Linkers for antibody drug conjugates

Abstract

Linkers of formula (L) and compounds of formula (II) and (III): wherein R, EO, v, w, P, R6, R7, (1), (2), Y1, U, D, BM, and q have the meanings defined herein, are provided. Processes for preparing the compounds and their uses in a variety of medical applications, in particular in the treatment of cancer, are also provided.

Description

[0001] COMPOUNDS

[0002] Field of the Invention

[0003] This invention relates to novel linkers, and to linker-payload compounds and binding molecule - linker - payload conjugates, such as antibody-drug conjugates (ADCs) containing such linkers, which are useful as anti-tumour agents. The invention also relates to the use of such conjugates as medicaments, to the use of such conjugates for in vitro, in situ and in vivo diagnosis or treatment of mammalian cells (or associated pathological conditions), to pharmaceutical compositions containing them, and to synthetic routes fortheir production.

[0004] Background

[0005] Antibody-drug conjugates (ADCs) have prompted a revolution in the treatment of cancer. ADCs, comprising monoclonal antibodies conjugated to cytotoxic agents (payloads) via a cleavable linker, have emerged as novel therapeutic options. Upon binding with the cell surface antigen targeted by the specific antibody, the ADC is internalized by the tumour cell and processed by the endo-lysosomal system. Within the tumour cognate antigen-positive cells are bound by the antibody, the payload is released through the cleavable linker technology and results in cytotoxic activity.

[0006] ADCs can induce tumour cell death through the so-called bystander effect, which occurs when the cytotoxic payload (warhead) diffuses across the cell membrane to neighbouring cells, thus inducing their apoptosis. ADCs interact with cancer and immune cells by eliciting mechanisms such as immunogenic cell death, antibody-dependent cell-mediated cytotoxicity and dendritic cell activation.

[0007] An example of an ADC approved as a human medicine is trastuzumab deruxtecan (Enhertu®), which contains deruxtecan as the linker-payload moiety and the DNA topoisomerase I inhibitor referred to herein as “Dxd” as the payload moiety. However, there remains a need in the art to develop improved linkers and payloads and ADCs containing them.

[0008] Summary of the Invention

[0009] In a first aspect, there is provided a linker of formula (L):

[0010]

[0011] wherein:

[0012] R is C1-6 alkyl;

[0013] EO is an ethylene glycol moiety, the terminal carbon of which is attached to the group -N(R)-and the terminal oxygen of which is attached to the group -(CH2)w- when w is 1 to 10, or to the - C(=O) group when w is 0;

[0014] v is 0 or 1 to 40;

[0015] w is 0 or 1 to 10;

[0016] P is a peptide moiety consisting of 1 to 10 amino acid residues;

[0017] R6and R7are each independently H or C1-6 alkyl, or Re and R? together with the carbon atom to which they are attached form a 3- to 10-membered carbocyclic ring;

[0018] position (1) is a first attachment point; and

[0019] position (2) is a second attachment point.

[0020] In a second aspect, there is provided a compound of formula (II):

[0021] (

[0022]

[0023] II) or a pharmaceutically acceptable salt thereof, wherein:

[0024] R, EO, v, w, P, Re, and R? are as defined herein;

[0025] Y1is a group capable of conjugating with a binding molecule to generate a conjugate;

[0026] U is O or S; and

[0027] D is a payload moiety linked through the U atom.

[0028] The compounds of formula (II) are also referred to generally herein as “linker-payload” compounds. This term generally covers the compounds either in their own right or when forming the linker-payload part of a binding molecule-linker-payload conjugate as defined below.

[0029] In a third aspect, there is provided a compound of formula (III):

[0030]

[0031] or a pharmaceutically acceptable salt thereof, wherein:

[0032] BM is a binding molecule or a fragment thereof;

[0033] R, EO, v, w, P, Re, and R? are as defined herein;

[0034] II is as defined herein;

[0035] D is as defined herein; and

[0036] q is an integer from 1 to 16.

[0037] The compounds of formula (III) are also referred to generally herein as “binding molecule-linker-payload” compounds. When the binding molecule BM is an antibody or a fragment thereof, the compounds are also referred to herein as “antibody-drug conjugates” or “ADCs”.

[0038] In a fourth aspect, there is provided a composition comprising a compound of formula (III’):

[0039] o R6(CH2)W - C II - P - N

[0040]

[0041] or a pharmaceutically acceptable salt thereof, wherein:

[0042] BM is a binding molecule or a fragment thereof;

[0043] R, EO, v, w, P, Re, and R? are as defined herein;

[0044] II is as defined herein;

[0045] D is as defined herein; and

[0046] q’ is an integer or a decimal from 1 to 16.

[0047] In another aspect of the invention, there is provided a pharmaceutical formulation including a compound of formula (II) or (III) as defined herein, or a pharmaceutically acceptable solvate or salt thereof, or a composition containing a compound of formula (III’) as defined herein ora pharmaceutically acceptable solvate or salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0048] In another aspect of the invention, there is provided a compound of formula (II) or (III) as defined herein, or a pharmaceutically acceptable solvate or salt thereof, or a composition containing a compound of formula (III’) as defined herein ora pharmaceutically acceptable solvate or salt thereof, for use as a medicament.

[0049] In a further aspect of the present invention, there is provided a compound of formula (II) or (III) as defined herein, or a pharmaceutically acceptable solvate or salt thereof, or a composition containing a compound of formula (III’) as defined herein ora pharmaceutically acceptable solvate or salt thereof, for use in the treatment of cancer.

[0050] In a further aspect of the present invention, there is provided use of a compound of formula (II) or (III) as defined herein, or a pharmaceutically acceptable solvate or salt thereof, or a composition containing a compound of formula (III’) as defined herein ora pharmaceutically acceptable solvate or salt thereof, in the manufacture of a medicament for the treatment of cancer.

[0051] In a further aspect of the present invention, there is provided a method of treatment of cancer, which method comprises administration of a therapeutically effective amount a compound of formula (II) or (III) as defined herein, or a pharmaceutically acceptable solvate or salt thereof, or a composition containing a compound of formula (III’) as defined herein or a pharmaceutically acceptable solvate or salt thereof, to a patient suffering from, or susceptible to, such a condition.

[0052] Advantages

[0053] It has been found by the present inventors that conjugates comprising the linkers of the present invention exhibit potent anti-tumour activity, particularly although not exclusively against breast cancer. These linkers can be incorporated into a variety of binding molecule-linker-payload conjugates, such as ADCs, which have the potential for development as pharmaceuticals for the treatment of cancers.Brief Description of the Figures

[0054] Figure 1 shows the potency of the Binding Molecule-Linker-Payload compounds of Formulae 111-1, HI-2, III-3, HI-4, and IH-5 when tested according to Biological Example 1 in the SK-BR-3 breast cancer cell line;

[0055] Figure 2 shows the potency of the potency of the Binding Molecule-Linker-Payload compounds of Formulae II I-6 and HI-7 when tested according to Biological Example 1 in the SK-BR-3 breast cancer cell line; and

[0056] Figure 3 shows the potency of the potency of the Binding Molecule-Linker-Payload compounds of Formulae 111-11, 111-12, and 111-14 when tested according to Biological Example 1 in the SK-BR-3 breast cancer cell line.

[0057] Definitions

[0058] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range.

[0059] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence.

[0060] The term "variant" refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.

[0061] The sequence may have one or more deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent molecule. These sequences are encompassed by the present invention. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the activity is retained.

[0062] For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0063] As used herein, “variant” is synonymous with “mutant” and refers to a polynucleotide or amino acid sequence which differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a gene or protein having a polynucleotide or amino acid sequence respectively, which is identical with the native gene or protein respectively. The nucleic acid sequence may be an RNA or DNA sequence or a variant thereof. The term "polynucleotide" includes an RNA or DNA sequence. It may be single or double stranded. It may, for example, be genomic, recombinant, mRNA or cDNA.

[0064] The terms “selectively binds / selectively binding” and “specifically binds / specifically binding” may be used interchangeably herein.

[0065] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0066] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0067] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of'.

[0068] The terms “identity” and “% sequence identity” as used herein, may refer to the proportion of nucleotides or amino acids (expressed in percent) of a contiguous nucleotide sequence or contiguous amino acid sequence respectively which across the sequence, are identical to a reference sequence. The identity is calculated by counting the number of aligned nucleobases or amino acids that are identical (a Match) between the sequence of interest and a reference sequence, and dividing that number by the total number of nucleotidesamino acids respectively and multiplying by 100. Therefore, Percentage of Identity = (Matches x 100) / Length of aligned region. Insertions and deletions are not allowed in the calculation the percentage of identity. Chemical modifications of nucleotides may be disregarded provided that the functional capacity to form Watson-Crick base pairing is retained.

[0069] Identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U. S. A.; Devereux et al., 1984, Nucleotide sequences Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching. For example, the percentage identity between two polypeptide sequences may be readily determined by BLAST which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0070] Once the software has produced an optimal alignment, it is possible to calculate % identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0071] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 40, i.e., 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, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl, n-triacontyl, n-tetracontyl, and the like. The alkyl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0072] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integerwhich is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, such as comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carboncarbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1 -octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1 -decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1 -undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 55-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1 -dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. The alkenyl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0073] The term "alkynyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon triple bonds. Preferably, the alkynyl group comprises from 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkynyl group comprises from 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon triple bonds, such as comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary alkynylgroups include ethynyl, 1-propynyl, 2-propynyl 1 -butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1 -octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl, 6-nonynyl, 7-nonynyl, 8-nonynyl, 1 -decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, 1 -undecynyl, 2-undecynyl, 3-undecynyl, 4-undecynyl, 55-undecynyl, 6-undecynyl, 7-undecynyl, 8-undecynyl, 9-undecynyl, 10-undecynyl, 1 -dodecynyl, 2-dodecynyl, 3-dodecynyl, 4-dodecynyl, 5-dodecynyl, 6-dodecynyl, 7-dodecynyl, 8-dodecynyl, 9-dodecynyl, 10-dodecynyl, 11-dodecynyl, and the like. The alkynyl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0074] The terms "cycloalkyl" and “cycloalkenyl” represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl. Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, and cyclodecenyl. The cycloalkyl or cycloalkenyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). The cycloalkyl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0075] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. The aryl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0076] The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. The heteroaryl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0077] The term "heterocyclyl" means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms selected from O, S, and N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1, the 5 maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. The heterocyclyl groups may each be substituted by one or more substituents selected from List 1, defined below.

[0078] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises from 1 to 40, / .e., 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, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene ( / .e., 1,1-ethylene, 1,2-ethylene), propylene ( / .e., 1,1 -propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), the butylene isomers (e.g., 1,1 -butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1 -iso-butylene, 1,2-iso-butylene, and 1,3-iso-butylene), the pentylene isomers e.g., 1,1 -pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso-pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1 -hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1 -isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1 -octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1 -isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-butylene can also be called butan-1,2-diyl). The alkylene groups may be substituted with one or more, preferably 1 to 5, such as 1, 2, or 3 substituents selected from List 1.

[0079] The terms "alkenylene" and “alkynylene” refers to a diradical of a straight or branched hydrocarbon containing at least one carbon-carbon double or triple bond respectively.

[0080] Preferably, the alkenylene or alkynylene group comprises from 2 to 40, i.e., 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, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 2 to 30, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 or 2 to 4 carbon atoms. Exemplary alkenylene groups include vinylene, propenylene and the butenylenes. Exemplary alkynylene groups include ethynylene, propynylene and the butynylenes. The alkenylene or alkynylene groups may be substituted with one or more, preferably 1 to 5, such as 1, 2, or 3 substituents selected from List 1.

[0081] The term "cycloalkylene", “cycloalkenylene” and “cycloalkynylene” represent cyclic nonaromatic versions of "alkylene", “alkenylene” and “alkynylene” with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms). More preferably cycloalkylene and cycloalkenylene have 3 to 10, especially 3 to 7, carbon atoms and cycloalkynylene have 8 to 10 carbon atoms. Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, and cyclodecylene. Exemplary cycloalkenylene groups include cyclopropenylene, cyclobutenylene, cyclopentenylene,cyclohexenylene, cycloheptenylene, cyclooctenylene, cyclononenylene, and cyclodecenylene. Exemplary cycloalkynylene groups include cyclooctynylene, cyclononynylene, and cyclodecynylene. The cycloalkylene, cycloalkenylene or cycloalkynylene group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). The cycloalkylene, cycloalkenylene or cycloalkynylene groups may be substituted with one or more, preferably 1 to 5, such as 1, 2, or 3 substituents selected from List 1.

[0082] The term "heterocycloalkylene", “heterocycloalkenylene” and “heterocycloalkynylene” represent non-aromatic heterocyclic versions of "cycloalkylene", “cycloalkenylene” and “cycloalkynylene” with 3 to 14 ring atoms, or 3 to 10 ring atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms), wherein 1 to 5, such as 1 to 4, such as 1, 2 or 3, such as 1 or 2, ring atoms are heteroatoms selected from N, O and S, and the other ring atoms are carbon atoms. More preferably heterocycloalkylene and heterocycloalkenylene have 3 to 10, especially 3 to 7, ring atoms and heterocycloalkynylene has 8 to 10 ring atoms, wherein 1, 2 or 3, such as 1 or 2, ring atoms are heteroatoms selected from N, O and S, and the other ring atoms are carbon atoms. The heterocycloalkylene, heterocycloalkenylene or heterocycloalkynylene group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). Examples of heterocycloalkylene groups include aziridylene, oxiranylene, thiiranylene, azetidinylene, oxetanylene, thietanylene, pyrrolidinylene, tetrahydrofuranylene, tetrhydrothiophenylene, pyrazolidinylene, imidazolinylene, dioxolanylene, thiazolidinylene, isoxazolidinylene, piperidinylene, piperazinylene, morpholinylene, 1,4-dioxanylene, thiomorpholinylene, 1,4-oxathianylene, 1,4-dithianylene, 1,3,5-trioxanylene, 1,3,5-thithianylene, azepanylene, oxepanylene, thiepanylene, diazepanylene, oxazapenylene, thiazepanylene, azocanylene, oxocanylene, thiocanylene, azonanylene, oxonanylene and thionanylene. Examples of heterocycloalkenylene groups include 1-, 2- or 3-pyrrolinylene, 2-or 3-pyrazolinylene, imidazolinylene, 2H or 4H-pyranylene, 3,4-dihydropyranylene, 1,4-thiazinylene, 1,4-oxazinylene, azepinylene, oxepinylene, thiepinylene, diazepinylene, oxazepinylene, thiazepinylene, azocinylene, oxocinylene, thiocinylene, azoninylene, oxoninylene and thioninylene. The heterocycloalkylene, heterocycloalkenylene or heterocycloalkynylene groups may be substituted with one or more, preferably 1 to 5, such as 1, 2, or 3 substituents selected from List 1.

[0083] “Halogen” means fluorine, chlorine, bromine or iodine.Substituents

[0084] In the compounds of the present invention, the substituents may have the meanings defined herein. The general definitions of the substituents are equally applicable to the linkers of formula (L), the payload-linker compounds of formula (II), and the binding molecule- linker payload compounds (such as antibody-drug conjugates) of formula (III), including those compounds of formula (III’) present in the compositions of the present invention as described herein, as appropriate.

[0085] R

[0086] In one embodiment, R is C1-6 alkyl. In some embodiments, R is C1-4 alkyl. In some embodiments, R is C1-3 alkyl. In some embodiments, R is methyl or ethyl. In some embodiments, R is methyl.

[0087] EO

[0088] EO is an ethylene glycol moiety, the terminal carbon of which is attached to the group -N(R)-and the terminal oxygen of which is attached to the group -(CH2)w- when w is 1 to 10, or to the - C(=O) group when w is 0. As is known to the person skilled in the art, the ethylene glycol moiety is a moiety of formula (-CH2-CH2-O-).

[0089] vand w

[0090] v is O or 1 to 40, such as 0, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40. In some embodiments, v is 0 or 1 to 30. In some embodiments, v is 0 or 1 to 20. In some embodiments, v is 0 or 1 to 10. In some embodiments, v is 0 or 1 to 8. In some embodiments, v is 0 or 1 to 7. In some embodiments, v is 0 or 1 to 6. In some embodiments, v is 0 or 1 to 5. In some embodiments, v is 0 or 1 to 4. In some embodiments, v is 0 or 1 to 3. In some embodiments, v is 1 to 4. In some embodiments, v is 2 to 10. In some embodiments, v is 2 to 8. In some embodiments, v is 2 to 7. In some embodiments, v is 2 to 6. In some embodiments, v is 2 to 5. In some embodiments, v is 2 to 4. In some embodiments, v is 2. In some embodiments, v is 0.

[0091] w is 0 or 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, w is 0 or 1 to 8. In some embodiments, w is 0 or 1 to 6. In some embodiments, w is 0 or 1 to 5. In someembodiments, w is 0 or 1 to 4. In some embodiments, w is 1 to 4. In some embodiments, w is 1 to 3. In some embodiments, w is 0, 1 or 2. In some embodiments, w is 0 or 1. In some embodiments, w is 0. In some embodiments, w is 1.

[0092] In some embodiments, at least one of v and w is not 0.

[0093] Peptide moiety (P)

[0094] P is a peptide moiety as defined herein. In this specification, the term “peptide” may take its normal meaning of a chain of amino acids linked by peptide bonds, but may also take the meaning of a single amino acid residue.

[0095] In one embodiment, the peptide moiety P comprises 1 to 10 amino acid residues. In one embodiment, the peptide moiety P consists essentially of 1 to 10 amino acid residues. In one embodiment, the peptide moiety P consists of 1 to 10 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 1 to 6 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 1 to 5 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 2 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 3 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 4 amino acid residues. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of 5 amino acid residues.

[0096] In one embodiment, the peptide moiety P comprises, consists essentially of or consists of any of AA1, AA1-Gly, Val-Cit, Vai-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn, Glu-Val-Cit, Glu-Val-AA1, Glu-Glu, Glu-Gly-Cit, Glu-Gly-AA1, or Gly-Gly-AA1-Gly where AA1represents any amino acid residue, for example those as defined below.

[0097] In one embodiment, the peptide moiety P comprises, consists essentially of or consists of any of AA2, AA2-Gly, Val-Cit, Vai-Ala, Val-AA2, Val-AA2-Gly, AA2-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn, Glu-Val-Cit, Glu-Val-AA2, Glu-Glu, Glu-Gly-Cit, Glu-Gly-AA2, or Gly-Gly-AA2-Gly where AA2represents an aromatic amino acid residue. In this specification “aromatic amino acid residue” encompasses any amino acid residue wherein the side chain is an aryl or heteroaryl group, as defined above, which may be substituted with any substituent selected from List 1, as defined above.In one embodiment, the peptide moiety P comprises, consists essentially of or consists of any of AA1, AA1-Gly, Val-Cit, Vai-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn, Glu-Val-Cit, Glu-Val-AA1, Glu-Glu, Glu-Gly-Cit, Glu-Gly-AA1, Gly-Gly-Phe-Gly (GGFG) or Gly-Gly-Pya-Gly, where AA1represents any amino acid residue as defined below, and Pya represents a (2-pyridyl)alanine, (3-pyridyl)alanine or (4-pyridyl)alanine residue.

[0098] In one embodiment, the peptide moiety P comprises, consists essentially of or consists of a H N A?

[0099] Val-AA1-Gly moiety, where AA1represents

[0100]

[0101] In one embodiment, the peptide moiety P comprises, consists essentially of or consists of a GGFG peptide moiety (SEQ ID NO:33).

[0102] In one embodiment, the peptide moiety P comprises, consists essentially of or consists of a GG(Pya)G peptide moiety wherein Pya represents a (2-pyridyl)alanine, (3-pyridyl)alanine or (4-pyridyl)alanine residue.

[0103] In one embodiment, the peptide moiety P comprises, consists essentially of or consists of a GG(Pya)G peptide moiety wherein Pya represents a (2-pyridyl)alanine residue. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of a GG(Pya)G peptide moiety wherein Pya represents a (3-pyridyl)alanine residue. In one embodiment, the peptide moiety P comprises, consists essentially of or consists of a GG(Pya)G peptide moiety wherein Pya represents a (4-pyridyl)alanine residue.

[0104] AA1

[0105] In one embodiment, the amino acid residue represented by AA1is selected from

[0106]

[0107]

[0108] position 1 is connected, optionally via a further conjugation moiety, to the portion of the molecule bearing the binding molecule or fragment thereof, or the group Y which is capable of conjugating to the binding molecule or fragment thereof, and position 2 is connected, optionally via a further conjugation moiety, to the portion of the molecule bearing the payload compound.

[0109] R6and R7

[0110] In one embodiment, R6is H or C1-6 alkyl. In one embodiment, R6is H or C1-4 alkyl. In one embodiment, R6is H or C1-3 alkyl. In one embodiment, R6is H, methyl or ethyl. In one embodiment, R6is H.

[0111] In one embodiment, R7is H or C1-6 alkyl. In one embodiment, R7is H or C1-4 alkyl. In one embodiment, R7is H or C1-3 alkyl. In one embodiment, R7is H, methyl or ethyl. In one embodiment, R7is H.

[0112] In one embodiment, R6and R7together with the carbon atom to which they are attached form a 3- to 10-membered carbocyclic ring. In one embodiment, R6and R7together with the carbon atom to which they are attached form a 3- to 8-membered carbocyclic ring. In one embodiment, R6and R7together with the carbon atom to which they are attached form a 3-to 6-membered carbocyclic ring.

[0113] In one embodiment, R6and R7are each H.Y1

[0114] Y1is a group capable of conjugating with a binding molecule to generate a conjugate. The precise nature of Y1is not limited provided that it is capable of performing this function. In one embodiment, the conjugation takes place by displacement of all of part of the group Y1by a suitable moiety (typically a thiol group) on the binding moiety. In one embodiment, the conjugation takes place by addition to the group Y1by a suitable moiety (typically a thiol group) on the binding moiety.

[0115] In one embodiment, Y1comprises a sulfone moiety. In one embodiment, Y1comprises a (C1-6 alkyl)sulfonyl moiety. In one embodiment, Y1comprises a (C1-4 alkyl)sulfonyl moiety. In one embodiment, Y1comprises a (C1-3 alkyl)sulfonyl moiety. In one embodiment, Y1comprises a methylsulfonyl or ethylsulfonyl moiety. In one embodiment, Y1comprises a methylsulfonyl moiety. In one embodiment, Y1comprises an ethylsulfonyl moiety.

[0116] U

[0117] In one embodiment, U is O or S. In one embodiment, U is O. In one embodiment, U is S.

[0118] X

[0119] In one embodiment, X is a bond, O, S, S(=O) or S(=O)2.

[0120] In one embodiment, X is O or S.

[0121] In one embodiment, X is O. In one embodiment, X is a bond. In one embodiment, X is S. In one embodiment, X is S(=O). In one embodiment, X is S(=O)2.

[0122] A

[0123] In one embodiment, when X is O, S, S(=O) or S(=O)2, A is C2-10 alkylene, C2-10 alkenylene, C2- alkynylene, C3-10 cycloalkylene, C3-10 cycloalkenylene, Cs- cycloalkynylene, C3-10 heterocycloalkylene, C3-10 heterocycloalkenylene, or Cs- heterocycloalkynylene.

[0124] In some embodiments, when X is O, S, S(=O) orS(=O)2, A is C4-10 alkylene, C2-10 alkenylene, C2- alkynylene, C3-10 cycloalkylene, C3-10 cycloalkenylene, Cs- cycloalkynylene, C3-10 heterocycloalkylene, C3-10 heterocycloalkenylene, or Cs- heterocycloalkynylene.In one embodiment, when X is a bond, A is C1-10 alkylene, C2-10 alkenylene, C2- alkynylene, C3-10 cycloalkylene, C3-10 cycloalkenylene, or Cs- cycloalkynylene, C3-10 heterocycloalkylene, C3-10 heterocycloalkenylene, or Cs- heterocycloalkynylene.

[0125] In some embodiments, when X is a bond, A is C2-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C3-10 cycloalkenylene, or Cs- cycloalkynylene, C3-10 heterocycloalkylene, C3-10 heterocycloalkenylene, or Cs- heterocycloalkynylene.

[0126] In one embodiment, A is C2-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C3-10 cycloalkenylene, or Cs- cycloalkynylene. In one embodiment, A is C2-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, or C3-10 cycloalkenylene. In one embodiment, A is C2-10 alkylene, C2-10 alkenylene, C3-10 cycloalkylene, or Cs-cycloalkenylene. In one embodiment, A is C2-10 alkylene or C3-10 cycloalkylene. In one embodiment, A is C2-10 alkylene. In one embodiment, A is C2-10 alkenylene. In one embodiment, A is C2-10 alkynylene. In one embodiment, A is C3-10 cycloalkylene. In one embodiment, A is C3-10 cycloalkenylene. In one embodiment, A is Cs- cycloalkynylene.

[0127] In one embodiment, A is C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, C3-10 cycloalkenylene, Cs- cycloalkynylene. In one embodiment, A is C1-10 alkylene, C2-10 alkenylene, C2-10 alkynylene, C3-10 cycloalkylene, or C3-10 cycloalkenylene. In one embodiment, A is C1-10 alkylene, C2-10 alkenylene, C3-10 cycloalkylene, or Cs-cycloalkenylene. In one embodiment, A is C1-10 alkylene or C3-10 cycloalkylene. In one embodiment, A is C1-10 alkylene.

[0128] In one embodiment, A is C1-10 alkylene. In one embodiment, A is C2-10 alkylene. In one embodiment, A is C1-9 alkylene. In one embodiment, A is C2-9 alkylene. In one embodiment, A is C1-8 alkylene. In one embodiment, A is C2-8 alkylene. In one embodiment, A is C1-7 alkylene. In one embodiment, A is C2-7 alkylene. In one embodiment, A is C1-6 alkylene. In one embodiment, A is C2-6 alkylene. In one embodiment, A is C1-5 alkylene. In one embodiment, A is C2-5 alkylene. In one embodiment, A is C1-4 alkylene. In one embodiment, A is C2-4 alkylene. In one embodiment, A is C1-3 alkylene. In one embodiment, A is C2-3 alkylene. In one embodiment, A is C1-2 alkylene. In one embodiment, A is methylene. In one embodiment, A is ethylene. In one embodiment, A is 1,1-ethylene. In one embodiment, A is 1,2-ethylene. In one embodiment, A is propylene. In one embodiment, A is 1,1 -propylene. In one embodiment, A is 1,2-propylene. In one embodiment, A is 1,3-propylene. In one embodiment, A is butylene. In one embodiment, A is 1,1-butylene. In one embodiment, A is 1,2-butylene. In one embodiment, A is 1,3-butylene. In one embodiment, A is 1,4-butylene.In one embodiment, A is pentylene. In one embodiment, A is 1,1 -pentylene. In one embodiment, A is 1,2-pentylene. In one embodiment, A is 1,3-pentylene. In one embodiment, A is 1,4-pentylene. In one embodiment, A is 1,5-pentylene. In one embodiment, A is hexylene. In one embodiment, A is 1,1-hexylene. In one embodiment, A is 1,2-hexylene. In one embodiment, A is 1,3-hexylene. In one embodiment, A is 1,4-hexylene. In one embodiment, A is 1,5-hexylene. In one embodiment, A is 1,6-hexylene. In one embodiment, A is unsubstituted C1-10 alkylene. In one embodiment, A is unsubstituted C2-10 alkylene. In one embodiment, A is unsubstituted C2-6 alkylene. In one embodiment, A is unsubstituted C2-4 alkylene.

[0129] In one embodiment, A is unsubstituted C4-10 alkylene. In one embodiment, A is unsubstituted C4-8 alkylene. In one embodiment, A is unsubstituted C4-6 alkylene.

[0130] In one embodiment, A is C3-10 cycloalkylene. In one embodiment, A is C3-9 cycloalkylene. In one embodiment, A is C3-8 cycloalkylene. In one embodiment, A is C3-7 cycloalkylene. In one embodiment, A is C3-6 cycloalkylene. In one embodiment, A is cyclopropylene. In one embodiment, A is 1,1 -cyclopropylene. In one embodiment, A is 1,2-cyclopropylene. In one embodiment, A is cyclobutylene. In one embodiment, A is 1,1 -cyclobutylene. In one embodiment, A is 1,2-cyclobutylene. In one embodiment, A is 1,3-cyclobutylene. In one embodiment, A is cyclopentylene. In one embodiment, A is 1,1 -cyclopentylene. In one embodiment, A is 1,2-cyclopentylene. In one embodiment, A is 1,3-cyclopentylene. In one embodiment, A is cyclohexylene. In one embodiment, A is 1,1 -cyclohexylene. In one embodiment, A is 1,2-cyclohexylene. In one embodiment, A is 1,3-cyclohexylene. In one embodiment, A is 1,4-cyclohexylene.

[0131] In one embodiment, A is C2-10 alkenylene, C2- alkynylene, C3-10 cycloalkenylene, Cs-cycloalkynylene, C3-10 heterocycloalkenylene, or Cs- heterocycloalkynylene.

[0132] In one embodiment, A is C2-10 alkenylene, C2- alkynylene, C3-10 cycloalkenylene, or Cs-cycloalkynylene. In one embodiment, A is C2-10 alkenylene or C2- alkynylene. In one embodiment, A is C2-10 alkenylene. In one embodiment, A is C2- alkynylene. In one embodiment, A is C3-10 cycloalkenylene. In one embodiment, A is Cs- cycloalkynylene.

[0133] In one embodiment, A is C2-10 alkenylene. In one embodiment, A is C3-10 alkenylene. In one embodiment, A is C2-9 alkenylene. In one embodiment, A is C2-8 alkenylene. In one embodiment, A is C2-7 alkenylene. In one embodiment, A is C2-6 alkenylene. In one embodiment, A is C2-5 alkenylene. In one embodiment, A is C2-4 alkenylene. In oneembodiment, A is C2-3 alkenylene. In one embodiment, A is ethenylene. In one embodiment, A is trans-ethenylene. In one embodiment, A is cis-ethenylene. In one embodiment, A is propenylene. In one embodiment, A is trans-propenylene. In one embodiment, A is cispropenylene. In one embodiment, A is butenylene. In one embodiment, A is trans-butenylene. In one embodiment, A is cis-butenylene. In one embodiment, A is pentenylene. In one embodiment, A is trans-pentenylene. In one embodiment, A is cis-pentenylene. In one embodiment, A is hexenylene. In one embodiment, A is trans-hexenylene. In one embodiment, A is cis-hexenylene.

[0134] In one embodiment, A is C2- alkynylene. In one embodiment, A is C3-10 alkynylene. In one embodiment, A is C2-9alkynylene. In one embodiment, A is C2-8 alkynylene. In one embodiment, A is C2-7 alkynylene. In one embodiment, A is C2-6 alkynylene. In one embodiment, A is C2-5 alkynylene. In one embodiment, A is C2-4 alkynylene. In one embodiment, A is C2-3 alkynylene. In one embodiment, A is ethynylene. In one embodiment, A is propynylene. In one embodiment, A is butynylene. In one embodiment, A is pentynylene. In one embodiment, A is hexynylene.

[0135] In one embodiment, A is C3-10 heterocycloalkenylene. In one embodiment, A is C3-9 heterocycloalkenylene. In one embodiment, A is C3-8 heterocycloalkenylene. In one embodiment, A is C3-7 heterocycloalkenylene. In one embodiment, A is C3-6 heterocycloalkenylene. In one embodiment, A is 1-, 2- or 3-pyrrolinylene. In one embodiment, A is 2- or 3-pyrazolinylene. In one embodiment, A is imidazolinylene. In one embodiment, A is 2H or4H-pyranylene. In one embodiment, A is 3,4-dihydropyranylene. In one embodiment, A is 1,4-thiazinylene. In one embodiment, A is 1,4-oxazinylene. In one embodiment, A is azepinylene. In one embodiment, A is oxepinylene. In one embodiment, A is thiepinylene. In one embodiment, A is diazepinylene. In one embodiment, A is oxazepinylene. In one embodiment, A is thiazepinylene. In one embodiment, A is azocinylene. In one embodiment, A is oxocinylene. In one embodiment, A is thiocinylene. In one embodiment, A is azoninylene. In one embodiment, A is oxoninylene. In one embodiment, A is thioninylene.

[0136] The alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocycloalkylene, heterocycloalkenylene, or heterocycloalkynylene groups may each be substituted by one or more substituents selected from List 1, defined below. In one embodiment, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocycloalkylene, heterocycloalkenylene, or heterocycloalkynylene groups may each be substituted by one or more substituents selected from List 1A, definedbelow. In one embodiment, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocycloalkylene, heterocycloalkenylene, or heterocycloalkynylene groups may each be substituted by one or more substituents selected from List 1 B, defined below. In one embodiment, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocycloalkylene, heterocycloalkenylene, or heterocycloalkynylene groups may each be substituted by one or more substituents selected from List 1 C, defined below. In one embodiment, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocycloalkylene, heterocycloalkenylene, or heterocycloalkynylene groups may each be substituted by one or more substituents selected from List 1 D, defined below.

[0137] In some embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocycloalkylene, heterocycloalkenylene, or heterocycloalkynylene groups are unsubstituted.

[0138] T

[0139] In one embodiment, T is C1-6 alkylene. In one embodiment, T is C1-5 alkylene. In one embodiment, T is C1-4 alkylene. In one embodiment, T is C1-3 alkylene. In one embodiment, T is C1-2 alkylene. In one embodiment, T is methylene.

[0140] R1

[0141] In one embodiment, R1 is H, C1-6 alkyl or C3-6 cycloalkyl. In one embodiment, R1 and R4 together with the carbon atoms to which they are attached form a 5- to 7-membered carbocyclic ring which is substituted by a group -NH-C(=O)-Rs. In the latter embodiment, the compounds of formulae (II) and (III) comprise payloads of formula (D-B1) as described in more detail below.

[0142] In one embodiment, R1 is H or C1-6 alkyl. In one embodiment, R1 is H or C1-5 alkyl. In one embodiment, R1 is H or C1-4 alkyl. In one embodiment, R1 is H or C1-3 alkyl. In one embodiment, R1 is H, methyl or ethyl. In one embodiment, R1 is H. In one embodiment, R1 is C1-6 alkyl. In one embodiment, R1 is C1-5 alkyl. In one embodiment, R1 is C1-4 alkyl. In one embodiment, R1 is C1-3 alkyl. In one embodiment, R1 is methyl or ethyl. In one embodiment, R1 is methyl. In one embodiment, R1 is ethyl.In one embodiment, Ri is C3-6-cycloalkyl. In one embodiment, Ri is Cs-s-cycloalkyl. In one embodiment, Ri is C3-4-cycloalkyl. In one embodiment, Ri is cyclopropyl. In one embodiment, Ri is Me, Et or Cs-e-cycloalkyl. In one embodiment, Ri is Me or Cs-e-cycloalkyl. In one embodiment, Ri is Et or Cs-e-cycloalkyl.

[0143] In one embodiment, either: Ri is H, C1-6 alkyl, or C3-6 cycloalkyl, and R4 is H or C1-6 alkyl, or: Ri and R4 together with the carbon atoms to which they are attached form a 5- to 7-membered carbocyclic ring which is substituted by a group -NH-C(=O)-Rs.

[0144] R2

[0145] In one embodiment, R2 is H, halogen, C1-6 alkyl, O-C1-6 alkyl or S-C1-6 alkyl. In one embodiment, R2 is H, halogen, or C1-6 alkyl. In one embodiment, R2 is H, halogen or C1-4 alkyl. In one embodiment, R2 is H, halogen or C1-3 alkyl.

[0146] In one embodiment, R2 is halogen or C1-6 alkyl. In one embodiment, R2 is halogen or C1-3 alkyl. In one embodiment, R2 is halogen or C1-2 alkyl.

[0147] In one embodiment, R2 is H.

[0148] In one embodiment, R2 is halogen. In one embodiment, R2 is F. In one embodiment, R2 is Cl. In one embodiment, R2 is Br. In one embodiment, R2 is I.

[0149] In one embodiment, R2 is C1-6 alkyl. In one embodiment, R2 is C1-5 alkyl. In one embodiment, R2 is C1-4 alkyl. In one embodiment, R2 is C1-3 alkyl. In one embodiment, R2 is methyl or ethyl. In one embodiment, R2is methyl. In one embodiment, R2 is ethyl.

[0150] In one embodiment, R2 is O-C1-6 alkyl. In one embodiment, R2 is O-C1-5 alkyl. In one embodiment, R2 is O-C1-4 alkyl. In one embodiment, R2 is O-C1-3 alkyl. In one embodiment, R2 is O-C1-2 alkyl. In one embodiment, R2 is O-methyl. In one embodiment, R2 is O-ethyl.

[0151] In one embodiment, R2 is S-C1-6 alkyl. In one embodiment, R2 is S-C1-5 alkyl. In one embodiment, R2 is S-C1-4 alkyl. In one embodiment, R2 is S-C1-3 alkyl. In one embodiment, R2 is S-C1-2 alkyl. In one embodiment, R2 is S-methyl. In one embodiment, R2 is S-ethyl.

[0152] R3In one embodiment, R3 is C1-6 alkyl. In one embodiment, R3 is C1-4 alkyl. In one embodiment, R3 is C1-3 alkyl.

[0153] In one embodiment, R3 is methyl. In one embodiment, R3 is ethyl.

[0154] R4

[0155] In one embodiment, R1 is H, C1-6 alkyl, or C3-6 cycloalkyl. In one embodiment, R4 is H or Ci-6 alkyl. In another embodiment, R1 and R4 together with the carbon atoms to which they are attached form a 5- to 7-membered carbocyclic ring which is substituted by a group -NH-C(=O)-Rs. In the latter embodiment, the compounds of formulae (II) and (III) comprise payloads of formula (D-B1) as described in more detail below.

[0156] In one embodiment, either: R1 is H, C1-6 alkyl, or C3-6 cycloalkyl, and R4 is H or C1-6 alkyl, or: R1 and R4 together with the carbon atoms to which they are attached form a 5- to 7-membered carbocyclic ring which is substituted by a group -NH-C(=O)-Rs.

[0157] In one embodiment, R4 is H. In one embodiment, R4 is C1-4 alkyl. In one embodiment, R4 is C1-3 alkyl. In one embodiment, R4 is methyl. In one embodiment, R4 is ethyl.

[0158] R5

[0159] Rs is C1-6 alkyl. In one embodiment, Rs is C1-4 alkyl. In one embodiment, Rs is C1-3 alkyl.

[0160] In one embodiment, Rs is methyl. In one embodiment, Rs is ethyl.

[0161] R8

[0162] In one embodiment, Rs is H, halogen, C1-6 alkyl, O-C1-6 alkyl or S-C1-6 alkyl. In one embodiment, Rs is H, halogen or C1-4 alkyl. In one embodiment, Rs is H, halogen or C1-3 alkyl.

[0163] In one embodiment, Rs is H.

[0164] In one embodiment, Rs is halogen. In one embodiment, Rs is F. In one embodiment, Rs is Cl. In one embodiment, Rs is Br. In one embodiment, Rs is I.In one embodiment, Rs is C1-6 alkyl. In one embodiment, Rs is C1-5 alkyl. In one embodiment, Rs is C1-4 alkyl. In one embodiment, Rs is C1-3 alkyl. In one embodiment, Rs is C1-2 alkyl. In one embodiment, Rs is methyl. In one embodiment, Rs is ethyl.

[0165] In one embodiment, Rs is O-C1-6 alkyl. In one embodiment, Rs is O-C1-5 alkyl. In one embodiment, Rs is O-C1-4 alkyl. In one embodiment, Rs is O-C1-3 alkyl. In one embodiment, Rs is O-C1-2 alkyl. In one embodiment, Rs is O-methyl. In one embodiment, Rs is O-ethyl.

[0166] In one embodiment, Rs is S-C1-6 alkyl. In one embodiment, Rs is S-C1-5 alkyl. In one embodiment, Rs is S-C1-4 alkyl. In one embodiment, Rs is S-C1-3 alkyl. In one embodiment, Rs is S-C1-2 alkyl. In one embodiment, Rs is S-methyl. In one embodiment, Rs is S-ethyl.

[0167] In one embodiment, Rs and R2 together with the carbon atoms to which they are attached form a 5-membered ring system. In one embodiment, Rs and R2 together represent -(CH2)3- such that Rs and R2 together with the carbon atoms to which they are attached form a benzocyclopentane (indane) ring system. In one embodiment, Rs and R2 together represent -O-(CH2)-O- such that Rs and R2 together with the carbon atoms to which they are attached form a benzodioxole ring system.

[0168] R’ and R”

[0169] In one embodiment, Rs is NR’R”. In one embodiment, R’ is H or C1-6 alkyl. In one embodiment, R’ is H or C1-4 alkyl. In one embodiment, R’ is H or C1-3 alkyl. In one embodiment, R’ is H, methyl or ethyl. In one embodiment, R’ is H.

[0170] In one embodiment, R” is H or C1-6 alkyl. In one embodiment, R” is H or C1-4 alkyl. In one embodiment, R” is H or C1-3 alkyl. In one embodiment, R” is H, methyl or ethyl. In one embodiment, R” is H.

[0171] n and m

[0172] n is 0, 1, or 2. m is 0, 1 or 2. m+n must be 1, 2 or 3.

[0173] In one embodiment, n is 0. In one embodiment, n is 1. In one embodiment, n is 2.

[0174] In one embodiment, m is 0. In one embodiment, m is 1. In one embodiment, m is 2.

[0175] Lists 1A, 1B, 1C and 1D“List 1” substituents are selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14-membered (such as 5- or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3-to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR’, -N(R’)2, -S(0)O-2R’, -S(O)I-2OR’, -OS(O)I.2R’, -OS(O)I-2OR’, -S(O)I-2N(R’)2, -OS(O)I-2N(R’)2, -N(R’)S(O)I-2R’, -N(R’)S(O)I-2OR’, -C(=X1)R’, -C(=X1)X1R’, -X1C(=X1)R’, and -X1C(=X1)X1R’, wherein X1is independently selected from O, S, NH and N(CHs); and each R’ is independently selected from the group consisting of H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, 5-or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CHF2, CH2F, -CN, azido, -NO2, -OH, -O(Ci-3alkyl), -S(Ci-3alkyl), -NH2, -NH(CI-3alkyl), -N(Ci- 3 alkyl)2, -NHS(O)2(CI-3alkyl), -S(O)2NH2-Z(CI-3alkyl)z, -C(=O)OH, -C(=0)0(C1.3alkyl), -C(=O)NH2-z(Ci-3alkyl)z, -NHC(=O)(C1-3 alkyl), -NHC(=NH)NHz.2(C1-3 alkyl)z, and -N(CI-3alkyl)C(=NH)NH2-z(Ci-3alkyl)z, wherein each z is independently 0, 1, or 2.

[0176] In some embodiment, the substituents are selected from List 1A, consisting of C1-3 alkyl, phenyl, halogen, -CF3, - CHF2, CH2F, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl.

[0177] In some embodiments, the substituents are selected from List 1B, consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), -CF3 -CHF2, and -CH2F.

[0178] In some embodiments, the substituents are selected from List 1 C, consisting of halogen (such as F, Cl, or Br). In some embodiments, the substituents are selected from List 1D, consisting of F.

[0179] Linkers - Formula (L)

[0180] The linkers of the invention are linkers of formula (L):

[0181]

[0182] wherein R, EO, v, w, P, Re, and R7, have the meanings defined herein, either in the broadest aspect or any of the preferred aspects defined herein,and wherein position (1) is a first attachment point; and position (2) is a second attachment point.

[0183] c? In the linkers of formula (L) as defined above, the variables R, EO, v, w, P, Re, and R7, may take any of the meanings defined above for these substituents, either in the J broadest embodiments or any of the narrower embodiments defined herein. 5

[0184] ^ Q TZ

[0185] In one embodiment, the linker of formula (L) is a linker of formula L1 to L5, as shown in Z ZI r—

[0186] Table 1 below: y O / \= - '

[0187] I \1i ' ■ '

[0188] Cpds Formula T. Z. \- L1

[0189] IZ

[0190] 0 0

[0191] 0 0

[0192] L2 ° H 9 H 9 H

[0193] _ A _ 0 _ _ N A N A N (2) if j z 1 — i

[0194] drN0 / =

[0195] w

[0196] L3 T- p Q

[0197] 0 H 9 H 9 H

[0198] A O N N N (2)

[0199] a

[0200] L4

[0201] L5 ° H 9 H 9 H

[0202] _ A _ 0 _ _ N A N A N (2)

[0203] d

[0204]

[0205] Table 1wherein:

[0206] position (1) is a first attachment point; and

[0207] position (2) is a second attachment point.

[0208] Payload Moiety (D)

[0209] The compounds of formula (II) or (III) may comprise any suitable payload moiety. The payload moiety may be referred to herein as substituent D. The payload moiety D is linked through the U atom to the linker.

[0210] Suitable payloads are well-known in the art (see e.g. Conilh, L. et al. 2023. Journal of Hematology & Oncology, 16(1), p.3). Suitable examples of payloads are also described herein and are also generally described in WO2022 / 068878, WO2022 / 170971 and W02004 / 010957, and national applications deriving therefrom.

[0211] In one embodiment, the payload is a cytotoxic drug. In one embodiment, the payload is an immunomodulator. In one embodiment, the cytotoxic drug is a tubulin polymerisation inhibitor, a DNA damaging agent, a TOPO1 inhibitor, an auristatin, a maytansinoid, a maytansine, or a calicheamicin. In one embodiment, the cytotoxic drug is a TOPO1 inhibitor. In one embodiment, the TOPO1 inhibitor is a camptothecin or an exatecan, or a derivative thereof.

[0212] In one embodiment, the payload is a topoisomerase inhibitor, preferably a topoisomerase 1 (TOPO1) inhibitor.

[0213] In one embodiment, the payload is exatecan, or a derivative thereof. In one embodiment, the payload is exatecan.

[0214] In one embodiment, the payload is an auristatin.

[0215] In one embodiment, D may comprise a structure shown as formula (D-A):

[0216]

[0217] or a pharmaceutically acceptable salt or solvate thereof,

[0218] wherein A, X, T, R2, R3, and Rs have the meanings defined herein, either in the broadest aspect or any of the preferred aspects defined herein.

[0219] In one embodiment, D may comprise a structure shown as formula (D-B):

[0220]

[0221] wherein A, X, Ri, R2, R3, R4 and Rs have the meanings defined herein, either in the broadest aspect or any of the preferred aspects defined herein.

[0222] In the embodiment when D comprises a structure of formula D-B, R1 and R4 together with the carbon atoms to which they are attached form a 5- to 7-membered carbocyclic ring which is substituted by a group -NH-C(=O)-Rs, the compounds are of formula (D-B1):

[0223]

[0224] wherein:

[0225] X, A, R2, R3, and Rs have the meanings defined herein, either in the broadest aspect or any of the preferred aspects recited herein,

[0226] n is 0, 1, 2; and m is 0, 1 or 2, with the proviso that m+n must be 1, 2 or 3.

[0227] In one embodiment, m is 0. In one embodiment, n is 1. In one embodiment, n is 2.

[0228] In one embodiment, n is 0. In one embodiment, n is 1. In one embodiment, n is 2.

[0229] In the payload compounds of formula (D-A) and formula (D-B) as defined above, including those of formula (DB-1), the variables X, A, T, R1, R2, R3, R4, Rs, and Rs may take any of the meanings defined above for these substituents, either in the broadest embodiments or any of the narrower embodiments defined herein.

[0230] In one embodiment, D is selected from a group of formula D-1 through D-17, as shown in Table 2 below, or a pharmaceutically acceptable salt or solvate thereof:

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] Linker-Payload Conjugates – Formula (II)

[0237] In one aspect of the invention, the linker-payload conjugates are compounds of formula (II):

[0238] II H

[0239] (CH2)W - C - P - N U - D

[0240]

[0241] (II)

[0242] or a pharmaceutically acceptable salt or solvate thereof,

[0243] wherein:

[0244] R, EO, v, w, P, R6, R7, Y1, U, and D are as defined herein, either in its broadest aspect or any of the preferred aspects recited herein.

[0245] In the linker-payload compounds of formula (II) as defined above, the variables R, EO, v, w, P, R6, R7, Y1, D, X, U, A, T, R1, R2, R3, R4, R5, and R8may take any of the meanings defined above for these substituents, either in the broadest embodiment or any of the narrower embodiments defined herein.

[0246] In the linker-payload conjugates of formula (II) of the present invention, the payload D may take any of the meanings as defined herein, either in the broadest embodiment or any of the narrower embodiments defined herein.

[0247] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-A) or formula (D-B) as defined herein, either in the broadest embodiment or any of the narrower embodiments defined herein.

[0248] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the linker is the linker of formula (L1) as defined herein.

[0249] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-A) as defined herein and the linker is the linker of formula (L1) as defined herein.In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-B), including those of formula (D-B1) as defined herein and the linker is the linker of formula (L1) as defined herein.

[0250] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the linker is the linker of formula (L2) as defined herein.

[0251] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-A) as defined herein and the linker is the linker of formula (L2) as defined herein.

[0252] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-B), including those of formula (D-B1) as defined herein and the linker is the linker of formula (L2) as defined herein.

[0253] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the linker is the linker of formula (L3) as defined herein.

[0254] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-A) as defined herein and the linker is the linker of formula (L3) as defined herein.

[0255] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-B), including those of formula (D-B1) as defined herein and the linker is the linker of formula (L3) as defined herein.

[0256] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the linker is the linker of formula (L4) as defined herein.

[0257] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-A) as defined herein and the linker is the linker of formula (L4) as defined herein.

[0258] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-B), including those of formula (D-B1) as defined herein and the linker is the linker of formula (L4) as defined herein.In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the linker is the linker of formula (L5) as defined herein.

[0259] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-A) as defined herein and the linker is the linker of formula (L5) as defined herein.

[0260] In one embodiment of the linker-payload conjugates of formula (II) of the present invention, the payload D is of the formula (D-B), including those of formula (D-B1) as defined herein and the linker is the linker of formula (L5) as defined herein.

[0261] In one embodiment, the compound of formula (II) is a compound of formula 11-1 through II- 20, as shown in Table 3 below, or a pharmaceutically acceptable salt or solvate thereof:

[0262]

[0263]

[0264]

[0265]

[0266] Table 3

[0267] Binding Molecule (BM)

[0268] The binding molecule-linker-payload compounds of the present invention comprises a binding molecule or fragment thereof that specifically binds to a target, typically a target expressed on the surface of a tumour cell.

[0269] In some embodiments, the binding molecule is not a polysaccharide.

[0270] In some embodiments the binding molecule may be an antibody or a fragment thereof. In some embodiments the antibody fragment is an antigen-binding fragment.

[0271] When the binding molecule is an antibody or a fragment thereof, the binding molecule-linker-payload compound may also be referred to as an “antibody-drug conjugate” (ADC). It will beunderstood by the skilled person that the term “antibody-drug conjugate” encompasses both specific antibody-drug conjugates of formula (III) wherein the binding molecule is an antibody, and having a DAR (as defined below) which is integral, and compositions (as defined below) comprising antibody-drug conjugates of formula (III’) wherein the binding molecule is an antibody, having different DARs, resulting in an average DAR (as defined below) which can be non-integral.

[0272] Targets which are expressed on the surface of a tumour cell may be identified by means of various molecular biological and immunological methods known in the art (see e.g. Carter, P., et al., 2004. Endocrine-related cancer, 11(4), pp.659-687). A target may be referred to as an antigen. An ideal expression profile is abundant and homogeneous antigen expression on the external surface of all tumour cells for multiple tumour types with the majority of patients for each tumour type, and absent from normal tissue. In some embodiments, the target is expressed on the surface of solid tumour cells.

[0273] In some embodiments, the target is expressed with higher expression on the surface of tumour cells as compared to healthy cells. In some embodiments, the target is expressed at a level sufficient to distinguish a tumour cell from healthy cells. Evaluation of antigen expression on the surface may be determined by any suitable method, for example by immunohistochemistry e.g. tissue microarray immunohistochemistry (see e.g. Hoos, A. and Cordon-Cardo, C., 2001. Laboratory investigation, 81(10), pp.1331-1338).

[0274] Example targets may include B7H3, TROP2, HER2, HER3, ALK receptor (pleiotrophin receptor); ovarian carcinoma antigen (CA125); prostate-specific antigen (PSA); melanoma-associated antigen p97; melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen; CD19; human B-lymphoma antigen-CD20; CD33; melanoma-specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3; epidermal growth factor receptor (EGFR); NY-BR-16; NY-BR-16; polymorphic epithelial mucin (PEM); malignant human lymphocyte antigen-APO-1; SSEA-1; SCP-1; SSEA-3; SSEA-4; sialyl Tn (STn); colon cancer antigen NY-CO-45; lung cancer antigen NY-LU-12 variant A; adenocarcinoma antigen ART1; Neuro-oncological ventral antigen 2 (NOVA2); tumour-associated antigens MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1); YKL-40; EpCAM; EREG; CA15-3; sialyl SSEA-1 (SLX); CLDN6; CLDN18.2 and Mesothelin. In some embodiments, the target is selected from B7H3, TROP2, HER2, and HER3.In some embodiments, the target expressed on the surface of a tumour cell is HER2, B7H3 or TROP2.

[0275] Wherein the binding molecule or fragment thereof is an antibody or fragment thereof that specifically binds to a target expressed on the surface of a tumour cell, this may be selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, a polyclonal antibody, a bispecific antibody, and fragments thereof, such as antigen-binding fragments thereof. In this respect, it will be understood by the skilled person that the term “antibody-drug conjugate” applies irrespective of whether the binding moiety is an antibody or a fragment thereof.

[0276] In some embodiments, the antibody or fragment thereof that specifically binds to a target expressed on the surface of a tumour cell is a full-length antibody, such as a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a bispecific antibody, a monoclonal antibody, or a polyclonal antibody. In some embodiments, the antibody or fragment thereof that specifically binds to a target expressed on the surface of a tumour cell is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody or fragment thereof that specifically binds to a target expressed on the surface of a tumour cell is a monoclonal antibody, for example a monoclonal human antibody, a monoclonal humanized antibody, or a monoclonal chimeric antibody.

[0277] In other embodiments, the antibody or fragment thereof that specifically binds to a target expressed on the surface of a tumour cell is an antibody fragment, such as a Fab, a Fab', a F(ab')2, an Fv, an scFv, a Fab'-SH, an sdAb, or a VHH.

[0278] In some embodiments the antibody or antigen-binding thereof is a VHH.

[0279] In some embodiments, the antibody or fragment thereof comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is a human IgG constant region, for example a human lgG1, lgG2, lgG3 or lgG4 constant region. In some embodiments, the immunoglobulin constant region is a human lgG1 constant region.

[0280] In some embodiments, the antibody or fragment thereof comprises an Fc region. In some embodiments, the Fc region is a human Fc region, for example a human lgG1, lgG2, lgG3 or lgG4 Fc region. In some embodiments, the antibody or fragment thereof comprises a human lgG1 Fc region.

[0281] In some embodiments the antibody or antigen-binding fragment thereof comprises a VHH coupled to an Fc region. In this embodiment the Fc region may be as defined above.In some embodiments, the antibody or fragment thereof comprises an Fc region comprising one or more mutations which reduce or abolish immune effect functions. Suitable mutations are known in the art and may include one or more amino acid substitutions (see e.g.

[0282] Wilkinson, I., et al., 2021. FcPLoS One, 16(12), p.e0260954; and Liu, R., et al., 2020.

[0283] Antibodies, 9(4), p.64). In some embodiments, the antibody or fragment thereof comprises an lgG1 Fc region comprising a L234A and / or a L235A mutation. In some embodiments, the antibody or fragment thereof comprises an I gG 1 Fc region comprising L234A and L235A mutations. Amino acids are numbered according to the Eu numbering (Eu-index) as set forth in Kabat (Kabat, E. A.; National Institutes of Health (U. S.) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991).

[0284] In some embodiments the Fc region is silenced to reduce, negate or abolish one or more Fc receptor binding and / or functionalities. In some embodiments, the Fc region of the binding molecule is modified to negate one or more Fc receptor functionalities. In some embodiments, the Fc region of the binding molecule is silenced in respect of one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16) and C1q functionality.

[0285] Thus, in some embodiments, the Fc region of as defined herein is a modified Fc region. Suitable silencing mutations are well known in the art. For example, the Fc region of the binding molecule may comprise a silencing modification selected from the STR mutation, the LALA mutation and the delta A mutation.

[0286] Anti-B7H3 antibodies and fragments thereof

[0287] In some embodiments, the target expressed on the surface of a tumour cell is B7H3. B7H3 is a protein that in humans is encoded by the CD276 gene and which may also be known as Cluster of Differentiation 276 (CD276) and B7 Homolog 3 (B7-H3).

[0288] In some embodiments, the antibody-drug conjugate comprises an anti-B7H3 antibody or fragment thereof.

[0289] In some embodiments the antibody-drug conjugate comprises an anti-B7H3 antibody or a fragment thereof that binds, such as specifically binds, B7H3.

[0290] The anti-B7H3 antibody or fragment thereof may specifically bind B7H3. The anti-B7H3 antibody may be a monoclonal antibody. In some embodiments, the anti-B7H3 antibody or fragment thereof is a humanized antibody. In some embodiments, the anti-B7H3 antibody or fragment thereof is a monoclonal humanized antibody. The anti-B7H3 antibody or fragmentthereof may be a bispecific antibody. The fragment thereof may be any antigen-binding fragment thereof, for example a Fab, a Fab', a F(ab')2, a Fv, a scFv, a Fab'-SH, an sdAb, or a VHH.

[0291] In some embodiments the antibody or antigen-binding fragment thereof comprises a VHH coupled to an Fc region.

[0292] In some embodiments, the anti-B7H3 antibody or fragment thereof is a full-length anti-B7H3 antibody.

[0293] The anti-B7H3 antibody or fragment thereof may comprise a variable region that specifically binds B7H3. In some embodiments, the anti-B7H3 antibody or fragment thereof comprises a heavy chain variable region and / or a light chain variable domain. In some embodiments, the anti-B7H3 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable domain. In some embodiments, the anti-B7H3 antibody or fragment thereof comprises a constant region, preferably derived from a human antibody, preferably the constant region is selected from the constant region of human lgG1, lgG2, lgG3 or lgG4. In some embodiments, the anti-B7H3 antibody or fragment thereof comprises a heavy chain and / or a light chain. In some embodiments, the anti-B7H3 antibody or fragment thereof comprises a heavy chain and a light chain. In some embodiments, the anti-B7H3 antibody or fragment thereof comprises or consists of two heavy chains and two light chains.

[0294] Anti-TROP2 antibodies and fragments thereof

[0295] In some embodiments, the target expressed on the surface of a tumour cell is TROP2.

[0296] TROP2 is a protein that in humans is encoded by the TACSTD2 and which may also be known as Tumour-associated calcium signal transducer 2, epithelial glycoprotein-1 antigen (EGP-1), or Trop-2.

[0297] In some embodiments, the antibody-drug conjugate comprises an anti-TROP2 antibody or fragment thereof.

[0298] In some embodiments the antibody-drug conjugate comprises an anti-TROP2 antibody or a fragment thereof that binds, such as specifically binds, TROP2.

[0299] The anti-TROP2 antibody or fragment thereof may specifically bind TROP2. The anti-TROP2 antibody may be a monoclonal antibody. In some embodiments, the anti-TROP2 antibody or fragment thereof is a humanized antibody. In some embodiments, the anti-TROP2 antibody or fragment thereof is a monoclonal humanized antibody. The anti-TROP2 antibody or fragment thereof may be a bispecific antibody. The fragment thereof may be any antigen-binding fragment thereof, for example a Fab, a Fab', a F(ab')2, a Fv, a scFv, a Fab'-SH, ansdAb, or a VHH. In some embodiments, the anti-TROP2 antibody or fragment thereof is a full-length anti-TROP2 antibody.

[0300] In some embodiments the antibody or antigen-binding fragment thereof comprises a VHH coupled to an Fc region.

[0301] The anti-TROP2 antibody or fragment thereof may comprise a variable region that specifically binds TROP2. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises a heavy chain variable region and / or a light chain variable domain. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable domain. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises a constant region, preferably derived from a human antibody, preferably the constant region is selected from the constant region of human lgG1, I gG2, lgG3 or lgG4. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises a heavy chain and / or a light chain. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises a heavy chain and a light chain. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises or consists of two heavy chains and two light chains.

[0302] Example anti-TROP2 antibody sequences

[0303] Example anti-TROP2 antibody CDRs, variable region sequences, and heavy and light chain sequences are provided below.

[0304] Kabat HCDR1 NYGMN (SEQ ID NO: 1)

[0305] Kabat HCDR2 WINTYTGEPTYTDDFKG (SEQ ID NO: 2)

[0306] Kabat HCDR3 GGFGSSYWYFDV (SEQ ID NO: 3)

[0307] Kabat LCDR1 KASQDVSIAVA (SEQ ID NO: 4)

[0308] Kabat LCDR2 SASYRYT (SEQ ID NO: 5)

[0309] Kabat LCDR3 QQHYITPLT (SEQ ID NO: 6)

[0310] IMGT HCDR1 GYTFTNYG (SEQ ID NO: 7)

[0311] IMGT HCDR2 INTYTGEP (SEQ ID NO: 8)

[0312] IMGT HCDR3 ARGGFGSSYWYFDV (SEQ ID NO: 9)

[0313] IMGT LCDR1 QDVSIA (SEQ ID NO: 10)

[0314] IMGT LCDR2 SAS (SEQ ID NO: 11)

[0315] IMGT LCDR3 QQHYITPLT (SEQ ID NO: 12)

[0316] VH QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 13)

[0317]

[0318] VL DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSA SYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGT KVEIK (SEQ ID NO: 14)

[0319] Heavy chain QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 15)

[0320] Light chain DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSA SYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 16)

[0321]

[0322] In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the Kabat numbering system.

[0323] In some embodiments, a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the Kabat numbering system.In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or a variant thereof having up to three amino acid substitutions, additions or deletions; and a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the Kabat numbering system.

[0324] In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the IMGT numbering system.

[0325] In some embodiments, a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 10, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 11; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the IMGT numbering system.

[0326] In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, or a variant thereof having up to three amino acid substitutions, additions ordeletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9, or a variant thereof having up to three amino acid substitutions, additions or deletions; and a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 10, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 11, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the IMGT numbering system.

[0327] In some embodiments, the CDR variants have up to two amino acid substitutions, additions or deletions. In some embodiments, the CDR variants have up to one amino acid substitution, addition or deletion. In some embodiments, the CDR variants have up to three amino acid substitutions. In some embodiments, the CDR variants have up to two amino acid substitutions. In some embodiments, the CDR variants have up to one amino acid substitution.

[0328] In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0329] In some embodiments, a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0330] In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof; and a light chain variable regionof the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6.

[0331] In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; and a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 10; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 11; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 12.In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 13; and a light chain variable region of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, a heavy chain variable region of the anti-TROP2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 13; and a light chain variable region of the anti-TROP2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 14.

[0332] In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0333] In some embodiments, a light chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a light chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0334] In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof; and a light chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof.

[0335] In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable regioncomprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and a light chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6.

[0336] In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; and a light chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 10; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 11; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 12.

[0337] In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 15; and a light chain of the anti-TROP2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, a heavy chain of the anti-TROP2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 15; and a light chain of the anti-TROP2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 16.In some embodiments, the anti-TROP2 antibody or fragment thereof comprises or consists of two heavy chains comprising or consisting of the amino acid sequence of SEQ ID NO: 15; and two light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-TROP2 antibody or fragment thereof comprises or consists of two heavy chains consisting of the amino acid sequence of SEQ ID NO: 15; and two light chains consisting of the amino acid sequence of SEQ ID NO: 16.

[0338] In some embodiments, the anti-TROP2 antibody or fragment thereof is sacituzumab or a fragment thereof. In some embodiments, the anti-TROP2 antibody or fragment thereof is sacituzumab.

[0339] Anti-HER2 antibodies and fragments thereof

[0340] In some embodiments, the target expressed on the surface of a tumour cell is HER2. HER2 is a protein that in humans is encoded by the ERBB2 gene and which may also be known as human epidermal growth factor receptor 2 (HER-2), Receptor tyrosine-protein kinase erbB-2 and CD340 (Cluster of Differentiation 340).

[0341] In some embodiments, the antibody-drug conjugate comprises an anti-HER2 antibody or fragment thereof.

[0342] In some embodiments the antibody-drug conjugate comprises an anti-HER2 antibody or a fragment thereof that binds, such as specifically binds, HER2.

[0343] The anti-HER2 antibody or fragment thereof may specifically bind HER2. The anti-HER2 antibody may be a monoclonal antibody. In some embodiments, the anti-HER2 antibody or fragment thereof is a humanized antibody. In some embodiments, the anti-HER2 antibody or fragment thereof is a monoclonal humanized antibody. The anti-HER2 antibody or fragment thereof may be a bispecific antibody. The fragment thereof may be any antigen-binding fragment thereof, for example a Fab, a Fab', a F(ab')2, a Fv, an ScFv, a Fab'-SH, an sdAb, or a VHH. In some embodiments, the anti-HER2 antibody or fragment thereof is a full-length anti-HER2 antibody.

[0344] In some embodiments the antibody or antigen-binding fragment thereof comprises a VHH coupled to an Fc region.

[0345] The anti-HER2 antibody or fragment thereof may comprise a variable region that specifically binds HER2. In some embodiments, the anti-HER2 antibody or fragment thereof comprises a heavy chain variable region and / or a light chain variable domain. In some embodiments, the anti-HER2 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable domain. In some embodiments, the anti-HER2 antibody or fragmentthereof comprises a constant region, preferably derived from a human antibody, preferably the constant region is selected from the constant region of human lgG1, lgG2, lgG3 or lgG4. In some embodiments, the anti-HER2 antibody or fragment thereof comprises a heavy chain and / or a light chain. In some embodiments, the anti-HER2 antibody or fragment thereof comprises a heavy chain and a light chain. In some embodiments, the anti-HER2 antibody or fragment thereof comprises or consists of two heavy chains and two light chains.

[0346] Example anti-HER2 antibody CDRs, variable region sequences, and heavy and light chain sequences are provided below.

[0347] Example anti-HER2 antibody sequences - Trastuzumab

[0348] Kabat HCDR1 DTYIH (SEQ ID NO: 17)

[0349] Kabat HCDR2 RIYPTNGYTRYADSVKG (SEQ ID NO: 18)

[0350] Kabat HCDR3 WGGDGFYAMDY (SEQ ID NO: 19)

[0351] Kabat LCDR1 RASQDVNTAVA (SEQ ID NO: 20)

[0352] Kabat LCDR2 SASFLYS (SEQ ID NO: 21)

[0353] Kabat LCDR3 QQHYTTPPT (SEQ ID NO: 22)

[0354] IMGT HCDR1 GFNIKDTY (SEQ ID NO: 23)

[0355] IMGT HCDR2 IYPTNGYT (SEQ ID NO: 24)

[0356] IMGT HCDR3 SRWGGDGFYAMDY (SEQ ID NO: 25)

[0357] IMGT LCDR1 QDVNTA (SEQ ID NO: 26)

[0358] IMGT LCDR2 SAS (SEQ ID NO: 27)

[0359] IMGT LCDR3 QQHYTTPPT (SEQ ID NO: 28)

[0360] VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 29)

[0361] VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK (SEQ ID NO: 30)

[0362] Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 31)

[0363] Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

[0364]

[0365] SSPVTKSFNRGEC (SEQ ID NO: 32)

[0366] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 17, or a variant thereof having up to three amino acid substitutions, additions ordeletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 19, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the Kabat numbering system.

[0367] In some embodiments, a light chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the Kabat numbering system.

[0368] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 17, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 19, or a variant thereof having up to three amino acid substitutions, additions or deletions; and a light chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the Kabat numbering system.

[0369] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25, ora variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the IMGT numbering system.

[0370] In some embodiments, a light chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the IMGT numbering system.

[0371] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25, or a variant thereof having up to three amino acid substitutions, additions or deletions; and a light chain variable region of the anti-HER2 antibody or fragment thereof comprises: (i) a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, or a variant thereof having up to three amino acid substitutions, additions or deletions; (ii) a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 27, or a variant thereof having up to three amino acid substitutions, additions or deletions; and (iii) a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the CDRs are defined according to the IMGT numbering system.

[0372] In some embodiments, the CDR variants have up to two amino acid substitutions, additions or deletions. In some embodiments, the CDR variants have up to one amino acid substitution, addition or deletion. In some embodiments, the CDR variants have up to three amino acid substitutions. In some embodiments, the CDR variants have up to two amino acid substitutions. In some embodiments, the CDR variants have up to one amino acid substitution.

[0373] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a heavychain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0374] In some embodiments, a light chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a light chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0375] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof; and a light chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 17; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 19; and a light chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22.In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; and a light chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 26; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28.

[0376] In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 29; and a light chain variable region of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 30. In some embodiments, a heavy chain variable region of the anti-HER2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 29; and a light chain variable region of the anti-HER2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 30.

[0377] In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 31.

[0378] In some embodiments, a light chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof. In some embodiments, a light chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0379] In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof; and a light chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof.

[0380] In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 17; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 19; and a light chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22.

[0381] In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the heavy chain variable region comprises: a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24; and a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; and a lightchain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions and / or substitutions in a framework region thereof, wherein the light chain variable region comprises: a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 26; a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 28.

[0382] In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 31; and a light chain of the anti-HER2 antibody or fragment thereof comprises or consists of the amino acid sequence of SEQ ID NO: 32. In some embodiments, a heavy chain of the anti-HER2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 31; and a light chain of the anti-HER2 antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 32.

[0383] In some embodiments, the anti-HER2 antibody or fragment thereof comprises or consists of two heavy chains comprising or consisting of the amino acid sequence of SEQ ID NO: 31; and two light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-HER2 antibody or fragment thereof comprises or consists of two heavy chains consisting of the amino acid sequence of SEQ ID NO: 31; and two light chains consisting of the amino acid sequence of SEQ ID NO: 32.

[0384] In some embodiments, the anti-HER2 antibody or fragment thereof is trastuzumab or a fragment thereof. In some embodiments, the anti-HER2 antibody is trastuzumab.

[0385] Anti-HER3 antibodies and fragments thereof

[0386] In some embodiments, the target expressed on the surface of a tumour cell is HER3. HER3 is a protein that in humans is encoded by the ERBB3 gene and which may also be known as human epidermal growth factor receptor 3 (HER-3) and Receptor tyrosine-protein kinase erbB-3.

[0387] In some embodiments, the antibody-drug conjugate comprises an anti-HER3 antibody or fragment thereof.

[0388] In some embodiments the antibody-drug conjugate comprises an anti-HER3 antibody or a fragment thereof that binds, such as specifically binds, HER3.The anti-HER3 antibody or fragment thereof may specifically bind HER3. The anti-HER3 antibody or fragment thereof may block HER3 ligand-dependent and HER3 ligandindependent signalling. The anti-HER3 antibody or fragment thereof may inhibit HER3 ligand-dependent and Her3 ligand-independent AKT phosphorylation.

[0389] The anti-HER3 antibody may be a monoclonal antibody. In some embodiments, the anti-HER3 antibody or fragment thereof is a humanized antibody. In some embodiments, the anti-HER3 antibody or fragment thereof is a monoclonal humanized antibody. In some embodiments, the anti-HER3 antibody or fragment thereof is a human antibody. In some embodiments, the anti-HER3 antibody or fragment thereof is a monoclonal human antibody. The anti-HER3 antibody or fragment thereof may be a bispecific antibody. The fragment thereof may be any antigen-binding fragment thereof, for example a Fab, a Fab', a F(ab')2, a Fv, a scFv, a Fab'-SH, an sdAb, or a VHH. In some embodiments, the anti-HER3 antibody or fragment thereof is a full-length anti-HER3 antibody.

[0390] In some embodiments the antibody or antigen-binding fragment thereof comprises a VHH coupled to an Fc region.

[0391] The anti-HER3 antibody or fragment thereof may comprise a variable region that specifically binds HER3. In some embodiments, the anti-HER3 antibody or fragment thereof comprises a heavy chain variable region and / or a light chain variable domain. In some embodiments, the anti-HER3 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable domain. In some embodiments, the anti-HER3 antibody or fragment thereof comprises a constant region, preferably derived from a human antibody, preferably the constant region is selected from the constant region of human lgG1, lgG2, lgG3 or lgG4. In some embodiments, the anti-HER3 antibody or fragment thereof comprises a heavy chain and / or a light chain. In some embodiments, the anti-HER3 antibody or fragment thereof comprises a heavy chain and a light chain. In some embodiments, the anti-HER3 antibody or fragment thereof comprises or consists of two heavy chains and two light chains.

[0392] Binding Molecule-Linker-Payload Conjugates – Formula (III)

[0393] The binding molecule - linker-payload conjugates of the invention are compounds of formula (III):

[0394]

[0395] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0396] BM is a binding molecule or a fragment thereof as defined herein, either in its broadest aspect or any of the preferred aspects recited herein;

[0397] R, EO, v, w, R6, R7, U, and D are as defined herein, either in its broadest aspect or any of the preferred aspects recited herein; andq is an integer from 1 to 16.

[0398] In compounds of formula (III) the binding molecule-drug ratio q is an integer. The ratio q is the number of linker-payload molecules attached to each binding molecule antibody or fragment thereof. As indicated above binding molecule-drug ratio q of the binding molecule-linker-payload conjugates used in the invention may vary. When the binding molecule is an antibody or a fragment thereof, the value q may also be termed a “drug-antibody ratio” (DAR).

[0399] In some embodiments, q is an integer from 1 to 16. In some embodiments, q is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, q is an integer from 4 to 12. In some embodiments, q is an integer from 4 to 12. In some embodiments, q is an integer from 6 to 10. In some embodiments, q is an integer from 5 to 9. In some embodiments, q is an integer from 4 to 8. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6. In some embodiments, q is 7. In some embodiments, q is 8.

[0400] In one embodiment, the compound of formula (III) is a compound of formula III-1 through III-20, as shown in Table 4 below, or a pharmaceutically acceptable salt or solvate thereof:

[0401]

[0402] -2

[0403] 5

[0404] -3

[0405] ?

[0406] ^

[0407] <

[0408] -4

[0409] 4

[0410] £ £ &^

[0411] -5

[0412] Q

[0413] Q

[0414] -6

[0415] /

[0416] o

[0417] OA WX~A

[0418] K

[0419] -7

[0420] _ _ _ _ _ _ _

[0421] -8

[0422] y ^ Y^NH^ANX^^O y IXI <HO\5> -9

[0423] ^ ^ ^N--Y Y^NHJI^0T W

[0424] H°\5

[0425]

[0426] -10 s'

[0427] b y

[0428] X. Ji I 6 O XxNHXN H-XO^° Y i TW b " XH°\5 -11

[0429] J X

[0430] -12

[0431] x XX xx Q ^ X X

[0432] -13

[0433] M XX XX

[0434] 0FAANXXHO\5 -14

[0435] Xi nr V^^^YT'W’

[0436] H°\5 -15

[0437] X / X^ Yb

[0438] X. JI 1 6 o \HY i /

[0439] b

[0440] X5 -16

[0441] X X^

[0442] -17 ^ JL ^ O ^ ^ N JL -^ N JL II |

[0443] x / xo^ ^H'brNHbN^>^svvX. ° X JI 1 6 ° X Y 1 TW b

[0444] H° X5

[0445]

[0446] 111-18

[0447] 0

[0448] 111-19

[0449] I i I

[0450] H H

[0451] HI-20

[0452] ^ ^

[0453]

[0454] Table ^ ^ 4

[0455] o'

[0456] Compositions comprising Binding Molecule-Drug Conjugates - Formula dll’)

[0457] Y c

[0458] In contrast to the compounds of formula (III) above, in the compositions of the present Q

[0459] _ _

[0460] invention of formula (III’) which may contain multiple compounds of formula (III) with different values of the binding molecule-drug ratio g’, the average binding molecule-drug ratio g’ may be non-integral. When the binding molecule is an antibody or a fragment thereof, the value 0

[0461] g’ may also be termed an average antibody-drug ratio (DAR).

[0462] Therefore, there is also provided a composition comprising a compound of formula (III’):

[0463] I I I I _ _ _ _ _ _

[0464] II H

[0465] (CH2)W - C - P - N U - D

[0466] R7

[0467]

[0468] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0469] BM is a binding molecule or a fragment thereof as defined herein, either in its broadest aspect or any of the preferred aspects recited herein;

[0470] R, EO, v, w, Re, R7, U, and D are as defined herein, either in its broadest aspect or any of the preferred aspects recited herein; and

[0471] g’ is an integer or a decimal from 1 to 16.In some embodiments, q’ is an integer or decimal from about 1 to about 16. In some embodiments, q’ is an integer or decimal of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15 or about 16. In some embodiments, q’ is an integer or decimal from about 4 to about 12. In some embodiments, q’ is an integer or decimal from about 4 to about 8.

[0472] In some embodiments, q’ is about 4.0. In some embodiments, q’ is about 4.1. In some embodiments, q’ is about 4.2. In some embodiments, q’ is about 4.3. In some embodiments, q’ is about 4.4. In some embodiments, q’ is about 4.5. In some embodiments, q’ is about 4.6. In some embodiments, q’ is about 4.7. In some embodiments, q’ is about 4.8. In some embodiments, q’ is about 4.9. In some embodiments, q’ is about 5.0. In some embodiments, q’ is about 5.1. In some embodiments, q’ is about 5.2. In some embodiments, q’ is about 5.3. In some embodiments, q’ is about 5.4. In some embodiments, q’ is about 5.5. In some embodiments, q’ is about 5.6. In some embodiments, q’ is about 5.7. In some embodiments, q’ is about 5.8. In some embodiments, q’ is about 5.9. In some embodiments, q’ is about 6.0. In some embodiments, q’ is about 6.1. In some embodiments, q’ is about 6.2. In some embodiments, q’ is about 6.3. In some embodiments, q’ is about 6.4. In some embodiments, q’ is about 6.5. In some embodiments, q’ is about 6.6. In some embodiments, q’ is about 6.7. In some embodiments, q’ is about 6.8. In some embodiments, q’ is about 6.9. In some embodiments, q’ is about 7.0. In some embodiments, q’ is about 7.1. In some embodiments, q’ is about 7.2. In some embodiments, q’ is about 7.3. In some embodiments, q’ is about 7.4. In some embodiments, q’ is about 7.5. In some embodiments, q’ is about 7.6. In some embodiments, q’ is about 7.7. In some embodiments, q’ is about 7.8. In some embodiments, q’ is about 7.9. In some embodiments, q’ is about 8.0.

[0473] Payload-Products of Enzymatic Hydrolysis

[0474] A further aspect of the invention relates to payload-products of the enzymatic cleavage of the linker-payloads described herein. In some embodiments, the enzymatic cleavage is by enzymatic hydrolysis.

[0475] In some embodiments, the linker group in the linker-payload has a single cleavage site for enzymatic hydrolysis. The cleavage site of the linker-payload depends on the enzyme employed for hydrolysis.In some embodiments, the linkergroup in the linker-payload has more than one cleavage site for enzymatic hydrolysis. Where there is more than one cleavage site, enzymatic hydrolysis of the linker-payload lead may lead to a mixture of payload-products.

[0476] In some embodiments, the linker group in the linker-payload has two cleavage sites for enzymatic hydrolysis, thus two different payload products are generated.

[0477] In some embodiments, the linker group in the linker-payload has two or more cleavage sites for enzymatic hydrolysis, namely a primary cleavage site, and one or more secondary cleavage site, and cleavage preferentially takes place at the primary cleavage site over the one or more secondary cleavage sites, thereby giving rise to a major payload-product and one or more minor payload-products.

[0478] In some embodiments, the linker group in the linker-payload has two cleavage sites for enzymatic hydrolysis, namely a primary cleavage site, and a secondary cleavage site, and cleavage preferentially takes place at the primary cleavage site over the secondary cleavage site, thereby giving rise to a major payload-product and a minor payload-product.

[0479] In some embodiments, the linker-payload is susceptible to enzymatic hydrolysis with a lysosomal enzyme such as a Cathepsin. In some embodiments, the lysosomal enzyme is Cathepsin B. In some embodiments, the lysosomal enzyme is Cathepsin L.

[0480] In some embodiments, the linker is a group selected from L1, L2, L3, L4 and L5 as described herein.

[0481] In some embodiments, the linkergroup is of formula L1, and the linker-payload has two cleavage sites susceptible to enzymatic hydrolysis with a lysosomal enzyme such as a Cathepsin, thereby generating two different payload-products.

[0482] In some embodiments, the linkergroup is of formula L2, and the linker-payload has one cleavage site susceptible to enzymatic hydrolysis with a lysosomal enzyme such as a Cathepsin, thereby generating a single payload-product.

[0483] In some embodiments, the linkergroup is of formula L3, and the linker-payload has a primary cleavage site and a secondary cleavage site, each of which is susceptible to enzymatic hydrolysis with a lysosomal enzyme such as a Cathepsin, thereby a generating a major payload-product and a minor payload-product.

[0484] In some embodiments, the linkergroup is of formula L4, and the linker-payload has a primary cleavage site and a secondary cleavage site, each of which is susceptible toenzymatic hydrolysis with a lysosomal enzyme such as a Cathepsin, thereby a generating a major payload-product and a minor payload-product.

[0485] In some embodiments, the linkergroup is of formula L5, and the linker-payload has a primary cleavage site and a secondary cleavage site, each of which is susceptible to enzymatic hydrolysis with a lysosomal enzyme such as a Cathepsin, thereby a generating a major payload-product and a minor payload-product.

[0486] In some embodiments, the payload-product of the enzymatic hydrolysis of the linker-payload is a compound selected from 1-1 to 1-17 as shown in Table 2’ below, and pharmaceutically acceptable salts thereof.

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493] In some embodiments, the payload-product formed on cleavage of the linker-payload retains a portion of the linker group. For example, in some embodiments, the linker-payload is cleaved such that a glycine moiety remains attached to the payload in the payload-product. In one embodiment, the payload-product of the enzymatic hydrolysis of the linker-payload is a selected from compounds l-T to 1-17’ shown in Table 2” below, and pharmaceutically acceptable salts thereof:

[0494]

[0495]

[0496]

[0497]

[0498]

[0499] General Methods

[0500] The compounds of the invention can be synthesised according to the Schemes set out below.Method 1. Synthesis of Payload Moieties of Formula (D-A)

[0501] The payload moieties of Formula (D-A) can be made according to Scheme 1 below.

[0502]

[0503] Scheme 1

[0504] In Scheme 1, A is C2-10 alkenylene or C2-10 alkynylene, T is alkylene where n = 1 to 6; PG1 is a carboxylic acid-protecting group, typically C1-6 alkyl, preferably tert-butyl; LG is a leaving group, preferably halogen or sulfonate, more preferably bromine. Compounds of formula (D-AI’) are those of formula (D-A) wherein X is O, and II is O.Step 1 comprises reaction of a compound of formula (IV) with a compound of formula (V) to generate a compound of formula (VI). The reaction is typically carried out in the presence of a strong base. In one embodiment, the strong base is a hydride, such as sodium hydride.

[0505] Step 2 comprises deprotection of the compound of formula (VI) to generate a compound of formula (VII). This can be carried out under suitable deprotection conditions well known to those skilled in the art. When the protecting group PG1 is tert-butyl, the reaction is typically carried out under acidic conditions, preferably formed using a silyl halide and hexafluoroisopropanol (HFIP). In one embodiment, the silyl halide is chlorotrimethylsilane.

[0506] Step 3 comprises coupling of the compound of formula (VII) with a compound of formula (VIII) to generate a compound of formula (D-A’). In one embodiment, the compound of formula (VII) is an amine salt, preferably as the mesylate salt. This coupling can be carried out under standard amide coupling conditions well known to those skilled in the art. The reaction is typically carried out using an activating agent, examples of which include 1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). The reaction is typically carried out in the presence of a base, typically an alkyl amine, such as N, N'-diisopropylethylamine (DIPEA).Method 2. Synthesis of Payload Moieties of Formula (D-B)

[0507] The payload moieties of Formula (D-B) can be made according to Scheme 2 below.

[0508]

[0509] Scheme 2

[0510] In Scheme 2, A is alkylene where n = 1 to 10; PG1 is a hydroxyl-protecting group, typically C1-6 alkyl, preferably methyl; LG1 is a leaving group, preferably halogen or sulfonate, more preferably bromine, LG2 and LG3 are leaving groups as defined below, and PG2 is a hydroxyl-protecting group, preferably a silyl group, more preferably tert-butyldimethylsilyl. Compounds of formula (D-B’) are those of formula (D-B) wherein X is O, and compounds of formula (D-B”) are those of formula (D-B) wherein X is a bond.Step 1 comprises reaction of a compound of formula (IX) with a compound of formula (X) to generate a compound of formula (XI). The reaction is typically carried out in the presence of an acid catalyst or a suitable salt. In one embodiment, the acid catalyst is a sulfonic acid, such as p-toluenesulfonic acid, or a salt thereof, preferably the pyridinium salt, pyridinium p-toluenesulfonate (PPTS).

[0511] Step 2 comprises deprotection of the compound of formula (XI) to generate a compound of formula (XII). This can be carried out under suitable deprotection conditions well known to those skilled in the art. When the protecting group PG1 is an alkyl group, the reaction is typically carried out in the presence of a strong acid, typically a hydrohalic acid. In one embodiment, the acid is hydrobromic acid.

[0512] Step 3 comprises reaction of a compound of formula (XII) with a compound of formula (XIII) to generate a compound of formula (D-B’). As part of this reaction, the protecting group PG2 of the compound of formula (XIII) is cleaved. The reaction is typically carried out in the presence of a base, typically a strong base. In one embodiment, the base is a guanidine, typically tetramethylguanidine (TMG).

[0513] Step 3’ comprises conversion of the hydroxyl group of the compound of formula (XII) into a leaving group O-LG2 to generate a compound of formula (Xllla). Typically LG2 is sulfonyl group, such as a trifluoromethylsulfonyl group.

[0514] Step 4 comprises coupling of the compound of formula (Xllla) with a compound of formula (Xlllc) under aryl coupling conditions to generate a compound of formula (XI 11 b). Typically LG3 is a trihaloborate such as trifluoroborate, and PG2 is a silyl group, such as a tert-butyldimethylsilyl group.

[0515] The reaction is typically carried out in the presence of a suitable aryl coupling catalyst and a base. Typical aryl coupling catalysts include palladium (II) salts such as PdCl2(dppf).

[0516] Typical bases include alkali metal carbonates such as caesium carbonate.

[0517] Step 5 comprises deprotection of the compound of formula (XI 11 b) to generate a compound of formula (D-B”). This can be carried out under suitable deprotection conditions well known to those skilled in the art. When the protecting group PG2 is a silyl group, the reaction is typically carried out in the presence of trifluoroacetic acid or a reagent that releases fluoride ions, such as tetrabutylammonium fluoride (TBAF).Method 2 A. Synthesis of Payload Moieties of Formula (D-B)

[0518] The payload moieties of Formula (D-B’”) wherein X is S may be made according to Scheme 2A. In Scheme 2A, A is alkylene where n = 1 to 10; PGi is a hydroxyl-protecting group, preferably a silyl group, more preferably tert-butyldimethylsilyl.

[0519]

[0520] Scheme 2A

[0521] Step 1 comprises substitution of a compound of formula (XIV) with a compound of formula (XV) to generate a compound of formula (XVI). The reaction is typically carried out in the presence of a base, typically a weak base. In one embodiment, the base is a carbonate salt, typically potassium carbonate.

[0522] Step 2 comprises reduction of the nitro group of a compound of formula (XVI) to generate the amine group of a compound of formula (XVII). As part of this reaction, the protecting group PGi of the compound of formula (XVI) is cleaved.

[0523] The reaction is typically carried out in the presence of a suitable reducing agent and an acid, typically a weak acid. Typical reducing agents include iron. Typical acids include ammonium chloride.

[0524] Step 3 comprises reaction of a compound of formula (XVII) with a compound of formula (X) to generate a compound of formula (D-B’”). The reaction is typically carried out in the presence of an acid catalyst or a suitable salt. In one embodiment, the acid catalyst is a sulfonic acid, such as p-toluenesulfonic acid, or a salt thereof, preferably the pyridinium salt, pyridinium p-toluenesulfonate (PPTS).Method 2B. Synthesis of Payload Moieties of Formula (D-B1)

[0525] The payload moieties of Formula (D-B1) wherein Ri and R4 together with the carbon atoms to which they are attached form a 6-membered carbocyclic ring may be made according to Scheme 2B. In this particular example, R5 is C1-6 alkyl, II is O, and X is O (compounds of formula (D-BT) or a bond (compounds of formula (D-B1”)), LG1, LG2 and LG3 are leaving groups, PG1 and PG2 are protecting groups, and n is 1 to 3.

[0526]

[0527] Scheme 2BStep 1 comprises reaction of a compound of formula (XIX) with an iodinating reagent under oxidising conditions to generate a compound of formula (XX). Typical reagents include iodine and a periodate, such as sodium periodate.

[0528] The reaction is typically carried out in the presence of a strong acid catalyst. In one embodiment, the acid catalyst is sulphuric acid.

[0529] Step 2 comprises reaction of the compound of formula (XX) with a suitable alkenoate under aryl coupling conditions to generate a compound of formula (XXI). Typical reagents include tert-butyl butenoate.

[0530] The reaction is typically carried out in the presence of a suitable aryl coupling catalyst and a triaryl phosphine. Typical aryl coupling catalysts include palladium (II) salts such as palladium (II) acetate. Typical triaryl phosphines include tri(o-tolyl)phosphine.

[0531] Step 3 comprises reaction of the compound of formula (XXI) to generate a compound of formula (XXII). This proceeds by (a) generation of a nitroso moiety alpha to the ester; (b) reduction of the nitroso, nitro group and alkene moieties; (c) attachment of the acyl group -C(=O)Rs to the amines.

[0532] Step 3(a) is typically carried out using a strong base such as potassium tert-butoxide, and an alkyl nitrite, such as isopentyl nitrite.

[0533] Step 3(b) is typically carried out using hydrogen in the presence of a suitable hydrogenation catalyst. Typical hydrogenation catalysts include palladium on carbon.

[0534] Step 3(c) is typically carried out using an acid anhydride Rs-C(=O)-O-C(=O)-R5, typically in the presence of the corresponding acid Rs-C(=O)-OH.

[0535] Step 4 comprises ring closure of the compound of formula (XXII) to generate a compound of formula (XXIII). Typical reagents include hydrochlorosilanes, such as chlorodimethylsilane. The reaction is typically carried out in the presence of a suitable catalyst, typically a Lewis acid catalyst such as indium (III) chloride.

[0536] Step 5 comprises selective deacylation of the anilino-amide of the compound of formula (XXIII) to generate a compound of formula (XXIV). Typical reagents include strong acids, such as hydrochloric acid.Step 6 comprises reaction of a compound of formula (XXIV) with a compound of formula (X) to generate a compound of formula (XXV). The reaction is typically carried out in the presence of an acid catalyst or suitable salt. In one embodiment, the acid catalyst is a sulfonic acid, such as p-toluenesulfonic acid, or a salt thereof, preferably the pyridinium salt, pyridinium p-toluenesulfonate (PPTS).

[0537] Step 7 comprises cleavage of the methyl ether bond of the compound of formula (XXV) to generate a compound of formula (XXVI). Typical reagents include strong acids, such as hydrobromic acid.

[0538] Step 8 comprises alkylation of the compound of formula (XXVI) with a compound of formula LGi-CH2-(CH2)n-OPGi to generate a compound of formula (XXVII). Typically LGi is a halogen atom, such as bromo, and PGi is a silyl group, such as a tert-butyldimethylsilyl group.

[0539] Step 9 comprises deprotection of the compound of formula (XXVII) to generate a compound of formula (D-BT). This can be carried out under suitable deprotection conditions well known to those skilled in the art. When the protecting group PGi is a silyl group, the reaction is typically carried out in the presence of trifluoroacetic acid or a reagent that releases fluoride ions, such as tetrabutylammonium fluoride (TBAF).

[0540] Step 10 comprises conversion of the hydroxyl group of the compound of formula (XXVI) into a leaving group O-LG2 to generate a compound of formula (XXVIII). Typically LG2 is sulfonyl group, such as a trifluoromethylsulfonyl group.

[0541] Step 11 comprises coupling of the compound of formula (XXVIII) with a compound of formula LG3-(CH2)n-OPG2 under aryl coupling conditions to generate a compound of formula (XXIX). Typically LG3 is a trihaloborate such as trifluoroborate, and PG2 is a silyl group, such as a tert-butyldimethylsilyl group.

[0542] The reaction is typically carried out in the presence of a suitable aryl coupling catalyst and a base. Typical aryl coupling catalysts include palladium (II) salts such as PdCl2(dppf).

[0543] Typical bases include alkali metal carbonates such as caesium carbonate.

[0544] Step 12 comprises deprotection of the compound of formula (XXIX) to generate a compound of formula (D-B1”). This can be carried out under suitable deprotection conditions wellknown to those skilled in the art. When the protecting group PG2 is a silyl group, the reaction is typically carried out in the presence of trifluoroacetic acid or a reagent that releases fluoride ions, such as tetrabutylammonium fluoride (TBAF).

[0545] Method 3. Synthesis of Linker-Payload Conjugates of Formula (II)

[0546] The linker-payload compounds of formula (II) may be formed by conjugating a payload compound D to molecules containing a whole or part of the linkers of Formula (L). This can be carried out by a number of methods well known to those skilled in the art.

[0547] In particular, the peptide moiety P of the linker of Formula (L) may be synthesised by standard peptide synthetic methods well known to those skilled in the art.

[0548] Method 3A. Synthesis of Linker-Payload Conjugates of Formula (II) comprising a payload moiety of formula (D-A)

[0549] By way of example, compounds of Formula (II) comprising a payload moiety of formula (D-A) may be made according to Scheme 3. The synthesis is shown for compounds wherein the linker is a linker of formula (L1), but is also applicable to compounds having other linkers of formula (L) including a peptide moiety P.

[0550]

[0551] Scheme 3

[0552] In Scheme 3, A is C2-10 alkenylene or C2-10 alkynylene, T is alkylene where n = 1 to 6; PG is an amine-protecting group, typically Fmoc. Compounds of formula (II’) are those of formula (II) wherein the payload moiety D is of formula (D-A).Step 1 comprises etherification of the compound of formula (XXX) by reaction with a compound of formula (XXXI), wherein PG is an amine-protecting group, to generate a compound of formula (XXXII). Typical amine-protecting groups PG include 9-fluorenylmethoxycarbonyl (Fmoc). The reaction is typically carried out in the presence of a suitable catalyst, typically a Lewis acid catalyst such as boron trifluoride.

[0553] Step 2 comprises coupling of the compound of formula (XXXII) with a compound of formula (VIII) to generate a compound of formula (XXXIII). In one embodiment, the compound of formula (VIII) is an amine salt, preferably as the mesylate salt. This coupling can be carried out under standard amide coupling conditions well known to those skilled in the art. The reaction is typically carried out using an activating agent, examples of which include 1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). The reaction is typically carried out in the presence of a base, typically an alkyl amine, such as N, N'-diisopropylethylamine (DIPEA).

[0554] Step 3 comprises deprotection of the compound of formula (XXXIII) to generate a compound of formula (XXXIV). This can be carried out under suitable deprotection conditions well known to those skilled in the art. When the protecting group PG is an Fmoc group, the reaction is typically carried out in the presence of a base, such as DBU.

[0555] Step 4 comprises coupling of the compound of formula (XXXIV) with a suitable intermediate to generate a compound of formula (II’). This can be carried out under standard amide coupling conditions well known to those skilled in the art. The reaction is typically carried out using an activating agent, examples of which include 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM). The reaction is typically carried out in the presence of a base, typically an alkyl amine, such as N, N'-diisopropylethylamine (DIPEA).

[0556] Method 3B. Synthesis of Linker-Payload Conjugates of Formula (II) comprising a payload moiety of formula (D-B)

[0557] By way of example, compounds of Formula (II) comprising a payload moiety of formula (D-B1) may be made according to Scheme 3A. This synthesis is equally applicable for compounds of Formula (II) comprising a payload moiety of formula (D-B) wherein Ri and R4 do not form a carbocyclic ring. The synthesis is shown for compounds wherein X is O, but is also applicable to compounds of formula (II) wherein X is S. The synthesis is shown for compounds wherein the linker is a linker of formula (L2), but is also applicable to other linkers of formula (L) including a peptide moiety P.o

[0558]

[0559] Scheme 3A

[0560] In Scheme 3A, PG is an amine-protecting group, typically Fmoc. Compounds of formula (HA) are those of formula (II) wherein the payload moiety D is of formula (D-B1).Step 1 comprises etherification of the compound of formula (D-B1) by reaction with a compound formula (XXX), wherein PG is an amine-protecting group, to generate a compound of formula (XXXV). Typical amine-protecting groups PG include 9-fluorenylmethoxycarbonyl (Fmoc). The reaction is typically carried out in the presence of a suitable catalyst, typically a Lewis acid catalyst such as boron trifluoride.

[0561] Step 2 comprises deprotection of the compound of formula (XXXV) to generate a compound of formula (XXXVI). This can be carried out under suitable deprotection conditions well known to those skilled in the art. When the protecting group PG is an Fmoc group, the reaction is typically carried out in the presence of a base, such as DBU.

[0562] Step 3 comprises coupling of the compound of formula (XXXVI) with a suitable intermediate to generate a compound of formula (HA). This can be carried out under standard amide coupling conditions well known to those skilled in the art. The reaction is typically carried out using an activating agent, examples of which include 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM).

[0563] Method 4. Synthesis of Binding Molecule-Linker-Payload Conjugates of Formula (III)

[0564] The binding molecule-linker-payload conjugates of formula (III) may be formed by conjugating a linker-payload of formula (II) to a binding molecule, such as an antibody. This can be carried out by a number of methods well known to those skilled in the art.

[0565] In one embodiment of such a method, the method comprises:

[0566] (a) reducing the disulfide bonds on the relevant amino acid residues (typically cysteine residues) on the binding molecule BM, to generate an activated form of the binding molecule in which the thiol groups on the relevant amino acids are free; and

[0567] (b) reacting the activated form of the binding molecule BM with the linker-payload conjugate of formula (II), thereby conjugating the linker-payload moiety to the binding molecule BM to form the compound of formula (III).

[0568] A generalised method for conjugation of a linker-payload of formula (II) to an I gG 1 antibody (shown generally by reference to its heavy and light chains) is shown in Scheme 4 below. In this particular synthetic route, the group Y is a sulfone group which is displaced by the thiol groups of the antibody to generate a thioether bond to the linker-payload moiety, and R represents generally the remainder of the linker-payload moiety.

[0569]

[0570] Scheme 4

[0571] Salts, Solvates, Polymorphs, Enantiomers, Isotopically Labelled Derivatives

[0572] Pharmaceutically acceptable salts of the compounds of formulae (D-A), (D-B), (II) and (III) include the acid addition and base salts thereof.

[0573] Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinafoate salts.

[0574] Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

[0575] Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.

[0576] For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0577] Pharmaceutically acceptable salts of payload moieties of formula (D-A) and (D-B) may be prepared by one or more of three methods:(i) by reacting the payload moieties of formula (D-A) and (D-B) with the desired acid or base; (ii) by removing an acid- or base-labile protecting group from a suitable precursor of the payload moieties of formula (D-A) and (D-B) or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or

[0578] (iii) by converting one salt of the payload moieties of formula (D-A) and (D-B) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.

[0579] All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.

[0580] The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term 'amorphous' refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order ('glass transition').

[0581] The term 'crystalline' refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order ('melting point').

[0582] The compounds of the invention may also exist in unsolvated and solvated forms.

[0583] The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

[0584] A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates - see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995). Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion. When the solvent or water is tightly bound,the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content will be dependent on humidity and drying conditions. In such cases, non- stoichiometry will be the norm.

[0585] Payload moieties of formula (D-A) and (D-B) containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can occur. This can take the form of proton tautomerism in payload moieties of formula (D-A) and (D-B) containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.

[0586] Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the payload moieties of formula (D-A) and (D-B), including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counterion is optically active, for example, d-lactate or / - lysine, or racemic, for example, d--tartrate or dl-arginine.

[0587] Cis / trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).

[0588] Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and / or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.

[0589] Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.

[0590] When any racemate crystallises, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.

[0591] While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, Stereochemistry of Organic Compounds by E. L. Eliel and S. H. Wilen (Wiley, 1994).

[0592] The present invention includes all pharmaceutically acceptable isotopically-l abelled compounds of formula (II) and (III), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.

[0593] Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as2H and3H, carbon, such as11C,13C and14C, chlorine, such as36Cl, fluorine, such as18F, iodine, such as123I and125I, nitrogen, such as13N and15N, and oxygen, such as15O,17O and18O.

[0594] Certain isotopically-labelled compounds of formula (II) and (III), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.3H, and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0595] Substitution with heavier isotopes such as deuterium, i.e.2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0596] Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.Isotopically-labelled compounds of formula I can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Intermediates using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

[0597] Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D2O, d6-acetone, d6-DMS.

[0598] Pharmaceutical Compositions

[0599] The present invention also provides a pharmaceutical composition which comprises a compound of formula (II) or (III) according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0600] In some embodiments, the compositions described herein may further comprise one or more selected from this list consisting of: an adjuvant, salt, active polypeptide, compound, component and active agent.

[0601] Compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition according to the invention may be produced using current good manufacturing practices (CGMP).

[0602] The pharmaceutical composition may be formulated to be suitable for administration to a patient in order to prevent and / or treat disease. Pharmaceutical compositions can be formulated for administration by different routes, for example, for oral, parenteral, topical, inhalative, intravenous, intramuscular, rectal, sublingual, transdermal, subcutaneous, intratumoral application routes, according to their chemical and physical properties.

[0603] The pharmaceutical composition may be in the form of a tablet, a coated tablet, powder, granulate, a pellet, a capsule, an effervescent tablet or a transdermal therapeutic system. The pharmaceutical composition may be in the form of a liquid composition, selected from the group consisting of a solution, a syrup, an infusion, an extract, a solution for intravenous application, or a solution for infusion. The pharmaceutical composition may be in the form of a semisolid composition such as an emulsion, a suspension, a cream, a lotion, a gel, a globule, a buccal tablet or a suppository.The term “carrier”, as used herein, may refer to a diluent, adjuvant, excipient, or vehicle. Such carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. A sterile saline solution is a preferred carrier.

[0604] Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0605] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition of the invention can be formulated as neutral or salt forms. Salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0606] In some embodiments, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.

[0607] In some embodiments, the composition may comprise an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[0608] In some embodiments, the composition may comprise one or more vesicles, nanoparticles, lipid nanoparticle (LNPs), liposomes or polymeric mixtures.

[0609] The composition may enable delivery of a nucleic acid(s) according to the invention and / or a vector according to the invention to a cell.

[0610] Kit

[0611] The present invention also provides a kit comprising the payload, payload-linker and binding molecule-linker-payload conjugates according to the invention. The present invention also provides a kit comprising the composition according to the invention.Dosage

[0612] For administration to human patients, the total daily dose of the compounds of the invention is typically in the range 1 mg to 5000 mg, preferably 10 mg to 2000 mg, more preferably 50 mg to 1500 mg, and still more preferably 100 to 500 mg depending, of course, on the mode of administration. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.

[0613] These dosages are based on an average human subject having a weight of about 60kg to 70kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.

[0614] For the avoidance of doubt, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

[0615] Medical Uses and Methods of Treatment

[0616] The compounds of the invention are indicated as pharmaceuticals. According to a further aspect of the invention there is provided a compound of the invention, as hereinbefore defined, for use as a pharmaceutical.

[0617] Compounds of the invention may be shown to be active e.g. in the biochemical assays described herein.

[0618] The disorders / conditions that the compounds of the invention may be useful in treating hence include cancer (such as lymphomas, solid tumours or a cancer as described hereinafter), inflammatory and autoimmune diseases, for example rheumatoid arthritis, psoriasis, psoriatic arthritis, osteoarthritis, ankylosing spondylitis, atopic dermatitis, inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, peanut allergy, asthma and celiac disease).

[0619] According to a further aspect of the present invention, there is provided a compound of the invention, as hereinbefore defined, for use in the treatment of cancer.

[0620] According to a further aspect of the present invention, there is provided use of a compound of the invention, as hereinbefore defined, in the manufacture of a medicament for the treatment of cancer.According to a further aspect of the present invention, there is provided a method of treatment of cancer, which method comprises administration of a therapeutically effective amount of a compound of the invention, as hereinbefore defined, to a patient suffering from, or susceptible to, such a condition.

[0621] According to a further aspect of the present invention, there is provided a compound of the invention, as hereinbefore defined, for use in the treatment of cancer.

[0622] As stated above, the compounds of the invention may be useful in the treatment of cancer. More, specifically, the compounds of the invention may therefore be useful in the treatment of a variety of cancer including, but not limited to: carcinoma such as cancer of the bladder, breast, colon, kidney, liver, lung (including non-small cell cancer and small cell lung cancer), oesophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, skin, squamous cell carcinoma, testis, genitourinary tract, larynx, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, non-small cell lung carcinoma, small cell lung carcinoma, lung adenocarcinoma, bone, adenoma, adenocarcinoma, follicular carcinoma, undifferentiated carcinoma, papilliary carcinoma, seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidney carcinoma, myeloid disorders, lymphoid disorders, hairy cells, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, colon-rectum, large intestine, rectum, brain and central nervous system, Hodgkin’s and leukaemia; hematopoietic tumours of lymphoid lineage, including leukaemia, acute lymphocitic leukaemia, acute lymphoblastic leukaemia, B-cell lymphoma, T-cell-lymphoma, mantle cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumours of myeloid lineage, including acute and chronic myelogenous leukaemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumours of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and other tumours, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma, thyroid follicular cancer and Kaposi’s sarcoma.

[0623] According to a further aspect of the present invention, there is provided use of a compound of the invention, as hereinbefore defined, in the manufacture of a medicament for the treatment of an autoimmune disease or an inflammatory disease.

[0624] According to a further aspect of the present invention, there is provided a method of treatment of an autoimmune disease or an inflammatory disease, which method comprisesadministration of a therapeutically effective amount of a compound of the invention, as hereinbefore defined, to a patient suffering from, or susceptible to, such a condition.

[0625] According to a further aspect of the present invention, there is provided a compound of the invention, as hereinbefore defined, for use in the treatment of an autoimmune disease or an inflammatory disease.

[0626] Examples of autoimmune disease and / or inflammatory diseases include but are not limited to coeliac disease, psoriasis, inflammatory bowel diseases such as colitis and Crohn’s disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, aplastic anaemia, myocarditis, autoimmune hepatitis, primary biliary cholangitis, alopecia areata, autoimmune urticaria, perphigus vulgaris, autoimmune polyendocrine syndrome (APS), autoimmune pancreatitis, type 1 diabetes, autoimmune thyroiditis, Sjogren’s syndrome, autoimmune haemolytic anaemia, gastritis vasculitis, granlumatosis with polyangitis, autoimmune uveitis, myasthenia gravis, acute disseminated encephalomyelitis, relapsing polychrondritis, Lambert-Eaton myasthenic syndrome, Hashimoto’s encephalopathy, Felty syndrome, autoimmune thyroiditis, Graves’ disease, nephritis, bullous pemphigoid, dermatitis, epidermolysis bullosa acquisita, linear IgA disease, autoimmune lymphoprolifeative syndrome, autoimmune neutropenia, autoimmune thrombocytopenis purpura, cold agglutinin disease, Evans syndrome, pernicious anaemia, Still’s disease, psoriatic arthritis, rheumatic fever, Guillain-Barre syndrome, Ord’s thyroiditis, ankylosing spondylitis, atopic dermatitis, systemic sclerosis (scleroderma), hidradenitis suppurativa, lupus nephritis, peanut allergy, and asthma.

[0627] Compounds of the invention are indicated both in the therapeutic and / or prophylactic treatment of the above-mentioned conditions.

[0628] “Patients” include mammalian (including human) patients. Hence, the method of treatment discussed above may include the treatment of a human or animal body.

[0629] The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient. The effect may be objective (e.g. measurable by some test or marker) or subjective (e.g. the subject gives an indication of or feels an effect).Examples

[0630] Abbreviations

[0631] AMU atomic mass units

[0632] DIC N, N'-Diisopropylcarbodiimide

[0633] DIPEA N, N'-Diisopropylethylamine

[0634] DMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride

[0635] EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0636] HATU Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium salt

[0637] ELSD Evaporative light scattering detection

[0638] HFIP 1,1,1,3,3,3-Hexafluoropropan-2-ol

[0639] HPLC High performance liquid chromatography

[0640] LRMS Low Resolution Mass Spectrum

[0641] PPTS Pyridinium p-toluenesulfonate

[0642] TEA Triethylamine

[0643] TFA Trifluoroacetic acid

[0644] TMG tetramethylguanidine

[0645] General Experimental Conditions

[0646] Compounds were purchased unless synthetic routes for their preparation are indicated. All synthetic reactions were conducted under positive nitrogen pressure with magnetic stirring unless otherwise stated. Centrifugation was done with a single speed (2,000 x g) Fisher Scientific mini-centrifuge, using 6 mm x 50 mm glass culture tubes. Chromatographic purifications were conducted on either a Biotage Select with uv / vis detection in the 220 nm -400 nm range or a Teledyne 300+ Next generation Flash chromatography unit with uv / vis detection in the 220 nm - 400 nm range and / or with ELSD detection. Flash chromatography uv / vis detection also used background solvent subtraction. UPLC-MS analyses were performed on a Waters Acquity system with mass detection in the 50 AMU - 2000 AMU range with a BEH C18 103 A, 1.4 μm, 2.1 mm x 100 mm column and eluting at 0.5 mL / min with deionized water containing 0.1% TFA and a gradient of acetonitrile containing 0.1% TFA 5 - 90% over 5 min then acetonitrile 100% for 1 min and column re-equilibration with 95:5 (deionized water containing 0.1% TFA: acetonitrile containing 0.1% TFA) for 1 min.Preparation 1: Synthesis of Intermediates 1, 2, 3, and 3a

[0647] The synthesis of Intermediates 1, 2, 3, and 3a is shown in Scheme 5 and described below.

[0648] O EDC, HOAT DIPEA, DCM lnt-1

[0649]

[0650] Scheme 5

[0651] Intermediate 1: To a stirring solution of 2-(methylthio)pyrimidine-5-carboxylic acid (493.1 mg, 2.78 mmol, Sigma-Aldrich) in 15 ml of dichloromethane was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (547.4 mg, 2.78 mmol), 1-hydroxy-7-azabenzotriazole (378.4 mg, 2.78 mmol) and then / V, / V-diisopropylethylamine (484 pL, 2.78 mmol) at room temperature. After 10 min, a solution of MeNH-PEG2-CH2COOtBu (500 mg, 2.14 mmol, 1PlusChem) in dichloromethane was added. After an additional 1.5 h, the reaction was diluted with dichloromethane and washed three times with 0.05 N HCI and three times with saturated sodium bicarbonate. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford 867 mg of Intermediate 1 as a yellow oil. The material was used as is in the next step without further purification. LC / MS for C17H27N3O5S [M+H]+calcd. 386.48 m / z, found 386.57 m / z, observed (M+H-tBu)+330.44 m / z.

[0652] Intermediate 2: To a stirring solution of crude Intermediate 1 from the previous step in 11 mL of dichloromethane was added 11 mL of trifluoroacetic acid at room temperature. After 20 h, the reaction was concentrated under reduced pressure to afford Intermediate 2. The material was used as is in the next step without further purification. LC / MS for C13H19N3O5S [M+H]+calcd. 330.37 m / z, found 330.42 m / z.Intermediate 3: To a stirring solution of crude Intermediate 2 from the previous step in 14 ml of deionized water was added OXONE (1.64 g, 5.35 mmol) at 0 °C. After 1.5 h, the product was isolated by solid-phase extraction with Waters Sep-Pak tC1835cc cartridges. The fractions containing product were concentrated under reduced pressure. The resulting material was taken up in a solution of 1:1 water / acetonitrile. The solution was dried via lyophilization to afford 721 mg of Intermediate 3 (93% yield) as a yellow residue. LC / MS for C13H19N3O7S [M+H]+calcd. 362.37 m / z, found 362.18 m / z.

[0653] Intermediate 3a: To a fritted syringe containing polymer-bound / V-Benzyl- / V'-cyclohexylcarbodiimide (1.0 g / mol, 0.332 mmol, Sigma-Aldrich) was added a solution of Intermediate 3 (60 mg, 0.166 mmol) in 8 mL of dichloromethane. To the resulting solution was added / V-hydroxysuccinimide (19.1 mg, 0.166 mmol). The reaction was incubated on a shaker for 1 h. Then the reaction was filtered to remove the polymer-bound material. The solution was concentrated via reduced pressure to afford 76 mg of Intermediate 3a (99% yield) as a residue. The material was used as is without further purification. LC / MS for C17H22N4O9S [M+H]+calcd. 459.44 m / z, found 459.19 m / z.

[0654] Preparation 2: Synthesis of Intermediate 5

[0655] The synthesis of Intermediate 5 is shown in Scheme 6 and described below.

[0656] Int-4b

[0657]

[0658] Int-4c Scheme 6

[0659] Intermediate 4a: To a stirring solution of Cbz-Val-OSu (1.0 g, 2.87 mmol, Chem-lmpex) in 15 mL of DMF was added H-Lys(Boc)-OH (742 mg, 3.01 mmol) followed by N, N-diisopropylethylamine (1.25 mL, 7.18 mmol) at room temperature. After 16 h, the reaction was concentrated, and the resulting material was taken up in ethyl acetate. The organic layer was washed with a saturated aqueous citric acid solution. Then the organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resultingsolids were dissolved in 5 mL of methanol and then precipitated into 80 mL of deionized water. The resulting mixture was spun at 3700 rpm for 10 min. The supernatant was removed, and the solids were dried via lyophilization to afford 1.26 g of Intermediate 4a (92% yield) as a white solid. LC / MS for C24H37N3O7 [M+H]+calcd. 480.57 m / z, found [M+Na]+502.32 m / z.

[0660] Intermediate 4b: To a stirring mixture of Intermediate 4a (1.26 g, 2.63 mmol) in 15 mL of ethyl acetate was added 4 M HCI in dioxane (6.57 mL, 26.37 mmol) at room temperature. After 1 h, the reaction was concentrated under reduced pressure to afford 1.2 g of crude Intermediate 4b as a white solid. The material was used as is without further purification. LC / MS for C19H29N3O5 [M+H]+calcd. 380.46 m / z, found 380.23 m / z.

[0661] Intermediate 4c: To a stirring mixture of Intermediate 4b (1.2 g, 2.89 mmol) in 20 mL of dichloromethane was added propionaldehyde (1.24 mL, 17.3 mmol) at room temperature. After 20 min, STAB (3.06 g, 14.43 mmol) was added. After an additional 2 h, 10 mL of saturated aqueous ammonium chloride was added. The reaction was stirred for 2 h and then extracted four times with dichloromethane. The combined organic layers were concentrated under reduced pressure. The resulting residue was dissolved in 4 mL of dichloromethane and precipitated into chilled 80 mL of diethyl ether. The mixture was spun at 3700 rpm for 10 min. The supernatant was removed and the solids were dried under reduced pressure to afford 1.05 g of Intermediate 4c (79% yield) as a white foam. LC / MS for C25H41N3O5 [M+H]+calcd. 464.62 m / z, found 464.33 m / z.

[0662] Intermediate 5: To a stirring solution of Intermediate 4c (1.0 g, 2.16 mmol) in 40 mL of methanol was added 2 mL of water and 2 mL of formic acid at room temperature. The flask was repeatedly purged and filled with nitrogen. To the resulting solution was added 5% palladium on carbon (500 mg). The reaction was stirred under nitrogen. After 16 h, the reaction was diluted with methanol and filtered through pre-rinsed Celite® with 0.5% formic acid in methanol. The filtrate was concentrated under reduced pressure and the residue was taken up in water with 20% acetonitrile. The solution was dried via lyophilization. The dried solids were then taken up in 1 N HCI and dried via lyophilization to afford 842 mg of Intermediate 5 (97% yield) as the HCI salt. LC / MS C17H35N3O3 [M+H]+calcd. 330.49 m / z, found [M+H]+330.57 m / z.Preparation 3: Synthesis of Intermediates 7, 10, and 11

[0663] The synthesis of Intermediates 7, 10, and 11 is shown in Scheme 7 and described below.

[0664]

[0665] Scheme 7

[0666] Intermediate 7: The tripeptide was synthesized using standard Fmoc-based solid phase peptide chemistry. The peptide was synthesized using automated microwave peptide synthesis with 0.25 mmol of commercially available 2-chlorotrityl-chloride resin loaded with a Fmoc-2PAL-OH residue. To a fritted syringe was added 0.25 mmol of commercially available 2-chlorotrityl-chloride resin (0.53 mmol / g, 472 mg). The resin was swelled in 8 mL of dichloromethane for 15 min. Then a solution of N-Fmoc-3-(2-pyridyl)-L-alanine (Fmoc-2-Pal-OH, 194.2 mg, 0.5 mmol) in 8 mL of dichloromethane containing / V, / V-diisopropylethylamine (174.2 ul, 1.0 mmol) was added to the resin. The reaction mixture was agitated for 20 h. The solution was drained from the fritted syringe, and the resin was treated twice with 10 mL of a solution composed of 92.5% dichloromethane, 5.0% methanol, and 2.5% N, N-diisopropylethylamine. Subsequent Fmoc-protected amino acid residues were coupled using standard conditions involving / V, / V-diisopropylcarbodiimide (DIC), / V, / V-diisopropylethylamine, and Oxyma. Each coupling reaction was conducted for 10 minutes at 50 °C, and the process was repeated twice to ensure complete coupling. Following the completion of the peptide sequence, Fmoc deprotection was carried out using a solution of 20% piperidine in DMF for 3 minutes at 60 °C. The peptide was cleaved from the resin by incubation with 8 mL of equal parts trifluoroacetic acid and dichloromethane for 3 h. After treatment, the solution wasdrained from the fritted syringe, and the collected solution was concentrated under reduced pressure. The dried residue was dissolved in 3 mL of a 1: 1 mixture of water and MeCN containing 0.05% v / v TFA, then dried via lyophilization to afford 55.5 mg of intermediate 7 (79.2% yield). LC / MS for C12H16N4O4 [M+H]+calcd. 281.28 m / z, found 281.22 m / z.

[0667] Intermediate 10: The tripeptide was synthesized using standard Fmoc solid phase peptide chemistry on an automated microwave peptide synthesizer. To a fritted syringe was added 0.25 mmol of commercially available 2-chlorotrityl-chloride resin (0.53 mmol / g, 472 mg). The resin was swelled with 8 mL of dichloromethane for 15 min. Then a solution of N-Fmoc-3-(3-pyridyl)-L-alanine (Fmoc-3-Pal-OH, 194.2 mg, 0.5 mmol) in 8 mL of dichloromethane containing / V, / V-diisopropylethylamine (174.2 ul, 1.0 mmol) was added to the resin. The reaction was agitated for 20 h. The solution was evacuated from the fritted syringe, after which the resin was treated two times with a 10 mL solution of 92.5% dichloromethane, 5.0% MeOH, and 2.5% / V, / V-diisopropylethylamine. Then Fmoc-protected amino acid residues were coupled using standard / V, / V’-diisopropylcarbodiimide (DIC), / V, / V-diisopropylethylamine, and Oxyma conditions. The coupling procedure was reacted for 10 min at 50 °C and the process was repeated to ensure complete coupling. After completion of the peptide sequence, Fmoc deprotection was performed using a solution of 20% piperidine in DMF for 3 min at 60 °C. The peptide was cleaved from the resin by incubation with 8 mL of equal parts trifluoroacetic acid and dichloromethane for 3 h. After treatment, the solution was drained from the fritted syringe, and the collected solution was concentrated under reduced pressure. The dried residue was dissolved in 3 mL of a 1: 1 mixture of water and MeCN containing 0.05% v / v TFA, then dried via lyophilization to afford 52.4 mg of intermediate 10 (74.8% yield). LC / MS for C12H16N4O4 [M+H]+calcd. 281.28 m / z, found 281.15 m / z.

[0668] Intermediate 11: The tripeptide was synthesized using standard Fmoc solid phase peptide chemistry on an automated microwave peptide synthesizer. To a fritted syringe was added 0.25 mmol of commercially available 2-chlorotrityl-chloride resin (0.53 mmol / g, 472 mg). The resin was swelled with 8 mL of dichloromethane for 15 min. Then a solution of / V-Fmoc-3-(4-pyridyl)-L-alanine (Fmoc-4-Pal-OH, 194.2 mg, 0.5 mmol) in 8 mL of dichloromethane containing / V, / V-diisopropylethylamine (174.2 pl, 1.0 mmol) was added to the resin. The reaction was agitated for 20 h. The solution was evacuated from the fritted syringe, after which the resin was treated two times with a 10 mL solution of 92.5% dichloromethane, 5.0% MeOH, and 2.5% / V, / V-diisopropylethylamine. Then Fmoc-protected amino acid residues were coupled using standard / V, / V'-diisopropylcarbodiimide (DIC), / V, / V-diisopropylethylamine, and Oxyma conditions. The coupling procedure was reacted for 10 min at 50 °C and the process was repeated to ensure complete coupling. After completion of the peptidesequence, Fmoc deprotection was performed using a solution of 20% piperidine in DMF for 3 min at 60 °C. The peptide was cleaved from the resin by incubation with 8 mL of equal parts trifluoroacetic acid and dichloromethane for 3 h. After treatment, the solution was drained from the fritted syringe, and the collected solution was concentrated under reduced pressure. The dried residue was dissolved in 3 mL of a 1: 1 mixture of water and MeCN containing 0.05% v / v TFA, then dried via lyophilization to afford 45.9 mg of Intermediate 11 (65.6% yield). LC / MS for C12H16N4O4 [M+H]+calcd. 281.28 m / z, found 281.18 m / z.

[0669] Preparation 4: Synthesis of Intermediates 6, 8, 9, 12 and 13

[0670] The synthesis of intermediates 6, 8, 9, 12, and 13 is shown in Scheme 8 and described below.

[0671]

[0672] Scheme 8

[0673] Intermediate 6: To a stirring solution of Intermediate 3a (272 mg, 0.594 mmol) in 3 mL of DMF was added Intermediate 5 (207 mg, 0.565 mmol) at room temperature. Then N-methylmorpholine (124 pl, 1.13 mmol) was added. After 16 h, the reaction was concentratedvia reduced pressure and purified via prep HPLC to afford 117 mg of Intermediate 6 (31% yield) as a white foam. LC / MS for C30H52N6O9S [M+H]+calcd. 673.84 m / z, found 673.84 m / z.

[0674] Intermediate 8: To a stirring solution of Intermediate 3a (76.0 mg, 0.164 mmol) in 1 mL of DMF was added H-Gly-Gly-Phe-OH (44.1 mg, 0.158 mmol, commercially available) at room temperature. After 16 h, the reaction was concentrated via reduced pressure and purified via prep HPLC to afford 52.2 mg of Intermediate 8 (53% yield) as a white foam. LC / MS for C26H34N6O10S [M+H]+calcd. 623.65 m / z, found 623.74 m / z.

[0675] Intermediate 9: To a stirring solution of Intermediate 3a (190 mg, 0.415 mmol) in 2 mL of DMF was added Intermediate 7 (110.9 mg, 0.396 mmol) at room temperature. Then / V-methylmorpholine (87 pl, 0.791 mmol) was added. After 16 h, the reaction was concentrated via reduced pressure and purified via prep HPLC to afford 53.5 mg of Intermediate 9 (22% yield) as a white foam. LC / MS for C25H33N7O10S [M+H]+calcd. 624.64 m / z, found 624.68 m / z.

[0676] Intermediate 12: To a stirring solution of Intermediate 3a (180 mg, 0.393 mmol) in 2 mL of DMF was added Intermediate 10 (104.8 mg, 0.374 mmol) at room temperature. Then / V-methylmorpholine (82.2 pl, 0.748 mmol) was added. After 16 h, the reaction was concentrated via reduced pressure and purified via prep HPLC to afford 41.4 mg of Intermediate 12 (18% yield) as a white foam. LC / MS for C25H33N7O10S [M+H]+calcd. 624.64 m / z, found 624.71 m / z.

[0677] Intermediate 13: To a stirring solution of Intermediate 3a (157 mg, 0.343 mmol) in 2 mL of DMF was added Intermediate 11 (91.6 mg, 0.327 mmol) at room temperature. Then / V-methylmorpholine (71.9 pl, 0.654 mmol) was added. After 16 h, the reaction was concentrated via reduced pressure and purified via prep HPLC to afford 67.2 mg of Intermediate 13 (33% yield) as a white foam. LC / MS for C25H33N7O10S [M+H]+calcd. 624.64 m / z, found 624.71 m / z.Example 1. Synthesis of Payload Moieties of Formula D-1

[0678] The compound of Formula D-1 may be made according to Scheme 9.

[0679] a) NaH b)

[0680]

[0681] Scheme 9. Synthesis of Compound D-1

[0682] Intermediate 46: 2-butyne-1,4-diol (3.1 g, 37 mmol, 3.8 eq) was weighed into a ~20 mL capacity vial equipped with a magnetic stir bar. Anhydrous DMF (5.0 mL) was added, and the vial was cooled in an ice / water bath for 5 min then, tBuOK (1.1 g 9.8 mmol, 1 eq) was added in 3 portions ~30 sec apart and the cooling bath was removed. Some of the tBuOK did not dissolve, anhydrous DMF (1 mL) was added. After 30 min, the reaction was cooled in an ice / water bath then a solution of tert-butyl 2-bromoacetate (1.6 mL, 11 mmol, 1.1 eq) in anhydrous DMF (1 mL) was added dropwise then the cooling bath was removed. A large amount of precipitate formed. After stirring for 30 min, the reaction was vacuum filtered while scrapping the filter periodically to remove solids. A BioTage Flash 120g C18 column was equilibrated with 95:5 deionized water (0.2% acetic acid):acetonitrile, flow rate 50 mL / min and the filtrate was injected onto the column. The column was eluted with 2 column volumes of 5% acetonitrile then with a gradient of 5% - 60% acetonitrile over column volumes. Peaks were detected by ELSD and 214 nm with solvent background subtraction. Fractions containing product were combined and solvent removed by rotary evaporation to give 1.0 g (70 % yield) of product as a semi-solid. LC / MS C6H8O4[M+H]+calcd. 201.1 m / z, found 201.2 m / z.

[0683] Intermediate 47: A vial was charged with Intermediate 46 (108.8 mg, 0.543 mmol) and 1,1,1,3,3,3-hexafluoropropan-2-ol HFIP (1.0 mL, 9.498 mmol) then chloro(trimethyl)silane(0.5 mL, 4 mmol) was added dropwise. The resulting mixture was stirred at room temperature for 3 h. Solvent was removed by rotary evaporation and the residue was mixed with toluene (~4 mL), then concentrated to dryness. The product was directly used in the next reaction step. HPLC / LRMS C6H8O4[M+H]+calcd. 145.0 m / z, found 145.1 m / z.

[0684] Compound D-1: DIPEA (1.3 mL, 7.5 mmol) was added to a mixture of exatecan mesylate (1.0344 g, 2.375 mmol), Intermediate 47 (0.460 g, 3.19 mmol), and HATU (1.3748 g, 3.616 mmol) in DMSO (20 mL) at room temperature. The resulting mixture was stirred at room temperature for 1 h. The mixture was purified with reverse column chromatography using 30-40% acetonitrile (0.1% formic acid) / water (0.1% formic acid). Fractions containing desired product were combined and solvent was removed by rotary evaporation to give 580 mg (43% yield) of solid. LC / MS C30H28FN3O7 [M+H]+calcd. 562.2 m / z, found 562.4 m / z.

[0685] Example 2. Synthesis of Payload Moieties of Formulae D-2, D-3, and D-4

[0686] The compounds of Formulae D-2, D-3, and D-4 may be made according to Scheme 10.

[0687]

[0688] Scheme 10. Synthesis of Compounds D-2, D-3, and D-4

[0689] Intermediate 17: Pyridinium p-toluenesulfonate (PPTS) (12 mg, 0.048 mmol), Intermediate 15 (commercially available, 60 mg, 0.30 mmol) and Intermediate 16 (commercially available, 80 mg, 0.0.3 mmol) were added to a 5 mL capacity vial equipped with a magnetic stir bar. Toluene (4 mL) was added, and the vial was sealed with a septum cap then placed in a heating block set to 105°C and stirred overnight. The vial was removed from the heating block and allowed to cool to room temperature. Solids were collected by vacuum filtration then triturated with CH2CI2 (1 mL) while cooling in an ice / water bath. Solids were collected by vacuum filtration then placed under high vacuum for 5 h, giving 60 mg (50% yield) ofproduct, as a solid that was used without further purification. HPLC / LRMS for C23H21FN2O5 [M+H]+calcd. 425.1 m / z, found 425.2 m / z.

[0690] Intermediate 18: Intermediate 17 (26 mg, 0.063 mmol) was added to a 3 mL capacity vial equipped with a magnetic stir bar. 48% hydrobromic acid (2 mL) was added and the vial was sealed under a stream of nitrogen with a septum cap. The vial was placed in a heating block set to 92°C and stirred overnight. The heating block was removed and after 5 min, the septum was carefully punctured with a fine needle to relieve pressure. The hydrobromic acid was carefully removed by rotary evaporation. The residue was then taken up in deionized water, which was then removed by rotary evaporation. The residue was taken up in dichloromethane (10 mL) then toluene (1 mL) was added. Solvent was rotary evaporated, and residue was dissolved again in a minimum volume of dichloromethane then rotary evaporated giving 19 mg (73% yield) of product as a solid. This was used in the next step without purification. HPLC / LRMS for C22H19FN2O5 [M+H]+calcd. 411.1 m / z, found 411.3 m / z.

[0691] Compound D-2: Intermediate 18 (2 mg, 0.0049 mmol) was weighed into a 3 mL capacity vial equipped with a magnetic stir bar then dissolved in anhydrous DMSO (0.40 mL). 2-bromoethoxy)(tert-butyl)dimethylsilane (Intermediate 19a, commercially available) (6 mg, 0.025 mmol) was added then the vial was sealed with a septum cap. The septum was fit with a nitrogen inlet and an oil bubbler outlet. After stirring under slow nitrogen flow for 5 min, TMG (6.0 pL, 0.048 mmol) was injected into the reaction via a 10 pL syringe. After stirring for 6 h additional 19a (6 pg, ~ 1 mg / pL, 0.025 mmol) and TMG (6.0 pL, 0.048 mmol) were sequentially injected into the vial and the reaction was stirred overnight. The reaction gives the desired silyl protected product, and the silyl group is also slowly removed under the reaction conditions. The vial was cooled in an ice / water bath then acetic acid (25 pL) was injected into the vial. A 12 gram BioTage C18 cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 11 mL / min. The reaction mixture was injected onto the column then the column was eluted with deionized water (containing 0.5% formic acid) and a linear gradient of 5% - 95% acetonitrile over 20 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation giving 1.4 mg (63% yield) of product as a solid. HPLC / LRMS for C24H23FN2O6 [M+H]+calcd. 455.2 m / z, found 455.1 m / z.Compound D-3: Prepared similarly to Compound D-2, using Intermediate 18 and (3-bromopropoxy)(tert-butyl)dimethylsilane (Intermediate 19b, commercially available), (54% yield). HPLC / LRMS for C25H25FN2O6 [M+H]+calcd. 469.2 m / z, found 469.3 m / z.

[0692] Compound D-4: Prepared similarly to Compound D-2, using Intermediate 18 and (4-bromobutoxy)(tert-butyl)dimethylsilane (Intermediate 19c, commercially available), (68% yield). HPLC / LRMS for C26H27FN2O6 [M+H]+calcd. 483.2 m / z, found 483.2 m / z.Example 3. Synthesis of Payload Moieties of Formulae D-5 through D-10

[0693] Compounds of formulae D-5 through D-10 may be made according to Scheme 11.

[0694]

[0695] Scheme 11. Synthesis of Compounds D-5, D-6, D-7, D-8, D-9, and D-10Example 4. Synthesis of Payload Moieties of Formula D-15

[0696] The compound of Formula D-15 may be made according to Scheme 12.

[0697]

[0698] Scheme 12. Synthesis of Compound D-15

[0699] Intermediate 17a: Pyridinium p-toluenesulfonate (PPTS) (12 mg, 0.048 mmol), Intermediate 15a (57 mg, 0.30 mmol, commercially available) and Intermediate 16 (80 mg, 0.0.3 mmol, commercially available) were added to a 5 mL capacity vial equipped with a magnetic stir bar. Toluene (4 mL) was added, and the vial was sealed with a septum cap then placed in a heating block set to 105°C and stirred overnight. The vial was removed from the heating block and allowed to cool to room temperature. Solids were collected by vacuum filtration then triturated with CH2CI2 (1 mL) while cooling in an ice / water bath. Solids were collected by vacuum filtration then placed under high vacuum for 5 h to afford 52 mg of Intermediate 17a (48% yield) as a solid that was used without further purification. LC / MS for C22H19FN2O5 [M+H]+calcd. 397.2 m / z, found 397.3 m / z.

[0700] Intermediate 18a: Intermediate 17a (24 mg, 0.061 mmol) was added to a 3 mL capacity vial equipped with a magnetic stir bar. 48% hydrobromic acid (2 mL) was added and the vial was sealed under a stream of nitrogen with a septum cap. The vial was placed in a heating block set to 92°C and stirred overnight. The heating block was removed and after 5 min, the septum was carefully punctured with a fine needle to relieve pressure. The hydrobromic acid was carefully removed by rotary evaporation. The residue was then taken up in deionized water, which was then removed by rotary evaporation. The residue was taken up in dichloromethane (10 mL) then toluene (1 mL) was added. Solvent was rotary evaporated, and residue was dissolved again in a minimum volume of dichloromethane then rotary evaporated to afford 17mg of Intermediate 18a (71% yield) as a solid. The material was used in the next step without purification. HPLC / LRMS for C21H17FN2O5 [M+H]+calcd. 411.1 m / z, found 411.3 m / z.

[0701] Compound D-15: Intermediate 18a (6 mg, 0.015 mmol) was weighed into a 3 mL capacity vial equipped with a magnetic stir bar then dissolved in anhydrous DMSO (0.60 mL). 2-bromoethoxy)(tert-butyl)dimethylsilane (18 mg, 0.075 mmol, commercially available) was added then the vial was sealed with a septum cap. The septum was fit with a nitrogen inlet and an oil bubbler outlet. After stirring under slow nitrogen flow for 5 min, TMG (18.0 pL, 0.14 mmol) was injected into the reaction via a 10 pL syringe. After stirring for 6 h additional 2-bromoethoxy)(tert-butyl)dimethylsilane (6 pg, ~ 1 mg / pL, 0.025 mmol) and TMG (6.0 pL, 0.14 mmol) were sequentially injected into the vial and the reaction was stirred overnight. The reaction gives the desired silyl protected product, and the silyl group is also slowly removed under the reaction conditions. The vial was cooled in an ice / water bath then acetic acid (75 pL) was injected into the vial. A 12 gram BioTage C18 cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 11 mL / min. The reaction mixture was injected onto the column then the column was eluted with deionized water (containing 0.5% formic acid) and a linear gradient of 5% -95% acetonitrile over 20 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to afford 5 mg of compound D-15 (75% yield) as a solid. LC / MS for C23H21FN2O6 [M+H]+calcd. 441.2 m / z, found 441.3 m / z.

[0702] Example 5. Synthesis of Payload Moieties of Formula D-16

[0703] The compound of Formula D-16 may be made according to Scheme 13.

[0704]

[0705] Scheme 13. Synthesis of Compound D-16Intermediate 40: Tert-Butyl dimethylsilyl protected mercaptoethanol (108 mg, 0.56 mmol, commercially available), was added to a solution of Intermediate 39 (99 g, 0.46 mmol, commercially available) and anhydrous DMF (2.3 mL) were added to a vial equipped with a magnetic stir bar and vigorously stirred at room temperature as potassium carbonate (128 mg, 1.36 mmol) was added. After 1 hour the reaction was extracted with 10% aqueous citric acid (20 mL) and Ethyl acetate (2 x 20 mL). The organic phase was concentrated by rotary evaporation. A 12 g Biotage silica column equipped with a corresponding samplet was equilibrated with hexanes at a flow rate of 12 mL / min, 220 nm detection. Concentrate was dissolved in minimum volume of EtOAc and loaded onto the column eluting with 5a linear gradient of ethyl acetate 2% - 10% over 25 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 170 mg (100% yield) of product as an off white solid. HPLC / LRMS for C11H12FNO4S [M+H]+calcd. 388.1 m / z, found 288.2 m / z.

[0706] Intermediate 41: Intermediate 40 (114.5 mg, 0.296 mmol) was weighed into a vial equipped with a magnetic stir bar to which a solution of ammonium chloride (11.9 mg, 0.22 mmol) in deionized water (0.6 mL) and ethanol (1.2 mL) was added followed by iron powder (99.4 g, 1.78 mmol). The flask was fit with a septum and placed under positive nitrogen pressure while stirring in a heating block that was set to 90°C. After 30 min 2 M HCI (250 uL) was added, stirred for 1 h then deionized water (10 mL) was added and the pH was brought to 5 using sat NaHCCh. The mixture was extracted with ethyl acetate (2 x 40 mL), washed with brine (5 mL) and dried over anhydrous sodium sulfate, filtered then evaporated to give 40 mg (56% yield) of product as semi-solid. HPLC / LRMS for C17H26FNO4SSi [M+H]+calcd.

[0707] 388.1 m / z, found 388.4 m / z.

[0708] Compound D-16: Pyridinium p-toluenesulfonate (PPTS, 5.4 mg, 21.6 mmol), Intermediate 41 (51.5 g, 0.21 mmol) and Intermediate 16 (57.9 g, 0.22 mmol, commercially available) were added to a small vial equipped with a magnetic stir bar. Toluene (1.3 mL) was added, and the vial was sealed with a pressure release septum cap then placed in a heating block set to 100 °C then stirred for 48 h. The vial was removed from the heating block, allowed to cool to room temperature and vortexed. The suspension was centrifuged, supernatant was removed then Toluene (1 mL) was added, mixed and centrifuged. Supernatant was removed then acetonitrile (0.5 mL) was added, triturated and centrifuged. Supernatant was removed and solids was dried under high vacuum for 5 h to give 16.9 mg (17 % yield) of slightly yellow solid. HPLC / LRMS for C24H23FN2O5S [M+H]+calcd. 471.1 m / z, found 471.3 m / z.Example 6. Synthesis of Linker-Payload Conjugates of Formulae 11-1 through 11-5

[0709] 14.

[0710] Compounds of formula 11-1 through II-5 may be made according to Scheme

[0711] o oHO''Hnt47 ox'V°HBF3-Et2O HATU, DIPEA, DMSO lnt-50 - - DMSO lnt-49 lnt-48

[0712] lnt-6 DMTMM, DIPEA, 85:15 DMF: H2O

[0713] lnt-8 DMTMM, DIPEA, 85:15 DMF: H2O

[0714] lnt-9 DMTMM, DIPEA, 85:15 DMF: H2O ox-b DBU THF lnt-12

[0715] HATU, DIPEA, lnt-51 DMSO lnt-13 DMTMM, DIPEA, 85:15 DMF: H2O o

[0716]

[0717] scheme 14. Synthesis of Compounds I through n-5.The syntheses of Compounds 11-1 through II-5 are described below.

[0718] Intermediate 49: Intermediate 47 (78.3 mg, 0.543 mmol) and Intermediate 48 (0.297 g, 0.806 mmol, commercially available) were dissolved in DMSO (3 mL). Boron trifluoride diethyl etherate (0.2 mL, 2 mmol) was added dropwise. The resulting mixture was stirred at room temperature for 26 h. Water (0.2 mL) was added to quench the reaction. The mixture was directly purified with reverse column chromatography using 10-100% acetonitrile (0.1% formic acid) / water (0.1% formic acid). Fractions containing desired product were combined and solvent was removed by rotary evaporation to give 88 mg (36% yield) of solid. LC / MS for C24H24N2O7 [M+H]+calcd. 453.2 m / z, found 453.1 m / z.

[0719] Intermediate 50: DIPEA (0.1 mL, 0.6 mmol) was added to a solution of exatecan mesylate (71.3 mg, 0.164 mmol), Intermediate 49 (88.67 mg, 0.196 mmol), and HATU (91.5 mg, 0.241 mmol) in DMSO (2 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The mixture was directly purified with reverse chromatography using 30-100% acetonitrile (0.1% formic acid / water (0.1% formic acid). Fractions containing product were pooled and solvent removed by rotary evaporation to give 63 mg (44% yield) of white solid. LC / MS for C48H44FN5O10 [M+H]+calcd. 870.3 m / z, found 870.5 m / z.

[0720] Intermediate 51: Intermediate 50 (149 mg, 0.171 mmol) was dissolved in THF (5 mL) at room temperature and DBU (43 pL, 0.34 mmol, 2 eq) was added. The resulting mixture was stirred at room temperature for 45 min. The suspension was vacuum filtered, and the solids were washed with cold THF (2 mL) to give 105 mg (95% yield) of white solid. LC / MS for C33H34FN5O8 [M+H]+calcd. 648.2 m / z, found 648.2 m / z.

[0721] Compound 11-1: DIPEA (20 pL, 0.115 mmol) was added to a mixture of Intermediate 51 (25.5 mg, 0.039 mmol), Intermediate 6 (29.2 mg, 0.43 mmol) and DMTMM (20.18 mg, 0.073 mmol) in 85:15 DMF: deionized water (2 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was directly purified with reverse column chromatography using 10-100% MeCN (0.1% formic acid) / water (0.1% formic acid). Fractions containing desired product were combined and solvent was removed by rotary evaporation to give 24 mg (47% yield) of solid. LC / MS for C59H66FN11O17S [M+H]+ calcd. 1252.3 m / z, found 1252.25 m / z.

[0722] Compound II-2: DIPEA (20 pL, 0.115 mmol) was added to a mixture of Intermediate 51 (21.4 mg, 0.033 mmol), Intermediate 8 (23.6 mg, 0.035 mmol) and DMTMM (18.19 mg, 0.066 mmol) in 85:15 DMF:deionized water (1.5 mL) at room temperature. The resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was purified withreverse column chromatography using 20-100% MeCN (0.1% formic acid) / water (0.1% formic acid). Fractions containing desired product were combined and solvent was removed by rotary evaporation to give 30.56 mg (74% yield) of solid. LC / MS C60H67FN10O17S [M+H]+calcd. 1251.5 m / z, found 1251.3 m / z.

[0723] Compound II-3: DIPEA (15 pL, 0.086 mmol) was added to a mixture of Intermediate 51 (21.6 mg, 0.035 mmol), Intermediate 9 (17 mg, 0.02 mmol), and DMTMM (10.62 mg, 0.034 mmol) in DMF / water (85 / 15) (1.0 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The reaction mixture purified, without work-up, by flash C18 chromatography eluting with deionized water (0.1% formic acid) and a linear gradient of acetonitrile 5-50% over 18 column volumes. Fractions containing desired product were combined and solvent was removed by rotary evaporation to give 12.3 mg (36% yield) of solid. LC / MS for C58H65FN12O17S [M+H]+calcd. 1253.3 m / z, found 1253.5 m / z.

[0724] Compound II-4: DIPEA (20 pL, 0.115 mmol) was added to a solution of Intermediate 51 (26.69 mg, 0.041 mmol), Intermediate 12 (17 mg, 0.027 mmol), and HATU (14.01 mg, 0.037 mmol) in DMSO (0.8 mL) at room temperature. The resulting mixture was stirred at room temperature for 1.33 h. The reaction mixture was directly purified by reverse chromatography eluting with deionized water (0.1% formic acid) and a linear gradient of acetonitrile (0.1% formic acid) from 10-40% to afford 15.7 mg of compound II-4 (46% yield) as a solid. LC / MS for C58H65FN12O17S [M+H]+calcd. 1253.3 m / z, found 1253.42 m / z.

[0725] Compound II-5: DIPEA (13 pL, 0.075 mmol) was added to a solution of Intermediate 51 (18.93 mg, 0.023 mmol), Intermediate 13 (11.30 mg, 0.018 mmol), and DMTMM (10.75 mg, 0.039 mmol) in 85:15 DMF:deionized water (1 mL) at room temperature. The resulting mixture was stirred at room temperature for 1.75 h. The reaction mixture was directly purified by reverse chromatography eluting with deionized water (0.1% formic acid) and a linear gradient of acetonitrile (0.1% formic acid) from 10-40% to afford 3.02 mg compound II-5 (13% yield) as a solid. LCMS for C58H65FN12O17S [M+H]+calcd. 1253.3 m / z, found 1253.42 m / z.

[0726] Example 7. Synthesis of Linker-Payload Conjugates of Formula 11-6 through 11-10 Compounds of formulae II-6 through 11-10 may be made according to Scheme 15.lnt-48

[0727]

[0728] Scheme 15. Synthesis of Compounds II-6, II-7, II-8, II-9, and 11-10The synthesis of Compounds II-6 and II-7 is shown in Scheme 15 and described below. Intermediate 20: Intermediate 48 (140 mg, 0.38 mmol) and Compound D-2 (113 mg, 0.25 mmol) were weighed into a 20 mL flask equipped with a magnetic stir bar. To which was added anhydrous DMSO (3.0 mL) and boron trifluoride diethyl etherate (100 pL, 0.81 mmol) at room temperature. The reaction was stirred overnight. A 30 gram BioTage C18 cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 24 mL / min. Deionized water (0.2 mL) was added to the reaction and after the solution was injected onto the column then eluted with deionized water (containing 0.5% formic acid) with a linear gradient of 5% - 95% acetonitrile over 20 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 107 mg (56% yield) of product as a solid. HPLC / LRMS for C42H39FN4O9 [M+H]+calcd. 763.3 m / z, found 763.4 m / z.

[0729] Intermediate 21: Intermediate 20 (80 mg, 0.105 mmol) was weighed into a small vial equipped with a magnetic stir bar. Anhydrous THF (2 mL) was added to the vial followed by TMG (30 pL, 0.26 mmol), then stirred at room temperature for 2 h. The resulting suspension was transferred to small test tubes and centrifuged to pellet solids. Supernatant was removed using a pipette then solids were resuspended by addition of anhydrous THF (1 mL). The suspension was centrifuged and supernatant was removed. The test tubes were placed in a vial that was put under high vacuum to remove residual solvent, giving 23.8 mg (76% yield) of product. HPLC / LRMS for C27H29FN4O7 [M+ H]+calcd. 541.2 m / z, found 541.2 m / z.

[0730] Compound II-6: Intermediate 21 (13 mg, 0.025 mmol) and Intermediate 6 (17 mg, 0.026 mmol) were dissolved in a solution of 85:15 DMF: water (1.0 mL) and TEA (20 pL, 0.14 mmol). To which, DMTMM (20 mg, 0.072 mmol) was added and stirred at room temperature for 30 min. A 30 gram BioTage C18 cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 24 mL / min. The reaction mixture was injected onto the column then eluted with deionized water (containing 0.5% formic acid) and a linear gradient of 5% - 65% acetonitrile over 32 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 7.5 mg (25% yield) of product as a solid.

[0731] HPLC / LRMS for C57H79FN10O15S [M+H]+calcd.1195.5 m / z, found 1195.5 m / z.

[0732] Compound II-7: Intermediate 21 (18.5 mg, 0.034 mmol) and Intermediate 8 (28 mg, 0.045 mmol, 1.3 eq) were dissolved in 85:15 DMF:deionized water (1 mL) to which DMTMM (17 mg, 0.062 mmol, 1.8 eq) and triethylamine (27 pL, 0.194 mmol, 5.7 eq) were added and stirred at room temperature for 10 min. UPLC analysis indicated complete reaction. A 30 gbiotage C18 column equipped with a corresponding C18 samplet was equilibrated with 3 CV of 95:5 deionized water (0.1% formic acid): acetonitrile at a flow rate of mL / min. The reaction mixture was injected onto the column then the column was run at 5% acetonitrile for 3 CV followed by a linear gradient of 5% - 60% acetonitrile over 24 CV. Fractions containing desired product 90% pure or higher by UPLC were combined, pool UPLC purity 95+%, and solvent was removed by rotary evaporation with a bath temperature of 23 °C. Residue was put under high vacuum for 5 h giving 16.6 mg of product as a solid (42 % yield).

[0733] HPLC / LRMS for C53H61FN10O16S [M+H]+calcd. 1145.4 m / z, found 1145.5 m / z.

[0734] Compounds of formulae 11-8 through 11-10 may be made by methods analogous to those described herein.Example 7A. Synthesis of Linker-Payload Conjugates comprising Payload Moieties of Formulae D-5 through D-7

[0735] Linker-Payload Conjugates comprising Payload Moieties of Formulae D-5 through D-7 may be made according to Scheme 15A.

[0736]

[0737] Scheme 15A. Synthesis of Linker-Payload Conjugates comprising Payload Moieties of Formulae D-5 through D-7Example 7B. Synthesis of Linker-Payload Conjugates comprising Payload Moieties of Formulae D-8 through D-10 Linker-Payload Conjugates comprising Payload Moieties of Formulae D-8 through D-10 may be made according to Scheme 15B.

[0738]

[0739] Scheme 15B. Synthesis of Linker-Payload Conjugates comprising Payload Moieties of Formulae D-8 through D-10Example 8. Synthesis of Linker-Payload Conjugates of Formula 11-11 through 11-15

[0740] Compounds of formulae 11-11 through 11-15 may be made according to Scheme 16.

[0741] o o

[0742]

[0743] Scheme 16. Synthesis of Compounds 11-11, 11-12, 11-13, 11-14, and 11-15The synthesis of Compounds 11-11, 11-12, and 11-14 is shown in Scheme 16 and described below.

[0744] Intermediate 44: Intermediate 48 (13 mg, 0.035 mmol, 1.2 eq, commercially available) and Compound D-15 (13 mg, 0.029 mmol) were weighed into vial equipped with a magnetic stir bar. To which was added anhydrous DMSO (0.25 mL) and boron trifluoride diethyl etherate (6.8 pL, 0.055 mmol, 1.94 eq) at room temperature. The reaction was stirred overnight. A 12 gram BioTage C18 flash cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 45 mL / min. Approximately half of the reaction mixture was transferred to a scintillation vial and 1:1 DMSO: Deionized water (0.2 mL) was added to it then vortexed for 1 min. The solution was quickly injected onto the column, which was eluted with 95:5 deionized water (containing 0.5% formic acid): acetonitrile 3 column volumes then with a linear gradient of 5% - 95% acetonitrile over 24 column volumes. The process was repeated for the other -half of the reaction solution. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 12 mg (57% yield) of product as a solid. HPLC / LRMS for C41H37FN4O9 [M+H]+calcd. 749.3 m / z, found 749.2 m / z.

[0745] Intermediate 45: Intermediate 44 (10.3 mg, 0.020 mmol) was weighed into a small vial equipped with a magnetic stir bar. Anhydrous THF (0.3 mL) was added to the vial followed by DBU (2.7 pL, 1.82 mmol, 0.93 eq), then stirred at room temperature for 2 h. The resulting suspension was vacuum filtered and washed with anhydrous THF (0.3 mL). Solid was placed under high vacuum for 4 h to give 8.5 mg (82.6 % yield) of product. HPLC / LRMS for C26H27FN4O7 [M+H]+calcd. 527.2 m / z, found 527.4 m / z.

[0746] Compound 11-11: DIPEA (150 pL, 0.862 mmol) was added to a mixture of Intermediate 6 (140.74 mg, 0.209 mmol), Intermediate 45 (130 mg, 0.247 mmol) and HATU (123 mg, 0.323 mmol) in DMSO (2.8 mL) was added. The resulting mixture was stirred at room temperature for 40 min. The reaction mixture was directly purified with reverse chromatography using 25-35% MeCN / water (both having 0.1% formic acid, 30 g) to afford 88.8 mg (36% yield) of Compound 11-11 as a solid. LCMS for C56H77FN10O15S [M+H]+calcd. 1181.4 m / z, found 1181.2 m / z.

[0747] Compound 11-12: DIPEA (20 pL, 0.115 mmol) was added to a mixture of Intermediate 8 (16.55 mg, 0.027 mmol), Intermediate 45 (13.2 mg, 0.025 mmol) and HATU (20.96 mg, 0.055 mmol) in DMSO (0.5 mL) was added. The resulting mixture was stirred at room temperature for 45 min. The reaction mixture was directly purified with reverse chromatography using 25-40% MeCN / water (both having 0.1% formic acid) (12 g column) toafford 5.97 mg (21% yield) of Compound 11-12 as a solid. LCMS for C52H59FN10O16S [M+H]+calcd. 1131.3 m / z, found 1131.6 m / z.

[0748] Compound 11-14: DIPEA (22 pL, 0.126 mmol) was added to a mixture of Intermediate 12 (19.42 mg, 0.031 mmol), Intermediate 45 (19.09 mg, 0.036 mmol) and HATU (25.82 mg, 0.068 mmol) in DMSO (0.7 mL) was added. The resulting mixture was stirred at room temperature for 35 min. The reaction mixture was directly purified with reverse chromatography using 10-35% MeCN / water (both having 0.1% formic acid) to afford 15.3 mg (43% yield) of Compound 11-14 as a solid. LCMS for C51H58FN11O16S [M+H]+calcd. 1132.4 m / z, found 1132.4.

[0749] Compounds of formulae 11-13 and 11-15 may be made by methods analogous to those described herein.Example 9. Synthesis of Linker-Payload Conjugates of Formula 11-16 through 11-20

[0750] Compounds of formulae 11-16 through II-20 may be made according to Scheme 17.

[0751]

[0752] Scheme 17. Synthesis of Compounds 11-16, 11-17, 11-18, 11-19, and II-20The synthesis of Compounds 11-17 and 11-19 is shown in Scheme 17 and described below.

[0753] Intermediate 42: Intermediate 48 (40.7 mg, 0.11 mmol, commercially available) and Compound D-16 (40 mg, 0.085 mmol) were weighed into a vial equipped with a magnetic stir bar. To which was added anhydrous DMSO (1.0 mL) and boron trifluoride diethyl etherate (28 pL, 0.22 mmol) at room temperature and stirred overnight. A 12 gram BioTage C18 flash cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 10 mL / min. The solution was quickly injected onto the column, which was eluted with 95:5 deionized water (containing 0.5% formic acid): acetonitrile 3 column volumes then with a linear gradient of 5% - 95% acetonitrile over 22 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 37 mg (56% yield) of product as a solid. HPLC / LRMS for C41H37FN4O9 [M+H]+calcd. 749.3 m / z, found 749.2 m / z.

[0754] Intermediate 43: Intermediate 42 (44 mg, 0.057 mmol) was weighed into a small vial equipped with a magnetic stir bar. Anhydrous THF (0.9 mL) was added to the vial followed by DBU (9.0 pL, 0.057 mmol), then stirred at room temperature for 2 h. The resulting suspension was centrifuged, supernatant removed then THF (0.5 mL) was added, triturated and centrifuge. Supernatant was removed and solid was placed under high vacuum for 4 h to give 25 mg (80% yield) of product. HPLC / LRMS for C27H29FN4O6S [M+H]+calcd. 557.2 m / z, found 557.1 m / z.

[0755] Compound 11-17: Intermediate 43 (21 mg, 0.037 mmol) and Intermediate 8 (40 mg, 0.06 mmol) were dissolved in a solution of 85:15 DMSO: water (0.75 mL) and TEA (44 pL, 0.316mmol). To which, DMTMM (22 mg, 0.080 mmol) was added and stirred at room temperature for 30 min. A 12 gram BioTage C18 flash cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of 10 mL / min. The reaction mixture was injected onto the column then eluted with deionized water (containing 0.5% formic acid) and a linear gradient of 5% -85% acetonitrile over 32 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 16 mg (37 % yield) of product as a solid. HPLC / LRMS for C53H61FN10O15S [M+H]+calcd.1161.4 m / z, found 1161.2 m / z.

[0756] Compound 11-19: Intermediate 43 (22 mg, 0.039 mmol) and Intermediate 12 (25 mg, 0.04 mmol) were dissolved in a solution of DMSO (0.50 mL) and DIPEA (14 pL, 0.08 mmol). To which, HATU (25.8 mg, 0.068 mmol) was added and stirred at room temperature for 50 min. A 12 gram BioTage C18 flash cartridge equipped with a corresponding samplet was equilibrated with 95:5 deionized water (containing 0.5% formic acid): acetonitrile, flow rate of10 mL / min. The reaction mixture was injected onto the column then eluted with deionized water (containing 0.5% formic acid) and a linear gradient of 5% - 85% acetonitrile over 32 column volumes. Fractions containing the desired product were combined and solvent was removed by rotary evaporation to give 10 mg (27 % yield) of product as a solid.

[0757] HPLC / LRMS for C52H59FN10O16S [M+H]+calcd.1148.4 m / z, found 1148.6 m / z.

[0758] Compounds of formulae 11-16, 11-18, and II-20 may be made by methods analogous to those described herein.

[0759] Example 10. Synthesis of Binding Molecule- Linker- Payload Conjugates of Formula 111-1 through 111-20

[0760] A method for preparing antibody-drug conjugates involves the coupling of a linker-payload to reduced, inter-chain disulfide-forming cysteine residues of an antibody. In the compounds of formulae III-1 through III-20, the antibody is trastuzumab.

[0761] The antibody was first reduced at a concentration of 2.0-8.0 mg / mL using a 5.5 molar equivalent of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in a reduction buffer comprising 20 mM potassium phosphate (K-Pi), 150 mM sodium chloride (NaCI), and 1 mM ethylenediaminetetraacetic acid (EDTA), adjusted to pH 6.9. The reduction reaction was performed at 37°C for 90 minutes to ensure complete reduction of disulfide bonds.

[0762] Following reduction, a linker-payload solution, prepared at a concentration of approximately 20 mM in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA), was added slowly to the reduced antibody at a molar excess of 10-20. The final concentration of DMSO or DMA in the reaction mixture was maintained at 10% of the total reaction volume. The reaction mixture is thoroughly mixed and incubated at room temperature (RT) for 2 hours to facilitate conjugation.

[0763] Upon completion of the conjugation reaction, the resulting antibody-drug conjugate (ADC) was purified to remove excess unreacted linker-payload by gel filtration. The purified ADC was formulated into a buffer comprising 20 mM histidine, 120 mM sodium chloride, 50 mM sucrose, and 0.02% polysorbate 20, adjusted to pH 6.0. The formulated ADC was subsequently filtered through a 0.2 pm filter under sterile conditions.

[0764] The resulting ADC is typically monomeric and achieves a drug-to-antibody ratio (DAR) of approximately 8 under these conditions. The ADC is characterized by the following methods:• Protein concentration is determined by bicinchoninic acid (BCA) protein assay. Aggregation is assessed using size-exclusion chromatography (SEC).

[0765] • Drug-to-antibody ratio (DAR) is quantified using liquid chromatography-mass spectrometry (LC-MS).

[0766] • Residual free toxin is measured using reverse-phase high-performance liquid chromatography (RP-HPLC)

[0767] The characterisation of the ADCs of Formulae 111-1, HI-2, HI-3, HI-4, HI-5, HI-6, HI-7, HI-11, HI-12, and HI-14 is summarised in Table 5.

[0768] ADC Cone, (mg / ml) DAR Monomer % Free toxin content

[0769] HI-1 2.15 8.0 100.0% <2%

[0770] HI-2 2.28 8.0 98.6% <2%

[0771] HI-3 6.07 8.0 Not determined <2%

[0772] HI-4 10.8 7.8 Not determined <2%

[0773] HI-5 12.3 7.9 Not determined <2%

[0774] HI-6 2.24 8.0 100.0% <2%

[0775] HI-7 2.83 8.0 99.7% <2%

[0776] HI-11 2.47 8.0 100.0% <2%

[0777] HI-12 6.66 7.9 98.1% <2%

[0778] HI-14 8.88 8.1 98.2% <2%

[0779]

[0780] Table 5

[0781] Biological Example 1. Cytotoxicity of Binding Molecule- Linker- Payload Conjugates of Formula III-1, III-2, III-3, III-4, III-5, III-6, III-7, III-11, III-12, and III-14

[0782] The cell line SK-BR-3 utilized in the cytotoxicity studies, was obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC-recommended protocols.

[0783] For the cytotoxicity assays, SK-BR-3 cells were seeded into 96-well culture microplates. Following overnight incubation, the cells were exposed to a series of ADC concentrations for 120 hours. The cytotoxic effect of the ADCs on cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), a widely used method that quantifies viable cells by measuring ATP levels in metabolically active cells, following the protocol provided by the manufacturer.The surviving fraction was determined by dividing the luminescence signal from wells treated with the ADCs by the luminescence signal from untreated control wells. These data were used to construct dose-response curves, and the half-maximal inhibitory concentration (IC50) values were calculated using a four-parameter variable slope model with GraphPad Prism software.

[0784] The results are shown in Figures 1 to 3 and in Table 6.

[0785] ADC IC50 (nM) in SKBR3 cells

[0786] III-1 0.58III-2 0.36III-3 0.17III-4 0.13III-5 0.15III-6 0.25III-7 0.24III-11 0.07III-12 0.11III-14 0.06

[0787]

[0788] Table 6

[0789] As shown in Table 6, all ADCs exhibited IC50 values in the sub-nanomolar range.

[0790] All publications mentioned in the above specification are herein incorporated by reference.

[0791] Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biochemistry, molecular biology, biotechnology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A linker of formula (L):wherein:R is C1-6 alkyl;EO is an ethylene glycol moiety, the terminal carbon of which is attached to the group -N(R)-and the terminal oxygen of which is attached to the group -(CH2)w- when w is 1 to 10, or to the - C(=O) group when w is 0;v is 0 or 1 to 40;w is 0 or 1 to 10;P is a peptide moiety consisting of 1 to 10 amino acid residues;R6and R7are each independently H or C1-6alkyl, or R6and R7together with the carbon atom to which they are attached form a 3- to 10-membered carbocyclic ring;position (1) is a first attachment point; andposition (2) is a second attachment point.

2. A linker according to claim 1, wherein R is methyl.

3. A linker according to claim 1 or 2, wherein P consists of 1 to 6 amino acid residues.

4. A linker according to any one of claims 1 to 3, wherein P comprises any of AA1, AA1-Gly, Val-Cit, Vai-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn, Glu-Val-Cit, Glu-Val-AA1, Glu-Glu, Glu-Gly-Cit, Glu-Gly-AA1, or Gly-Gly- AA1-Gly (GGFG) where AA1represents any amino acid residue.

5. A linker according to any one of claims 1 to 4, wherein P comprises any of AA1, AA1-Gly, Val-Cit, Vai-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn, Glu-Val-Cit, Glu-Val-AA1, Glu-Glu, Glu-Gly-Cit, Glu-Gly-AA1, Gly-Gly-Phe-Gly (GGFG) or Gly-Gly-Pya-Gly, where AA1represents any amino acid residue and Pya represents a (2-pyridyl)alanine, (3-pyridyl)alanine or (4-pyridyl)alanine residue.

6. A linker according to claim 4 or 5, wherein the amino acid residue represented by AA1isselected from NHBoc7. A linker according to claim 4 or 5, wherein P is a Val-AA1-Gly moiety, where AA1represents8. A linker according to claim 4 or 5, wherein P is a GGFG peptide moiety.

9. A linker according to claim 4 or 5, wherein P is a GG(Pya)G peptide moiety wherein Pya represents a (2-pyridyl)alanine, (3-pyridyl)alanine or (4-pyridyl)alanine residue.

10. A linker according to any one of claims 1 to 9, wherein v is 2.

11. A linker according to any one of claims 1 to 10, wherein w is 1.

12. A linker according to any one of claims 1 to 11, wherein R6and R7are each independently H.c? 13. A linker according to any one of claims 1 to 12, which is selected from the group consisting of linkers L1 to L5: J Cpds FormulaL1z—ooL2 IZ° H 9 H 9 H _ JL _ 0 _ _ N A N A N (2) odrN ZI\11" \ / / o wO=— / / \> Z 7LZ—L30 z — H 9 H J ^ 9° / =L O N A,..O=ZZE H ^. N JL| / XN (2)" TO T- QoL4L5 ° H 9 H 9 H _ JI _ 0 _ _ N A N A N (2)d14. A compound of formula (II):(II) or a pharmaceutically acceptable salt thereof, wherein:R, EO, v, w, P, R6, and R7are as defined in any one of claims 1 to 12;Y1is a group capable of conjugating with a binding molecule to generate a conjugate;U is O or S; andD is a payload moiety linked through the U atom.

15. A compound according to claim 14, wherein Y1is a leaving group.

16. A compound according to claim 15, wherein Y1comprises a sulfone moiety.

17. A compound according to any one of claims 14 to 16, wherein U is O.

18. A compound according to any one of claims 14 to 17, wherein D comprises a cytotoxic drug selected from a tubulin polymerisation inhibitor, a DNA damaging agent, a TOPO1 inhibitor, an auristatin, a maytansinoid, or a calicheamicin.

19. A compound according to any one of claims 14 to 18, wherein D comprises a TOPO1 inhibitor.

20. A compound according to claim 19, wherein the TOPO1 inhibitor is a camptothecin or an exatecan, or a derivative thereof.

21. A compound according to any one of claims 14 to 20, wherein D comprises a structure shown as formula (D-A):or a pharmaceutically acceptable salt or solvate thereof, wherein:A is C2-10alkenylene, C2-10alkynylene, C3-10cycloalkenylene, C8-10cycloalkynylene, C3-10heterocycloalkenylene, or C8-10heterocycloalkynylene;X is O or S;T is C1-6 alkylene;R8is H, halogen, C1-6alkyl, O-C1-6alkyl or S-C1-6alkyl;R2 is H, halogen, C1-6 alkyl, O-C1-6 alkyl or S-C1-6 alkyl;or R8and R2together with the carbon atoms to which they are attached form a 5-membered ring system; andR3 is Ci-6 alkyl.

22. A compound according to any one of claims 14 to 20, wherein D comprises a structure shown as formula (D-B):or a pharmaceutically acceptable salt or solvate thereof, wherein:X is a bond, O, S, S(=O) or S(=O)2;when X is O, S, S(=O) or S(=O)2, A is C2-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C3-10cycloalkenylene, C8-10cycloalkynylene, C3-10heterocycloalkylene, C3-10heterocycloalkenylene, or C8-10heterocycloalkynylene, each optionally substituted with one or more substituents selected from List 1;when X is a bond, A is C1-10alkylene, C2-10alkenylene, C2-10alkynylene, C3-10cycloalkylene, C3-10cycloalkenylene, C8-10cycloalkynylene, C3-10heterocycloalkylene, C3-10heterocycloalkenylene, or C8-10heterocycloalkynylene, each optionally substituted with one or more substituents selected from List 1;R1and R4are each independently H, C1-6alkyl, or C3-6cycloalkyl, or: R1and R4together with the carbon atoms to which they are attached form a 5- to 7-membered carbocyclic ring which is substituted by a group -NH-C(=O)-R5;R2 is H, halogen or C1-6 alkyl;R3 is C1-6 alkyl; andRs is C1-6 alkyl or-NR’R”, wherein R’ and R” are each independently H or C1-6 alkyl.

23. A compound according to any one of claims 14 to 22, wherein D is selected from a group of formula D-1 through D-17 or a pharmaceutically acceptable salt or solvate thereof:

24. A compound according to any one of claims 14 to 23, which is a compound of formula II-1 through II-20 or a pharmaceutically acceptable salt or solvate thereof:

25. A compound of formula (III):or a pharmaceutically acceptable salt thereof, wherein:BM is a binding molecule or a fragment thereof;R, EO, v, w, P, R6, and R7are as defined in any one of claims 1 to 12;U is as defined in claim 14 or 17;D is as defined in any one of claims 14 or 18 to 23; andq is an integer from 1 to 16.

26. A compound according to claim 25, wherein BM is an antibody or fragment thereof.

27. A compound according to claim 25 or 26, wherein BM specifically binds to a target expressed on the surface of a tumour cell.

28. A compound according to claim 27, wherein the target is selected from B7H3, TROP2, HER2 and HER3.

29. A pharmaceutical composition comprising a compound according to any one of claims 1-28 and a pharmaceutically acceptable carrier.

30. A compound according to any one of claims 1-28 for use in medicine.

31. A compound according to any one of claims 1-28 for use in treating cancer.

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