoFisher)

[0561] Activated carbon (Merck)

[0562] Sodium deoxycholate (Merck)

[0563] Propylene glycol (Merck)

[0564] - DMSO (Merck)

[0565] Protein concentrators (Millipore)

[0566] 0.22 pM syringe filter (Pall)

[0567] Enzymatic conjugation

[0568] The reagents set out in table A were combined in PBS buffer (prepared according to the manufacturer's instructions) in a sterile glass bottle with a size of at least 2 times the reaction volume. The reaction was agitated using a magnetic stirrer bar and was incubated for about 22 hours at room temperature. This reaction gives an ADC intermediate.

[0569] Table A: Concentration of MTGase reaction components

[0570] Purification

[0571] The ADC intermediate formed is then cleaned using protein A chromatography via bind-elute mode to remove excess linker and residual enzyme at room temperature.

[0572] A loading level of 9-16 mg / mL resin was used, and pool collection was started at 45 mAU / cm at an absorbance of 280nm and ended at 35 mAU / cm absorbance at 280 nm. The affinity chromatography steps are as shown in table B below:

[0573] Table B: Affinity chromatography sequence of steps

[0574] 1Mole equivalent per mole antibody

[0575] The eluted ADC intermediate was neutralized using 2M Tris at pH 7.4.

[0576] Click conjugation

[0577] Payload comprising moieties were conjugated to the ADC intermediate in a one-pot reaction by reaction of the members of the click-pair groups. The reaction is set on a roller machine at room temperature.

[0578] In one example method, conjugation of the first payload was achieved by combining 1 -3 mg / mL ADC intermediate with ca. 11 mM Sodium deoxycholate, 0.28-28 wt.% propylene glycol in TBS pH 7.5. To the solution was added 7-14 mol. Equivalents per antibody of linker-payload moiety. The reaction was set on a roller machine at room temperature for at least 12 hours.

[0579] Removal of free payload comprising moieties

[0580] Activated carbon was used to remove the free payload comprising moieties from the reaction mixture.

[0581] The reaction was carried out as follows:

[0582] Step 1 : Dilute activated carbon powder into 100 mg / mL in PBS;

[0583] Step 2: Based on the amount of ADC in the reaction mix, add 1 :1 ratio of activated carbon solution (weight : weight);

[0584] Step 3: Incubate reaction mixture with activated carbon at ambient temperature (25°C) and rotate for 1 hour.

[0585] Suspended activated carbon was removed from the mixture using a 0.22 pm PES filter and concentrated using a protein concentrator with 50 kDa MWCO. The sample was filtered with a 0.22 pm PES filter again before using it in subsequent examples.

[0586] The ADC is optionally further purified using HIC purification. i) Monomeric purity Monomeric purity was determined using size exclusion chromatography.

[0587] Samples were prepared by diluting them to 1 mg / mL in PBS.

[0588] The samples were analysed on a Thermo Ultimate 3000 UPLC / Waters ACQUITY H-Class PLUS Bio System equipped with a ACQUITY UPLC Protein BEH SEC Column, 200 A, 1 .7 pm, 4.6 mm X 150 mm column and a ACQUITY UPLC Protein BEH SEC Guard Column, 200 A, 1 .7 pm, 4.6 mm X 30 mm guard column.

[0589] The analysis method was as follows:

[0590] Mobile phase: 0.2M Potassium phosphate buffer, 0.2M Potassium Chloride, 15% (v / v) IPA, pH 6.8

[0591] Flow rate: 0.35 mL / min

[0592] Run time: 15 minutes

[0593] Column temperature: Room temperature UV detection: 280 nm

[0594] Injection load: 10 pg

[0595] The percentage purity results (% purity) were determined as

[0596] % purity = (Monomer peak area / Total peak area) x 100%. ii) Potency determination

[0597] The binding potency to the target antigen relative to a refence material was evaluated by ELISA.

[0598] The potency determination was carried out on a Molecular Devices, SpectraMax ID3 plate reader, equipped with a BioTek 405 TS plate washer.

[0599] The following reagents were used during the potency determination:

[0600] Wash buffer: PBS with 0.05% Tween 20

[0601] Blocking buffer: PBS with 1 % BSA

[0602] Coating antigen: His tag target antigen, 1 ug / mL

[0603] Secondary antibody: Anti-human IgG Fc antibody (HRP), 1 :7000 (abeam, #ab97725)

[0604] TMB solution: 1 -Step™ turbo TMB-ELISA Substrate Solution (Thermo scientific, #34022) Stop solution: ELISA stop solution (Invitrogen, SS04)

[0605] The test was carried out as follows, briefly, a 96-well plate was coated with 100 pl / well of coating antigen overnight (or up to 72hrs) at 4°C. The plate was then blocked by washing the plate three times with wash buffer (200 pl / well) and then blocking the plate with blocking buffer (200 pl / well) and subsequently incubating at room temperature for 1 hour. Samples were then added to the wells by washing the plate three times with wash buffer (200 pl / well) and then adding serially diluted samples (100 pl / well) and subsequently incubating at room temperature for 1 hour. Secondary antibody addition was carried out by washing the plate three times with wash buffer (200 pl / well) and adding diluted secondary antibody (1 :7000) to each well (100 pl / well) and incubating for 1 hour at room temperature in the dark. This was followed by TMB addition, which involved adding TMB equilibrated to room temperature (100 pl / well), the plate was then incubated at room temperature for 15 minutes in the dark. The reaction was then stopped by adding stop solution equilibrated to room temperature (100 pl / well) and the plates were read at 450 nm on a plate reader.

[0606] Results were generated by preparing a 4-parameter logistic dose-response curve to compute the ECso values of the samples and reference. The relative potency of the samples was determined by ECso of reference / ECso of sample x 100%.

[0607] Hi) Drug-to-Antibody Ratio (DAR)

[0608] DAR was determined by reversed phase liquid chromatography-mass spectrometry (RPLC-MS). DAR analysis was used to determine the average number of payloads and linkers attached to the Fc region of the ADC.

[0609] Samples were prepared by reducing 5 pg of ADC in 10 mM DTT at 40°C for 30 min and injecting a 5 pL aliquot for analysis.

[0610] The samples were analysed on a Waters ACQUITY UPLC H-Class PLUS Bio System instrument equipped with a Waters TUV and Xevo® G2-XS QTof Mass Spectrometer detector and an ACQUITY UPLC Protein BEH C4 column, 300 A, 1 .7 pm, 2.1 mm X 50 mm (#186004495).

[0611] The analysis method was as follows:

[0612] Column Temperature: 70°C

[0613] Mobile phase A: Water + 0.1 % formic acid

[0614] Mobile phase B: Acetonitrile + 0.1 % formic acid

[0615] Wavelength: 280 nm

[0616] Effective gradient: linear increase from 20 to 80 % of solvent B within 1 to 3.5 min at the flow rate of 0.4 mL / min

[0617] Xevo G2-XS: MS scan from 350 to 4000 m / z, ESI positive, sensitivity mode

[0618] The results were processed according to the following method. Analyte peaks time window: input time window range which covers the whole region of the peak and expected RT (i.e. mid-point of the time window range); background subtract results 5 % from baseline. The MaxEntl deconvolution parameters were as follows: input m / z range; output mass range; TOF resolution 20,000. The mass error tolerance was 100 ppm. The amino acid modifiers were input relevant modifiers such as -Lysine C-TERM, Pyroglutamic acid E N-TERM and linker-payload; select type as variable and maximum modification of 1. After processing and inspection of the deconvoluted spectra for identified species the overall DAR for each payload (e.g. exatecan and berzosertib), was calculated according to the following equation:

[0619] The results of this analysis are given in table C.

[0620] Table C

[0621] CM1 is a comparative linker of the structure: which is available from Conju-Probe under reference CP-2051 .

[0622] Example 8 — Material generation for additional ADCs

[0623] Enzymatic (MTG) reaction

[0624] The acyl acceptors used for the Topol i + berzo ADCs carry a branched click handle, which is linked to the naked antibody under the catalyzation of Ajinomoto MTG enzyme. MTG catalyzes site-specific transpeptidation between a primary amine in the acyl acceptor and the side chain of glutamine 295 (acyl donor) on a deglycosylated antibody such as Ab-N297A. Acyl acceptor LM-3 / 5 / 6 / 7 was used for dual payload ADC, which enabled a high Topol i and high Berzo drug-antibody ratio (DAR) of up to 4. Specifically, the following reagents were added into a falcon tube at a size of at least 2x reaction volume. The reaction is agitated by rotating at 30 rpm and is incubated for 17-20 hr at room temperature.

[0625] The formed ADC intermediate was then cleaned-up by Protein A chromatography via bind-elute mode to remove excess acyl acceptor and residual MTG following the process parameters listed below. Linker-Payload conjugation

[0626] For LM-3 / 5 / 6 / 7-linked ADC (dual payload ADC), both Topol i and ATRi payloads were conjugated to antibody through a single click addition manner. In this case, both Topol i and ATRi payloads were found in a single branched molecule (LP-2 / 4 / 5 / 6) linked to a DBCO click moiety. Upon addition, both payloads were conjugated onto the ADC intermediate at the site of azides via SPAAC reaction. The reaction was set on a roller machine at room temperature and stopped after 16 - 18 hours.

[0627] General synthetic schemes for LM3, LM5, LM6, and LM7 are shown in Figure 1. General synthetic schemes for LP-2, LP-4, LP-5, and LP-6 are shown in Figure 2A and Figure 2B. Process flowchart for ADC production is shown in Figure 3. The resulting ADCs are shown in Tables C-1 and C-2 below.

[0628] LP-0 is a linker payload of the structure:

[0629]

[0630] Table C-1 Table C-2 Example 9 — Cathepsin cleavage

[0631] The release of payloads was measured after cathepsin cleavage. Results are shown in Table C-3. Table C-3

[0632] Increase in arm length of linker payload alone or in combination with increased AA (acyl acceptor) arm length led to increased payload release (exa, ber); Increase in AA arm length alone did not lead to increase in payload release; Change to more labile cleavage group (VA>VC) increases extent of payload released (both exa and ber) regardless of AA arm.

[0633] Example 10 - Efficacy of ADCs

[0634] The efficacy of ADCs was analysed using CellTiter-Glo® 2D Cell Viability Assay (Promega, #G9242). Cells of a cell line which expresses tumour associated antigen (TAA) were seeded in 96-well white opaque plates in 175 pl of cell culture media, and incubated at 37°C, 5% CC>2for 24 h. The cells were seeded at a density of 9000 cells / well.

[0635] The following ADC was evaluated:

[0636] ADC1

[0637] The agents were added to the cells in culture in aliquots of 25 pl, either alone or in combination, (from 0.33 uM, 10 points of 3-fold serial dilution).

[0638] The cells were then incubated for 5 days at 37°C and 5% CO2 Post-incubation, 50 pl of CellTiter-Glo reagent was added to the plates and incubated for 25 mins with gentle agitation at 600 rpm. Cell viability was measured via Luminescence using Victor Nivo, PerkinElmer (2 replicates).

[0639] Background luminescence was subtracted, based on luminescence detected from wells having media only ( / .e. no cells). Precent inhibition was calculated using the formula 100- {(lum. of cells treated with test article / lum. of cells treated with buffer control) *100}, where lum. = luminescence. The average and SEM was calculated by GraphPad PRISM 10. Graphs were plotted using GraphPad PRISM 10, fitting the data points to a four-parameter logistic model.

[0640] The results are shown in figure 4.

Claims

1. Claims1 . A linker moiety comprising:(a) an amino group for conjugation to an antigen-binding moiety;(b) at least one click group for connecting a payload comprising moiety;(c) a urea group:wherein RNis selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the urea group; and b indicates where the at least one click group is linked to the urea group.

2. The linker moiety according to claim 1 , wherein RNis H.

3. The linker moiety according to either claim 1 or claim 2, wherein the amino group is linked to the urea group by a first spacer group (A1) which is of the formula:-(CH2)xa-(C2H4O)xb-(CH2)xc-ureawhere xa is 0 or 1 , xb is 0-12, and xc is 0 to 6, wherein at least one of xa and xb is 1 , andursaindicates where A1 is bound to the urea group.

4. The linker moiety according to claim 3, wherein A1 is selected from -(CH2)s- , -(C2H4)-O-(C2H4)- and -(C2H4O)3-(C2H4)-.

5. The linker moiety according to any one of claims 1 to 4, wherein one click group is linked to the urea group.

6. The linker moiety according to any one of claims 1 to 4, wherein two click groups are linked to the urea group.

7. The linker moiety according to claim 6, wherein the two click groups are linked to the urea group by a second spacer group (B1) of formula (B1-1):RL1is -(C2H4O)xi3-(CH2)xd-(C(=O))xi4- where xl3 is 0 to 4, xd is 0 to 3, xl4 is 0 or 1 ,RNB1is -(C2H4O)xei-(CH2)xfi-(NH)xgi-(C(=O)CH2)xhi- where xe1 is 0 to 4, xf1 is 0 to 2, xg1 is 0 or 1 , and xh1 is 0 or 1 , RNB2is H or -(C2H4O)xe2-(CH2)xf2-(NH)xg2-(C(=O)CH2)xh2- where xe2 is 0 to 4, xf2 is 0 to 2, xg2 is 0 or 1 , and xh2 is 0 or 1 .

8. The linker moiety according to claim 7, wherein the second spacer group (B1) is:

10. A linker between:(a) at least one payload; and(b) an antigen-binding moiety; comprising a moiety derived from a compound according to any one of claims 1 to 9.

11. A conjugate comprising:(a) at least one payload;(b) an antigen-binding moiety; wherein the linker between the payload and the antibody comprises a moiety derived from a compound according to any one of claims 1 to 9.

12. The conjugate according to claim 11 , wherein the at least one payload is comprised in a payload comprising moiety comprising more than one payload, such as two payloads.

13. The conjugate according to claim 12, wherein the payload comprising moiety comprises a branching group which is:

14. The conjugate according to claim 13, wherein the link between two payloads and the click group comprises:where each of Q1and Q2are independently:, where Qxis such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue;where a1 = 0 to 5, b1 = 0 to 8, c1 = 0 or 1 , d1 = 0 to 5. each of X2and X3are independently:where a2 = 0 to 5 and b2 = 0 to 8; a3 = 0 to 5, b3 = 0 to 8, c2 = 0 or 1 , d2 = 0 to 5, and e1 = 0 to 8.

15. The conjugate according to claim 14, wherein the link between the payload moieties and the click group comprises, or is:

16. The conjugate according to any one of claims 12 to 15, wherein the more than one payload are(a) a payload which is a DNA damage response (DDR) inhibitor, and (b) a payload which is a DNA topoisomerase I (TOP1) inhibitor.

17. The use of a conjugate according to any one of claims 11 to 16 in the manufacture of a medicament for treating cancer.

18. A conjugate according to any one of claims 11 to 16 for use in the treatment of cancers.

19. A linker-payload molecule comprising at least a payload for conjugation to an antigen-binding moiety, wherein the linker for conjugation to the antibody comprises a moiety derived from a compound according to any one of claims 1 to 9.

20. A modified antigen-binding moiety comprising a moiety derived from a compound according to any one of claims 1 to 9.

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