Antibody drug conjugates

Abstract

The invention relates to binding molecule-payload conjugates (BPCs), and specifically antibody-drug conjugates (ADCs), which comprise a binding molecule and one or more payload moieties, wherein the binding molecule specifically binds to MUC16.

Description

[0001] ANTIBODY DRUG CONJUGATES

[0002] FIELD OF THE INVENTION

[0003] The invention relates to binding molecule-payload conjugates (BPCs), and specifically antibody-drug conjugates (ADCs) and VHH-Fc fusion protein-drug conjugates, which comprise a binding molecule and one or more payload moieties, wherein the binding molecule specifically binds to MUC16.

[0004] BACKGROUND TO THE INVENTION

[0005] Mucin-16 (MUC16) is a protein that in humans is encoded by the MUC16 gene. MUC16 is a member of the mucin family glycoproteins. MUC16 is a highly glycosylated mucin composed of a large extracellular domain (CA-125), which is cleaved and released, and a retained domain (MUC-CD). MUC-CD comprises a non-repeating extracellular domain (MUC16 ectodomain) proximal to a cleavage site, a transmembrane domain and a cytoplasmic tail with potential phosphorylation sites. Distal to the cleavage site, the released extracellular domain (CA-125) contains 16-20 tandem repeats of 156 amino acids, each with many potential glycosylation sites.

[0006] MUC16 is overexpressed in several malignancies including ovarian, breast, lung, and pancreatic cancers and CA-125 is an established serum marker for the detection and progression of ovarian cancers. It has been reported that overexpression of MUC16 can promote unfavourable characteristics of cancer cells, including enhanced proliferation, tumour metastasis and facilitation of tumour immune escape via suppression of natural killer (NK) and macrophages. MUC16 is therefore a potentially attractive target for immune-based therapies, however cleaved CA-125 antigen present in circulation presents a significant challenge.

[0007] Antibody-drug conjugates (ADCs) comprise biologically active small molecule compounds conjugated to monoclonal antibodies or antibody fragments by chemical methods, so as to fully utilize antibodies’ binding specificity to normal cell and to tumour cell surface antigens, and small molecule’s high anti-tumour biological activity, while avoiding defects such as the low specific efficacy of the former as well as toxic side effects of the latter. Compared with traditional chemotherapeutic or targeted drugs, antibody-drug conjugates can more accurately bind to tumour cells and reduce their effects on normal cells.

[0008] There is an unmet need for safe and effective therapeutics targeting MUC16-positive cancers.SUMMARY OF THE INVENTION

[0009] The present inventors have identified binding molecule-payload conjugates (BPC), in particular antibody drug conjugates (ADC) and VHH-Fc fusion protein-drug conjugates, demonstrating an excellent efficacy and tolerability profile against MUC16-expressing cancers.

[0010] The invention provides a BPC comprising a binding molecule and one or more payload moieties; wherein:

[0011] a) the binding molecule is an antibody or a fragment thereof and comprises one or more of heavy chain complementarity determining regions (HCDRs) 1-3 and one or more of light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein:

[0012] HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3, LCDR1 comprises an amino acid sequence according to SEQ ID NO: 4, LCDR2 comprises an amino acid sequence according to SEQ ID NO: 5, and LCDR3 comprises an amino acid sequence according to SEQ ID NO: 6, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences; and

[0013] b) the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers.

[0014] In some embodiments, the binding molecule comprises all of the HCDRs 1-3 and all of the LCDRs 1-3.

[0015] In some embodiments, the binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 7, or a variant having at least 80% sequence identity thereto.

[0016] In some embodiments, the binding molecule comprises a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% sequence identity thereto.

[0017] In some embodiments, the binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 7, or a variant having at least 80% sequence identitythereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% sequence identity thereto.

[0018] The BPC according to the invention may be an ADC, wherein the binding molecule comprises a heavy chain and a light chain.

[0019] In some embodiments, the binding molecule comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% sequence identity thereto.

[0020] In some embodiments, the binding molecule comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% sequence identity thereto.

[0021] In some embodiments, the binding molecule comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% sequence identity thereto, and a light chain comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% sequence identity thereto.

[0022] The invention further provides a binding molecule-payload conjugate (BPC) comprising a binding molecule and one or more payload moieties; wherein:

[0023] a) the binding molecule comprises a VHH domain comprising one or more of complementarity determining regions (CDRs) 1-3 according to the IMGT numbering scheme, wherein:

[0024] CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences; and

[0025] b) the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers.

[0026] In some embodiments, the binding molecule comprises all of the CDRs 1-3.

[0027] In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 23, or a variant having at least 80% sequence identity thereto.

[0028] In some embodiments, the binding molecule comprises a Fc region. In some embodiments, the binding molecule is a VHH-Fc fusion protein.

[0029] The Fc region may be a modified Fc region.In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 29. In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 30. In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 29 and an amino acid sequence according to SEQ ID NO: 30.

[0030] In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 24, or a variant having at least 80% sequence identity thereto.

[0031] The one or more payload moieties may be covalently linked to the binding molecule via a linker.

[0032] In some embodiments, the linker comprises or is a peptide linker. In some embodiments, the peptide linker is selected from AA1, AA1-Gly, Val-Cit, Val-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn and Gly-Gly-Phe-Gly.

[0033] The one or more payload moieties may be independently selected from a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a colored agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumor cell growth, promotes tumor cell apoptosis or necrosis.

[0034] In some embodiments, the payload moiety is a drug. In some embodiments, the drug is a cytotoxic drug, immune modulator, or a STING inhibitor. In some embodiments, the cytotoxic drug is a tubulin inhibitor, a DNA damaging agent, a type I topoisomerase (TOPO1) inhibitor, an auristatin, a maytansinoid, or a calicheamicin.

[0035] In some embodiments, the conjugate has the structure represented by formula I:

[0036]

[0037] or a tautomer, a mesomer, a racemate, an enantiomer or a diastereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein,BM is the binding molecule;

[0038]

[0039] each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carboncarbon double bond, and amido (preferably selected from a direct bond, a carboncarbon triple bond, and a carbon-carbon double bond);

[0040] Rx and Ry are each independently selected from H and C1-4 alkyl;

[0041] each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;

[0042] y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6);

[0043] each y2 is independently selected from any integer between 0 and 15 (such as 6-15); each y3 is independently selected from 1, 2, and 3;

[0044] each y4 is independently selected from 0 and 1; position 1 is attached to BM via an S atom, and position 2 is attached to L2 or L3;

[0045]

[0046] and 6 (such as 4, 5, and 6); each y2 is independently selected from any integer between 0 and 10 (such as 6-10); each y3 is independently selected from 1 or 2; each y4 is independently selected from 0 and 1, position 1 is attached to Li, and position 2 is attached to L3;

[0047] L3is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from natural amino acid residues, nonnatural amino acid residues, or selected from amino acid residue represented by AA1or stereoisomer thereof;

[0048] o

[0049] H / \

[0050]

[0051] Ra / Rb

[0052] aa1; in the amino acid residue represented by AA1, any one of Raand Rbis H, pm1 Rm1a pm1b ■y n ^il — Rn1 Rn1a—Rn1b^ V / rl Wr1a lzWfl b -X and the other is

[0053]

[0054] H,,nand; or, Raand Rb, together with the carbon atom to which they are both attached, form a 5-6 membered heterocyclic ring, and said 5-6 membered heterocyclic ring is piperidine ring or piperazine ring.

[0055] r, r1, r1aand r1bare each independently 0, 1, 2, 3, 4 or 5;

[0056] Rm1, Rn1, Rm1a, Rn1a, Rm1band Rn1bare each independently H, C1-6 alkyl or -COORx1, wherein, Rx1is C1-6 alkyl;

[0057] or, Rm1and Rn1, Rm1aand Rn1a, and Rm1band Rn1b, together with the nitrogen atom to which they are both attached, form a 5-6 membered heterocyclic ring, and of said 5-6 membered heterocyclic ring, the heteroatom is selected from 1 or 2 N atoms; the said 5-6 membered heterocyclic ring is optionally substituted with one or more R0’;

[0058] Rzis selected from C1-6 alkyl;

[0059] R° and R0’ are each independently selected from C1-6 alkyl, -NRm2Rn2or 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl; of said 5-6 membered heterocyclyl, the heteroatom is selected from 1 or 2 N atoms;

[0060] Rm2and Rn2are each independently selected from H and C1-6 alkyl;

[0061]

[0062] position 2 is attached to W;

[0063] Ri and R2are each independently selected from H, halogens and C1-4 alkyl; or, Ri and R2, together with the carbon atom to which they are both attached form a 5-6 membered heterocyclic ring, the heterocyclic ring contains 1, 2, or 3 of O, S, or N or any combination thereof;

[0064] R3 is selected from H and C1-4 alkyl; or R3 and X, together with the carbon atom to which they are both attached, form a 5-6 membered carbon ring;

[0065] W is absent or present, when W is present, W is selected from -O-, -S-, -NR4-,

[0066]

[0067]

[0068] and, position 1 is attached to X, and position 2 is attached to L4 or L3;

[0069] fj7 R7

[0070] ('jh* MU, X is selected from optionally substituted -

[0071]

[0072] (CH2)ni-,v R?,R7, v position 1 is attached to the parent ring and position 2 is attached to W or L4; the substituent is selected from one or two C1-4 alkyls;

[0073] R4, Rs, and R? are each independently selected from H and C1-4 alkyl;

[0074] n, n1, n2, n3 are each independently selected from any integer between 0 and 6; and q represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

[0075] In some embodiments, the linker-payload comprises the structure:o

[0076]

[0077] In some embodiments, the conjugate is

[0078] o

[0079]

[0080] or a pharmaceutically acceptable salt thereof wherein

[0081] BM is the binding molecule as defined herein; and

[0082] q represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

[0083] In some embodiments, the connection number q is selected from the group consisting of integers 1 to 8. In some embodiments, the connection number q is selected from the group consisting of integers 4 to 8. In some embodiments, the connection number q is 8.

[0084] The invention also provides a method for producing a BPC according to the invention, comprising contacting a binding molecule as defined herein with a suitable linker-payload compound.

[0085] The invention also provides a composition comprising a BPC according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.The invention also provides a method of treating or diagnosing a disease, comprising administering the BPC or composition according to the invention to a subject.

[0086] The invention also provides the BPC or composition according to the invention for use in a method of therapy or a diagnostic method.

[0087] The method may be a method of treating, preventing or diagnosing cancer.

[0088] In some embodiments, the cancer expresses MUC16. In some embodiments, the expression of MUC16 is increased compared to the expression of MUC16 by the same non-cancerous tissue or cells.

[0089] In some embodiments, the cancer is ovarian cancer, pancreatic cancer, breast cancer, lung cancer, oesophageal cancer, prostate cancer, bladder cancer or endometrial cancer.

[0090] DESCRIPTION OF THE FIGURES

[0091] Figure 1 - (a) Binding of BNT-ADC-003, of BNT-mAb-003, or BNT-ADC-002-IC to human MUC16-ECD by ELISA, (b-c) Binding of BNT-mAb-003 and human IgG1 isotype control to human MUC16-ECD by ELISA. Binding of BNT-ADC-003 and rituximab to (d) FcyRI; (e) FcyRlla (H131); (f); FcyRlla (R131) (g); FcyRllb (h) FcyRllla (F158); (i) FcyRllla (V158); (j) FcyRlllb; (k) FcRn at pH 6.0; and (I) FcRn at pH 7.4 by SPR. (m) Binding of BNT-ADC-003, rituximab and human IgG4 isotype control to human C1q by ELISA.

[0092] Figure 2 - Binding of BNT-ADC-003, BNT-mAb-003 or BNT-ADC-002-IC to (a) OVCAR-3 and (b) SW1990 cells by FACS.

[0093] Figure 3 - Cytotoxicity of BNT-ADC-003, BNT-mAb-003, or BNT-payload-002 against (a) OVCAR-3 and (b) SW1990 cells.

[0094] Figure 4 - (a, b) Internalisation of BNT-ADC-003, BNT-mAb-003, BNT-ADC-002-IC or BNT-payload-002 in OVCAR-3 cells.

[0095] Figure 5 - Complement dependent cytotoxicity of BNT-ADC-003, BNT-mAb-003, or BNT-ADC-002-IC against (a, b) OVCAR-3 and (c) SW1990 cells.

[0096] Figure 6 - Antibody dependent cellular cytotoxicity of BNT-ADC-003, BNT-mAb-003, or BNT-ADC-002-IC against (a, c, d) OVCAR-3 and (b) SW1990 cells.

[0097] Figure 7 - In vivo efficacy study in OVCAR-3 CDX model, (a) T umour volume (b) body weight and (c) percent survival following treatment with PBS control, BNT-ADC-002-IC (1 mg / kg),BNT-mAb-003-IC (1 mg / kg), or BNT-ADC-003 (0.3, 1, 3 or 8 mg / kg). Survival curve was calculated based on tumour volume 1000 mm3.

[0098] Figure 8 - In vivo efficacy study in OVCAR-3 CDX model, (a) T umour volume (b) body weight and (c) percent survival following treatment with PBS control, BNT-ADC-002-IC (3 mg / kg), or BNT-ADC-003 (1, 3 or 8 mg / kg). Survival curve was calculated based on tumour volume 1000 mm3.

[0099] Figure 9 - In vivo efficacy study in SW1990 CDX model, (a) Tumour volume (b) body weight and (c) percent survival following treatment with PBS control, BNT-ADC-002-IC (3 mg / kg), BNT-mAb-003 (3 mg / kg) or BNT-ADC-003 (1, 3 or 8 mg / kg). Survival curve was calculated based on tumour volume 1000 mm3.

[0100] Figure 10 - In vivo mouse pharmacokinetic study. Concentration of ADC in plasma detected by (a) Fc+Fc ELISA and (b) Fc+anti-payload ELISA.

[0101] Figure 11 - (a) Average and (b) individual body weight of mice following a single intravenous dose of PBS or BNT-ADC-003 (20, 60 or 180 mg / kg).

[0102] DETAILED DESCRIPTION OF THE INVENTION MUC16

[0103] The present invention provides a binding molecule-payload conjugate (BPC) comprising a binding molecule and one or more payload moieties, wherein the binding molecule specifically binds to MUC16.

[0104] In some embodiments, MUC16 is considered the antigen of the binding molecule according to the present invention.

[0105] In some embodiments “specifically binds to” may indicate that the binding molecule binds to the antigen, i.e. MUC16, in preference to other antigens.

[0106] In embodiments, it will be understood herein that “specifically binds to” refers to the antibodylike binding of the binding molecule, which may be via heavy chain complementarity determining regions (HCDRs) 1-3 and / or light chain complementarity determining regions (LCDRs) 1-3, to the target MUC16. Thus, in embodiments, it will be understood herein that the term “specifically” does not exclude the binding molecule from having other targets.

[0107] The term “MUC16” (or “Mucin-16”) relates to the protein that in humans is encoded by the MUC16 gene. MUC16 is a highly glycosylated mucin composed of a large extracellulardomain (CA-125) containing 16-20 tandem repeats of 156 amino acids, which is cleaved and released, and a retained domain (MLIC-CD). MUC16 is predicted to harbour ~56 SEA (sea urchin sperm protein, enterokinase and agrin) domains, and is thought to be cleaved at a site in the SEA domains. MLIC-CD comprises a non-repeating extracellular domain (MUC16 ectodomain), a transmembrane domain and a cytoplasmic tail. The N-terminal and tandem repeat domains are highly O-glycosylated.

[0108] An exemplary human MUC16 sequence is provided in SEQ ID NO: 31.

[0109] MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIWTEHTLPFTSPDKTLASPTSSWGRT TQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSSTEGNFTKEASTYTLT VETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVTDSHTPGR TNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEPGSAGHSRISTSAPLSSSASVLDNKISETSI FSGQSLTSPLSPGVPEARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMD IPSTHIAKTLASEWLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESE MTATLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGRQSSSTAAHGSSDILRATTSSTSKASSWTSESTA QQFSEPQHTQWVETSPSMKTERPPASTSVAAPITTSVPSWSGFTTLKTSSTKGIWLEETSADTLIGESTAGPTT HQFAVPTGISMTGGSSTRGSQGTTHLLTRATASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMV SSVIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKISTSIPLLSSASVLEDKVSATSTFSHHKATS SITTGTPEISTKTKPSSAVLSSMTLSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLSTSAETTDSPNIHPTG TLTSESSESPSTLSLPSVSGVKTTFSSSTPSTHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPT MDTWPTRSAQFSSSHLVSELRATSSTSVTNSTGSALPKISHLTGTATMSQTNRDTFNDSAAPQSTTWPETSPRFK TGLPSATTTVSTSATSLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTNGPGSMAVASTNIPIGKGYITEG RLDTSHLPIGTTASSETSMDFTMAKESVSMSVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGT SSALTPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVTMSSTFSTQRVTTSMIMDTVETSRWNMPNLPSTTS LTPSNIPTSGAIGKSTLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSESPSTIKLTMASWKP GSYTPLTFPSIETHIHVSTARMAYSSGSSPEMTAPGETNTGSTWDPTTYITTTDPKDTSSAQVSTPHSVRTLRTT ENHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTQLHYTKLAEKSSGFETQSAPGPVSWIPTSP TIGSSTLELTSDVPGEPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSPSSPMSTFAIFPPMSTPSHELS KSEADTSAIRNTDSTTLDQHLGIRSLGRTGDLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLKDQT SIPASASPSHLTEVYPELGTQGRSSSEATTFWKPSTDTLSREIETGPTNIQSTPPMDNTTTGSSSSGVTLGIAHL PIGTSSPAETSTNMALERRSSTATVSMAGTMGLLVTSAPGRSISQSLGRVSSVLSESTTEGVTDSSKGSSPRLNT QGNTALSSSLEPSYAEGSQMSTSIPLTSSPTTPDVEFIGGSTFWTKEVTTVMTSDISKSSARTESSSATLMSTAL GSTENTGKEKLRTASMDLPSPTPSMEVTPWISLTLSNAPNTTDSLDLSHGVHTSSAGTLATDRSLNTGVTRASRL ENGSDTSSKSLSMGNSTHTSMTYTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLTLPFSSIPVEEVISTG ITSGPDINSAPMTHSPITPPTIVWTSTGTIEQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSVSSGSS TSSPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLSEALSSGHSGVSNPSSTTTEFPLFSAASTSA AKQRNPETETHGPQNTAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLTSESTANPSLGTASSA GTKLTRTISLPTSESLVSFRMNKDPWTVSIPLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTV SFSPSPDTEVTTISHFPEKTTHSFRTISSLTHELTSRVTPIPGDWMSSAMSTKPTGASPSITLGERRTITSAAPT TSPIVLTASFTETSTVSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDVTKTASISPTSISGMT ASSSPSLFSSDRPQVPTSTTETNTATSPSVSSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTF STMVSTDTASGENPTSSNSWTSVPAPGTWTSVGSTTDLPAMGFLKTSPAGEAHSLLASTIEPATAFTPHLSAAV VTGSSATSEASLLTTSESKAIHSSPQTPTTPTSGANWETSATPESLLWTETSDTTLTSKILVTDTILFSTVSTP PSKFPSTGTLSGASFPTLLPDTPAIPLTATEPTSSLATSFDSTPLVTIASDSLGTVPETTLTMSETSNGDALVLK TVSNPDRSIPGITIQGVTESPLHPSSTSPSKIVAPRNTTYEGSITVALSTLPAGTTGSLVFSQSSENSETTALVD SSAGLERASVMPLTTGSQGMASSGGIRSGSTHSTGTKTFSSLPLTMNPGEVTAMSEITTNRLTATQSTAPKGIPV KPTSAESGLLTPVSASSSPSKAFASLTTAPPTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTA SSSLSKSPEKNPRARMMTSTKAI SASSFQSTGFTETPEGSASPSMAGHEPRVPTSGTGDPRYASESMSYPDPSKA SSAMTSTSLASKLTTLFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPSMARQPNILVHLQTSA LTLSPTSTLNMSQEEPPELTSSQTIAEEEGTTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISV PAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEETGSSPAGTSPGSPEMSTTLKIMSSKEPSISPEIRSTVR NSPWKTPETTVPMETTVEPVTLQSTALGSGSTSISHLPTGTTSPTKSPTENMLATERVSLSPSPPEAWTNLYSGT PGGTRQSLATMSSVSLESPTARSITGTGQQSSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTS PNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAEQQTSRSVDEAYSSTSSWSDQTSGSDITLGASPDVTNTL YITSTAQTTSLVSLPSGDQGITSLTNPSGGKTSSASSVTSPSIGLETLRANVSAVKSDIAPTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTPNPEVGMSTMDSTPATERRTTSTEHPSTWSSTAASDSWTVTDMTSN LKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIGTSLVTPSSDASAVKTETSTSERTLSPSDTTASTPIS TFSRVQRMSISVPDILSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLSTLDWDTGRSLSSATAT TSAPQGATTPQELTLETMISPATSQLPFSIGHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTQLPTTTSAHP GQVPRSAATTLDVIPHTAKTPDATFQRQGQTALTTEARATSDSWNEKEKSTPSAPWITEMMNSVSEDTIKEVTSS SSVLRTLNTLDINLESGTTSSPSWKSSPYERIAPSESTTDKEAIHPSTNTVETTGWVTSSEHASHSTIPAHSASS KLTSPWTTSTREQAIVSMSTTTWPESTRARTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTE ITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMILAMQTSPPGATSLSAPTLDTSATASWTGTPLAT TQRFTYSEKTTLFSKGPEDTSQPSPPSVEETSSSSSLVPIHATTSPSNILLTSQGHSPSSTPPVTSVFLSETSGL GKTTDMSRISLEPGTSLPPNLSSTAGEALSTYEASRDTKAIHHSADTAVTNMEATSSEYSPIPGHTKPSKATSPL VTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVASSATETSTVITHVSSGDATTHVTKTQATFSSG TSISSPHQFITSTNTFTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMPYLTETPLAVTPDFMQ SEKTTLISKGPKDVSWTSPPSVAETSYPSSLTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTS MEPVTNSPQNLNNPSNEILATLAATTDIETIHPSINKAVTNMGTASSAHVLHSTLPVSSEPSTATSPMVPASSMG DALASI SI PGSETTDIEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATKMEVPSFDATFIPTPA QSTKFPDIFSVASSRLSNSPPMTISTHMTTTQTGSSGATSKIPLALDTSTLETSAGTPSWTEGFAHSKITTAMN NDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVAFTPQWHSTSSPVSMSSVLTSSLVKTAGKVDTSLETVTSS PQSMSNTLDDISVTSAATTDIETTHPSINTWTNVGTTGSAFESHSTVSAYPEPSKVTSPNVTTSTMEDTTISRS IPKSSKTTRTETETTSSLTPKLRETSISQEITSSTETSTVPYKELTGATTEVSRTDVTSSSSTSFPGPDQSTVSL DISTETNTRLSTSPIMTESAEITITTQTGPHGATSQDTFTMDPSNTTPQAGIHSAMTHGFSQLDVTTLMSRIPQD VSWTSPPSVDKTSSPSSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRTRLEPVTSSLPNFSST SDKILATSKDSKDTKEIFPSINTEETNVKANNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSS ETTNIKREPTYFLTPRLRETSTSQESSFPTDTSFLLSKVPTGTITEVSSTGVNSSSKISTPDHDKSTVPPDTFTG EIPRVFTSSIKTKSAEMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEVTTLMGMGPGNVSWMT TPPVEETSSVSSLMSSPAMTSPSPVSSTSPQSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITH ERPATYKDTAHTEAAMHHSTNTAVTNVGTSGSGHKSQSSVLADSETSKATPLMSTTSTLGDTSVSTSTPNISQTN QIQTEPTASLSPRLRESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLDRPTIAPDISTGMITR LFTSPIMTKSAEMTVTTQTTTPGATSQGILPWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPV EETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALLTSVLVTTTNVLGTTSPEPVTSSPPNLSSPTQERLTT YKDTAHTEAMHASMHTNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSVSTSTPAFFETRIQTE STSSLIPGLRDTRTSEEINTVTETSTVLSEVPTTTTTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTF PFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEGTHSPVTQRFPHSEETTTMSRSTKGVSWQSPPSVEETS SPSSPVPLPAITSHSSLYSAVSGSSPTSALPVTSLLTSGRRKTIDMLDTHSELVTSSLPSASSFSGEILTSEAST NTETIHFSENTAETNMGTTNSMHKLHSSVSIHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLT PELKEDSTALVMNSTTESNTVFSSVSLDAATEVSRAEVTYYDPTFMPASAQSTKSPDISPEASSSHSNSPPLTIS THKTIATQTGPSGVTSLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKTSPFSAEEANSLSSQA PLLVTTSPSPVTSTLQEHSTSSLVSVTSVPTPTLAKITDMDTNLEPVTRSPQNLRNTLATSEATTDTHTMHPSIN TAVANVGTTSSPNEFYFTVSPDSDPYKATSAWITSTSGDSIVSTSMPRSSAMKKIESETTFSLIFRLRETSTSQ KIGSSSDTSTVFDKAFTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFAGLTKSEERTIATQTG PHRATSQGTLTWDTSITTSQAGTHSAMTHGFSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLLSLPAITSPSP VPTTLPESRPSSPVHLTSLPTSGLVKTTDMLASVASLPPNLGSTSHKIPTTSEDIKDTEKMYPSTNIAVTNVGTT TSEKESYSSVPAYSEPPKVTSPMVTSFNIRDTIVSTSMPGSSEITRIEMESTFSLAHGLKGTSTSQDPIVSTEKS AVLHKLTTGATETSRTEVASSRRTSIPGPDHSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGT FTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNKDPEILSWTIPPSIEKTSFSSSLMPSPAMTSPPVSSTLPKTIH TTPSPMTSLLTPSLVMTTDTLGTSPEPTTSSPPNLSSTSHEILTTDEDTTAIEAMHPSTSTAATNVETTSSGHGS QSSVLADSEKTKATAPMDTTSTMGHTTVSTSMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEV PTGTTAEVSRTEVTSSGRTSIPGPSQSTVLPEISTRTMTRLFASPTMTESAEMTIPTQTGPSGSTSQDTLTLDTS TTKSQAKTHSTLTQRFPHSEMTTLMSRGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPPISSTLPVTISSSPLP VTSLLTSSPVTTTDMLHTSPELVTSSPPKLSHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSSGHESPSSAL ADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLSPKLRETGSSVETSSAIETSAVLSEVSIGAT TEISRTEVTSSSRTSISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGSTSESTFTLDTSTTPS LVITHSTMTQRLPHSEITTLVSRGAGDVPRPSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSL LTPGQVKTTEVLDASAEPETSSPPSLSSTSVEILATSEVTTDTEKIHPFSNTAVTKVGTSSSGHESPSSVLPDSE TTKATSAMGTISIMGDTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEANTVLSKVSTGATTEVS RTEAISFSRTSMSGPEQSTMSQDISIGTIPRISASSVLTESAKMTITTQTGPSESTLESTLNLNTATTPSWVETH SIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPAVTSPHPVSTTFLAHIPPSPLPVTSLLTSGP ATTTDILGTSTEPGTSSSSSLSTTSHERLTTYKDTAHTEAVHPSTNTGGTNVATTSSGYKSQSSVLADSSPMCTT STMGDTSVLTSTPAFLETRRIQTELASSLTPGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVTSIPGPA QSTISPDISTRTVSWFSTSPVMTESAEITMNTHTSPLGATTQGTSTLDTSSTTSLTMTHSTISQGFSHSQMSTLM RRGPEDVSWMSPPLLEKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLAKTTDMLHKSSEPVTN SPANLSSTSVEILATSEVTTDTEKTHPSSNRTVTDVGTSSSGHESTSFVLADSQTSKVTSPMVITSTMEDTSVSTSTPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGVPTGATAEVSRTEVTSSSRTSISGFAQLTVS PETSTETITRLPTSSIMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQRFSHSEMTTLVSRSPG DMLWPSQSSVEETSSASSLLSLPATTSPSPVSSTLVEDFPSASLPVTSLLNPGLVITTDRMGISREPGTSSTSNL SSTSHERLTTLEDTVDTEDMQPSTHTAVTNVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDF FETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDIST EAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWP SLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALLTLGPVKTTDMLRTSSEPETSSPPNLSSTSAE ILATSEVTKDREKIHPSSNTPWNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMT QPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRIST PLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKT SPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPPSMNITSDESLATSKA TMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVTSPWTSSTIKDIVSTTIPASSEITRIEMESTSTL TPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTEST EMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSHPSVEEASSASFSLSS PVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILAITEVTTDTEKLEM TNWTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETS SATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSG ATSQGTFTLDSSSTASWPGTHSATTQRFPQSWTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYS TPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLAASKATTETEAIHVFENTAASHVET TSATEELYSSSPGFSEPTKVISPWTSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTET STVLYKMPSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEWTRLSTSPIMTESAEITITTQTGYSLATSQV TLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSSTLLDSS PSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHE SHSSVLADSETTITIPSMGITSAVDDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYG VPTSATTEVSMTEIMSSNRIHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLAT TTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSLFVEKTSSSSSLLSLPVTTSPSVSSTLPQSIPSSSFSV TSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSE PSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGFRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSK TDFTSSAKTSSPDWPPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETIST GTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHE ITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSSPILDTRMTESVELGTVTSAYQVPSLSTRLTR TDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYT PTLGTLTPLNASMQMASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSRS GAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAIPTNISPSELD ALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATPSIATSPGAETSSAIP IMTVSPGAEDLVTSQVTSSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSL AAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEVP GWTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGWTSLVTSSRAVTSTTIP ILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGA EASSTVPTVSPEVPGWTSLVTSSSGVNSTSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGWTP LVTSSRAVTSTTIPILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTSLVTSSRAVTSTTIPTLTISSD EPETTTSLVTHSEAKMISAIPTLAVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSW PTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPSTVTSPEAESSSAISTTISPGIPGVLTSLVTS SGRDISATFPTVPESPHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDV PDMVTSQVTSSGTDTSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPGASKMLTSLVISSGTDSTTTF PTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLV PSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSPGVDTRSGVPTTTIPP SIPGWTSQVTSSATDTSTAIPTLTPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTP VSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALLS THPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPL STHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPS VTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGS LFTPLTTPGMSTLASESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMVP FTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSATAVDAICTHRP DPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTA GPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDA ICTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSL SSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKD GAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTSSTPGTSTVDLG TSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTL LRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSG CRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNT STPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSV GLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHR SSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPL FKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYV NGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTTGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQ GLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLD RDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTITNLQYEEDMRRPGSRKFNT TERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITEL GPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPMTASPLLVLFTINFTITNLRYEENMHHPG SRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLT HSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEE NMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYW ELSQLTHNITELGPYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTIT NLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDR EQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGWSEEPFTLNFTINNLRYMADMGQPGSLKFN ITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITR LGPYSLDKDSLYLNGYNEPGPDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFN STEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQ LGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFC LVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYL NFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLAR RVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGVITCLICGVL VTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ ( SEQ ID NO: 31 )

[0110] The putative cleavage site is shown in bold / italics. The transmembrane domain is highlighted in bold / underline.

[0111] In some embodiments, the binding molecule according to the present invention binds to an epitope within the retained domain, MLIC-CD, i.e. C-terminal of the cleavage site in MUC16.

[0112] In some embodiments, the binding molecule according to the present invention binds to an epitope within the MUC16 ectodomain.

[0113] In some embodiments, the binding molecule according to the present invention binds to an epitope within the 55thand 56thSEA domains of MUC16.

[0114] In some embodiments, the binding molecule according to the present invention binds to an epitope within the 56thSEA domain of MUC16.

[0115] In some embodiments, the binding molecule according to the present invention binds to an epitope within SEQ ID NO: 31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31.

[0116] In some embodiments, the binding molecule according to the present invention binds to an epitope within SEQ ID NO: 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32.In some embodiments, the binding molecule according to the present invention binds to an epitope within SEQ ID NO: 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.

[0117] APQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKA LFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQP GTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTR NGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLP ( SEQ ID NO: 32 ) MESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSV PNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLP ( SEQ ID NO: 33 )

[0118] Suitable assays and techniques for measuring / quantifying binding activity of the binding molecule according to the invention may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art. For example, it will be understood that EC50 is a measure of the concentration of a binding molecule that induces a specific response that is 50% between the maximum response and the baseline response. As such, EC50 can be used to assess the ability of a binding molecule to bind to a target.

[0119] Binding molecule

[0120] The binding molecule according to the present invention may be an antibody or a fragment thereof.

[0121] The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds and includes any molecule comprising an antigen binding portion thereof. The term “antibody” includes monoclonal antibodies and fragments or derivatives of antibodies, including, without limitation, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, scFvs and antigenbinding antibody fragments such as Fab and Fab' fragments and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein.

[0122] Within an antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, and each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0123] A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH p-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL p-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1977, J. Biol. Chem. 252:6609-6616; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved p-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). Alternatively, IMGT numbering may be used. These terminologies are well recognised in the art. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948; Morea et al., 2000, Methods 20:267-279). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those skilled in the art.

[0124] For example, CDR positions defined according to either the Kabat, Chothia, or IMGT designations, are set forth in the Table below.

[0125] Kabat IMGT Chothia

[0126] HCDR1 31-35 27-38 26-32

[0127] HCDR2 50-65 56-65 52-56

[0128] HCDR3 95-102 105-117 95-102

[0129]

[0130] LCDR1 24-34 27-38 24-34LCDR2 50-56 56-65 50-56

[0131]

[0132] LCDR3 89-97 105-117 89-97

[0133] In some embodiments, the binding molecule is an antibody or a fragment thereof comprising one or more of HCDRs 1-3 and one or more of LCDRs 1-3 according to the IMGT numbering scheme, wherein:

[0134] i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 4, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 5, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 6, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0135] In some embodiments, the binding molecule is an antibody or a fragment thereof comprising HCDRs 1-3 and LCDRs 1-3 according to the IMGT numbering scheme, wherein:

[0136] i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 4, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 5, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 6, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0137] Table 1: CDR sequences (according to IMGT numbering scheme)

[0138] SEQ ID NO: 1 HCDR1 GFSLTTYG

[0139] SEQ ID NO: 2 HCDR2 IWSDANT

[0140] SEQ ID NO: 3 HCDR3 AKHYYGSHYAMDY

[0141] SEQ ID NO: 4 LCRD1 ESVDNFGQSF

[0142] SEQ ID NO: 5 LCRD2 LAS

[0143]

[0144] SEQ ID NO: 6 LCRD3 QQNNEDPWTIn some embodiments, the binding molecule is an antibody or a fragment thereof comprising one or more of HCDRs 1-3 and one or more of LCDRs 1-3 according to the Kabat numbering scheme, wherein:

[0145] i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 11, ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 12, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 13, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 14, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 15, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 16, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0146] In some embodiments, the binding molecule is an antibody or a fragment thereof comprising HCDRs 1-3 and LCDRs 1-3 according to the Kabat numbering scheme, wherein:

[0147] i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 11, ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 12, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 13, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 14, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 15, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 16, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0148] Table 2: CDR sequences (according to Kabat numbering scheme)

[0149] SEQ ID NO: 11 HCDR1 TYGVT

[0150] SEQ ID NO: 12 HCDR2 VIWSDANTNYHSALIS

[0151] SEQ ID NO: 13 HCDR3 HYYGSHYAMDY

[0152] SEQ ID NO: 14 LCRD1 RASESVDNFGQSFMH

[0153] SEQ ID NO: 15 LCRD2 LASNLES

[0154]

[0155] SEQ ID NO: 16 LCRD3 QQNNEDPWT

[0156] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations. In some embodiments, HCDR1 may comprise one, two or three amino acid mutations. In some embodiments, HCDR2 may comprise one, two or three amino acid mutations. In some embodiments, HCDR3 may comprise one, two or three amino acidmutations. In some embodiments, LCDR1 may comprise one, two or three amino acid mutations. In some embodiments, LCDR2 may comprise one, two or three amino acid mutations. In some embodiments, LCDR3 may comprise one, two or three amino acid mutations.

[0157] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the binding molecule according to the invention from binding to MUC16. In other words, a binding molecule according to the invention comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to MUC16. In some embodiments, the mutation suitably maintains the same capacity (e.g. affinity) to bind to MUC16 as the parent binding molecule. The term “parent binding molecule” in this context refers to the binding molecule without the mutation in question.

[0158] In some embodiments, the binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 7, or a variant having at least 80% sequence identity thereto.

[0159] In some embodiments, the binding molecule comprises a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% sequence identity thereto.

[0160] In some embodiments, the binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 7, or a variant having at least 80% sequence identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% sequence identity thereto.

[0161] In some embodiments, the VH has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 7.

[0162] In some embodiments, the VL has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8.

[0163] Table 3: VH / VL and HC / HL sequencesSEQ ID NO: 7 VH QVQLQESGPGLVKPSQTLSLTCTVSGFSLTTYGVTWIRQPPG KGLEWIGVIWSDANTNYHSALISRVTISKDTSKNQVSLKLSSVT AADTAVYYCAKHYYGSHYAMDYWGQGTTVTVSS SEQ ID NO: 8 VL DIVLTQSPASLAVSPGQRATITCRASESVDNFGQSFMHWYQQ KPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTINPVEAD DTANYYCQQNNEDPWTFGGGTKVEIK SEQ ID NO: 9 HC QVQLQESGPGLVKPSQTLSLTCTVSGFSLTTYGVTWIRQPPG KGLEWIGVIWSDANTNYHSALISRVTISKDTSKNQVSLKLSSVT AADTAVYYCAKHYYGSHYAMDYWGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG N VFSCSVM H EALH N H YTQKSLSLSPG SEQ ID NO: 10 LC DIVLTQSPASLAVSPGQRATITCRASESVDNFGQSFMHWYQQ KPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTINPVEAD DTANYYCQQNNEDPWTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT

[0164]

[0165] KSFNRGEC

[0166] In some embodiments, the binding molecule comprises one or more immunoglobulin constant domains. In some embodiments, the immunoglobulin constant domains comprise a constant light chain domain (CL). In some embodiments, the immunoglobulin constant domains comprise a constant heavy 1 (CH1) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 2 (CH2) domain. In some embodiments, the immunoglobulin constant domains comprise a CH2 and a CH3 domain. In some embodiments, the CH2 and CH3 domains are considered to be an Fc (fragment crystallisable) region. In some embodiments, the immunoglobulin constant domains comprise a CL, CH1, CH2 and CH3 domain.

[0167] Table 4: Exemplary constant region sequences

[0168] SEQ ID NO: 25 Human CL-kappa RTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 26 Human lgG1 CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYIC

[0169]

[0170] NVNHKPSNTKVDKRVSEQ ID NO: 27 Human lgG1 Hinge EPKSCDKTHTCPPCP

[0171] SEQ ID NO: 28 Partial lgG1 hinge DKTHTCPPCP

[0172] SEQ ID NO: 29 Human lgG1 CH2 APESTRGPSVFLFPPKPKDTLMISRTPE (comprising STR VTCWVDVSHEDPEVKFNWYVDGVEVH mutation) NAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 30 Human lgG1 CH3 AK447 GQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGN

[0173]

[0174] VFSCSVMHEALHNHYTQKSLSLSPG In one embodiment the binding molecule is an antibody. In one embodiment the binding molecule is a monoclonal antibody.

[0175] The binding molecule may be an antibody comprising a heavy chain(s) and a light chain(s).

[0176] The term “heavy chain” refers to a large protein subunit of an immunoglobulin. Heavy chains can be of any immunoglobulin isotype (for example IgG, IgE, IgM, IgD, IgA or IgY), subtype (for example lgG1, lgG2, lgG2a, lgG2b, lgG2c, lgG3, lgG4, lgA1 or lgA2) or allotype.

[0177] The term “light chain” refers to a small protein subunit of an immunoglobulin. Light chains can be of any type (for example kappa or lambda), subtype or allotype.

[0178] Antibodies described herein include polyclonal and monoclonal antibodies and include IgA such as lgA1 or lgA2, IgG such as lgG1, lgG2, lgG3, or lgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an lgG1 antibody, more particularly an lgG1, kappa or lgG1, lambda isotype (i.e. lgG1, K, A), an lgG2a antibody (e.g. lgG2a, K, A), an lgG2b antibody (e.g. lgG2b, K, A), an lgG3 antibody (e.g. I gG3, K, A) or an lgG4 antibody (e.g. I gG4, K, A). In preferred embodiments the antibody is an lgG1, preferably lgG1, lambda.

[0179] The antibody may be of any species (for example human, monkey, camel, llama, goat, sheep, rabbit, mouse, rat, mouse, hamster or chicken) or it may be a hybrid derived from more than one species. It may be naturally occurring or it may be non-naturally occurring (i.e. an isolated antibody). The antibody may be created by genetic engineering (for example a chimeric antibody, humanised antibody, camelised antibody, intrabody, bispecific antibody).

[0180] In some embodiments, the binding molecule comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% sequence identity thereto.In some embodiments, the binding molecule comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% sequence identity thereto.

[0181] In some embodiments, the binding molecule comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% sequence identity thereto, and a light chain comprising amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% sequence identity thereto.

[0182] In some embodiments, the heavy chain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 9.

[0183] In some embodiments, the light chain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10.

[0184] In one embodiment, the binding molecule is a single domain antibody.

[0185] The term “single domain antibody” refers to an antibody fragment comprising a single monomeric variable antibody domain. Single domain antibodies are able to selectively bind antigen in a similar manner to classical antibodies, for example due to interaction between the antigen and complementarity determining regions (CDRs).

[0186] Single domain antibodies may be antibody fragments derived from heavy chain-only antibodies, such as those found in camelid and shark species. The binding moiety of heavy chain-only antibodies derived from camelids is referred to as the Variable Heavy domain of Heavy chain (VHH) domain. Shark species produce a homodimeric heavy chain-only antibody known as immunoglobulin new antigen receptor (IgNAR). The antigen binding domain of IgNAR is referred to as VNAR. Alternatively, single domain antibodies may be derived from the variable domains, typically the VH domain, of antibodies.

[0187] Single domain antibodies do not require a VL domain for antigen recognition or stable expression, and their smaller size may permit deeper tumour penetration compared to larger antibodies.

[0188] In some embodiments, the binding molecule is a single domain antibody (sdAb), for example a VHH, VH or VNAR.

[0189] In some embodiments, the binding molecule comprises a VHH domain.In some embodiments, the binding molecule comprises a VHH domain comprising one or more of complementarity determining regions (CDRs) 1-3 according to the IMGT numbering scheme, wherein:

[0190] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0191] In some embodiments, the binding molecule comprises a VHH domain comprising CDRs 1-3 according to the IMGT numbering scheme, wherein:

[0192] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0193] In some embodiments, the binding molecule comprises a VHH domain comprising one or more of CDRs 1-3 according to the Kabat numbering scheme, wherein:

[0194] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 20, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 21, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 22, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0195] In some embodiments, the binding molecule comprises a VHH domain comprising CDRs 1-3 according to the Kabat numbering scheme, wherein:

[0196] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 20, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 21, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 22, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0197] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations. In some embodiments, CDR1 may comprise one, two or three amino acidmutations. In some embodiments, CDR2 may comprise one, two or three amino acid mutations. In some embodiments, CDR3 may comprise one, two or three amino acid mutations.

[0198] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the binding molecule according to the invention from binding to MUC16. In other words, a binding molecule according to the invention comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to MUC16. In some embodiments, the mutation suitably maintains the same capacity (e.g. affinity) to bind to MUC16 as the parent binding molecule. The term “parent binding molecule” in this context refers to the binding molecule without the mutation in question.

[0199] In some embodiments, the binding molecule comprises one or more immunoglobulin constant domains. In some embodiments, the immunoglobulin constant domains comprise a constant light chain domain (CL). In some embodiments, the immunoglobulin constant domains comprise a constant heavy 1 (CH1) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 2 (CH2) domain. In some embodiments, the immunoglobulin constant domains comprise a CH2 and a CH3 domain. In some embodiments, the CH2 and CH3 domains are considered to be an Fc (fragment crystallisable) region. In some embodiments, the immunoglobulin constant domains comprise a CL, CH1, CH2 and CH3 domain.

[0200] In some embodiments, the binding molecule comprises an Fc region.

[0201] The binding molecule may comprise a VHH-Fc fusion protein. The binding molecule may consist of a VHH-Fc fusion protein.

[0202] In some embodiments, the binding molecule comprises a VHH-Fc fusion protein comprising one or more of CDRs 1-3 according to the IMGT numbering scheme, wherein:

[0203] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.In some embodiments, the binding molecule comprises a VHH-Fc fusion protein comprising CDRs 1-3 according to the IMGT numbering scheme, wherein:

[0204] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0205] In some embodiments, the binding molecule comprises a VHH-Fc fusion protein comprising one or more of CDRs 1-3 according to the Kabat numbering scheme, wherein:

[0206] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 20, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 21, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 22, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0207] In some embodiments, the binding molecule comprises a VHH-Fc fusion protein comprising CDRs 1-3 according to the Kabat numbering scheme, wherein:

[0208] i. CDR1 comprises an amino acid sequence according to SEQ ID NO: 20, ii. CDR2 comprises an amino acid sequence according to SEQ ID NO: 21, and iii. CDR3 comprises an amino acid sequence according to SEQ ID NO: 22, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0209] In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 23, or a variant having at least 80% sequence identity thereto.

[0210] In some embodiments, the binding molecule comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 23.

[0211] In some embodiments, the binding molecule comprises an amino acid sequence according to SEQ ID NO: 24, or a variant having at least 80% sequence identity thereto.

[0212] In some embodiments, the binding molecule comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 24.Table 5: VHH-Fc sequences

[0213] SEQ ID NO: 17 CDR1 (IMGT) GFALDYYT

[0214] SEQ ID NO: 18 CDR2 (IMGT) ISSSGDTT

[0215] SEQ ID NO: 19 CDR3 (IMGT) TAATFVPCGGSLYYQGYEYDY

[0216] SEQ ID NO: 20 CDR1 (Kabat) YYTIG

[0217] SEQ ID NO: 21 CDR2 (Kabat) CISSSGDTTNYADSVKG

[0218] SEQ ID NO: 22 CDR3 (Kabat) ATFVPCGGSLYYQGYEYDY

[0219] SEQ ID NO: 23 VHH domain EVQLVESGGGLVQPGGSLRLSCAASGF ALDYYTIGWFRQAPGKEREGVSCISSSG DTTNYADSVKGRFTISKDNSKNTVYLQM NSLRPEDTAVYYCTAATFVPCGGSLYY QGYEYDYWGQGTMVTVSS SEQ ID NO: 24 VHH-lgG1Fc-STR-fusion EVQLVESGGGLVQPGGSLRLSCAASGF ALDYYTIGWFRQAPGKEREGVSCISSSG DTTNYADSVKGRFTISKDNSKNTVYLQM NSLRPEDTAVYYCTAATFVPCGGSLYY QGYEYDYWGQGTMVTVSSGSDKTHTC PPCPAPESTRGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSP

[0220]

[0221] G

[0222] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0223] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0224] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a globalalignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0225] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0226] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp. W636-W641) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0227] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0228] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0229] Constant regions

[0230] The binding molecule may comprise an Fc region. In some embodiments, the binding molecule is an antibody or antigen-binding fragment thereof comprising an Fc region. In some embodiments, the binding molecule is a single domain antibody comprising an Fc region, for example a VHH-Fc fusion protein.

[0231] In some embodiments, the binding molecule, e.g. via the Fc region, binds to one or more or all of the Fc receptors. The Fc receptors may comprise one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16), C1q and FcRn. In some embodiments, the binding molecule, e.g. via the Fc region, binds to FcyRI.

[0232] It will be understood that the Fc region may interact with Fc receptors presented on the surface of a cell and / or may interact with proteins of the complement system. The Fc receptors may be Fc gamma receptors, e.g. FcyRI. The proteins of the complement system may include C1q.

[0233] In some embodiments, the Fc region, e.g. via the elbow region between CH2 and CH3, is able to bind neonatal Fc receptor (FcRn). Interaction of Fc region with FcRn may increase the halflife of the binding molecule compared to a binding molecule lacking Fc region.

[0234] In other embodiments the Fc region of the binding molecule is silenced to reduce, negate or abolish one or more Fc receptor binding and / or functionalities. Binding molecules comprising such Fc regions may be described as “Fc-inert” or “Fc-silenced”. 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.

[0235] Thus, in some embodiments, the Fc region of the binding molecule 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 or the LALA mutation. In some embodiments, the binding molecule comprises an Fc region comprising an STR mutation.

[0236] In some embodiments, the Fc region of the binding molecule may not be capable of binding to immune cells and / or recruiting immune cells.

[0237] The binding of the modified Fc region to FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRI II (CD16), C1q and FcRn may be reduced compared to a wild-type Fc region.

[0238] In some embodiments, the binding of the modified Fc region to FcyRI may be reduced compared to a wild-type Fc region.

[0239] By “reduced binding” is meant at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% reduced binding compared to a wild-type Fc region.

[0240] In some embodiments, the binding molecule comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 29.

[0241] In some embodiments, the binding molecule comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 30.

[0242] In some embodiments, the binding molecule comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 29 and an amino acid sequence according to SEQ ID NO: 30.

[0243] Binding molecule-payload construct

[0244] The present invention provides a binding molecule-payload conjugate (BPC) comprising a binding molecule as defined herein and one or more payload moieties wherein the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers. Where more than one payload moiety is covalently linked to the binding molecule, optionally via one or more linkers, the payload moieties may be the same or different, and / or the linkers may be the same or different.

[0245] It will be understood that the term “payload” may be interchangeable with “cargo”.It is to be understood herein that the term “BPC” is analogous to the term “ADC”, as in “antibody-drug conjugate”, except that the term “binding molecule” as used herein encompasses antibodies and fragments thereof, and other antigen binding molecules, and is not limited to full-length antibodies perse.

[0246] ADCs are a class of targeted therapeutics that can improve the selectivity and the cytotoxic activity of cancer drugs. Upon binding of an ADC to a target antigen present on the surface of a cell, the ADC may become internalised and trafficked to intracellular compartments (e.g. a lysosome) where the payload is released from the ADC. When the payload is a drug, the release of the drug from the ADC may allow the drug to exert its effect on the cell. The payload may be released from the ADC by proteolysis of a cleavable linker (if present) or by degradation of the antibody of the ADC.

[0247] In some embodiments, the payload moiety is selected from a drug, a detectable marker, a radioisotope (which may be a radiotherapeutic agent and / or a radioimaging agent), a fluorescent agent, a luminescent agent, a colored agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumor cell growth, promotes tumor cell apoptosis or necrosis.

[0248] In some embodiments, the payload moiety is a drug. Preferably, the payload moiety is a cytotoxic drug, an immune modulator, or a STING agonist or inhibitor

[0249] The cytotoxic drug may be a tubulin inhibitor, a DNA damaging agent, a TOPO1 inhibitor, an auristatin, a maytansinoid, or a calicheamicin.

[0250] In some embodiments, the cytotoxic drug is a topoisomerase inhibitor, which may be a type I topoisomerase (TOPO1) inhibitor or a type II topoisomerase (TOPO2) inhibitor. In some embodiments, the TOPO1 inhibitor is a camptothecin or an exatecan. Camptothecins may include topotecan, irinotecan, and belotecan.

[0251] In some embodiments, the cytotoxic drug is a tubulin inhibitor (also known as a microtubule inhibitor). In some embodiments, the cytotoxic drug is an auristatin. In some embodiments, the auristatin is monomethyl auristatin E (MMAE). In some embodiments, the auristatin is monomethyl auristatin F. In some embodiments, the cytotoxic drug is a maytansinoid.In some embodiments, the BPC comprises more than one payload moiety. In some embodiments, the BPC comprises two payload moieties. In some embodiments, the BPC contains two payload moieties.

[0252] In some embodiments, the at least two different payloads may be independently selected from a drug, a detectable marker, a radioisotope (which may be a radiotherapeutic agent and / or a radioimaging agent), a fluorescent agent, a luminescent agent, a colored agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumor cell growth, promotes tumor cell apoptosis or necrosis.

[0253] Where more than one payload moiety is covalently linked to the binding molecule, optionally via one or more linkers, in one embodiment a first payload moiety is a topoisomerase inhibitor (as defined and exemplified above) and a second payload moiety is a tubulin inhibitor (as defined and exemplified above).

[0254] It will be understood that a cytotoxic drug, chemotherapeutic drug, or chemotherapeutic entity may refer to a drug or molecule that:

[0255] • is destructive to a cell;

[0256] • induces apoptosis in a cell;

[0257] • inhibits or prevents the function of a cell;

[0258] • inhibits or prevents a cell from proliferating; and / or

[0259] • reduces the viability of a cell.

[0260] In some embodiments, the invention comprises a BPC compound as defined herein. In other embodiments, the invention comprises a tautomer of the BPC as defined herein. In other embodiments, the invention comprises a mesomer of the BPC as defined herein. In other embodiments, the invention comprises a racemate of the BPC as defined herein. In other embodiments, the invention comprises an enantiomer of the BPC as defined herein. In other embodiments, the invention comprises a diastereoisomer of the BPC as defined herein. In other embodiments, the invention comprises a composition which is a mixture of the BPCs as defined herein. In other embodiments, the invention comprises a pharmaceutically acceptable salt of the BPC as defined herein.

[0261] Linker and payload moieties

[0262] Typically, in the BPCs of the present invention, one or more payload moieties is covalently linked to the binding molecule via a linker to form the BPC. The moiety linked to the bindingmolecule is generally referred to herein as the “linker-payload moiety”. Typically, when the BPC is administered to the subject, the linker-payload moiety is released by breakdown of the BPC in vivo. The linker-payload moiety then typically breaks down to release the payload in vivo.

[0263] The drug-antibody ratio (DAR) is the number of linker-payload molecules attached to each binding molecule (it can be understood that the term “drug-antibody ratio” applies equally whether the binding molecule is, for example, an antibody or a fragment thereof). As indicated above, the drug-antibody ratio (DAR) of the BPCs according to the invention may vary. In this specification the terms “drug-antibody ratio”, “DAR” and “connection number” are synonymous.

[0264] DAR may be calculated by methods known in the art. For example, DAR may be calculated using reverse-phase high-performance liquid chromatography liquid chromatography-mass spectrometry (RP-HPLC and LC-MS).

[0265] Test sample comprising BPC may be diluted and reduced by dithiothreitol (DTT) prior to separation using a gradient elution mode by a RP-HPLC with ultraviolet (UV) detection at 280 nm.

[0266] The DAR in the test sample may be calculated according to the following formula:

[0267] (LC1)% (HC3)%D

[0268]

[0269] AR~ (NDLC)% + (LC1)%X 2 +(NDHC)% + (HC3)% *2

[0270] wherein NDLC = Light chain without conjugation of linker-payload; LC1 = Light chain conjugated with 1 linker-payload; NDHC = Heavy chain without conjugation of linkerpayload; HC3 = Heavy chain conjugated with 3 linker-payloads.

[0271] In some embodiments, the DAR is an integer from 1 to 16. In some embodiments, the DAR is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, the DAR is an integer from 4 to 12. In some embodiments, DAR is an integer from 6 to 10. In some embodiments, the DAR is an integer from 7 to 9. In some embodiments, the DAR is 6. In some embodiments, the DAR is 7. In some embodiments, the DAR is 8. In some embodiments, the DAR is 9. In some embodiments, the DAR is 10.

[0272] In some embodiments, the DAR is an integer from 1 to 8. In some embodiments, the DAR is an integer from 4 to 8. In some embodiments, the DAR is an integer from 2 to 4. In some embodiments, the DAR is an integer from 1 to 2.In some embodiments, the payload moiety is a TOPO1 inhibitor and the DAR is an integer from 4 to 8. In some embodiments, the payload moiety is MMAE and the DAR is an integer from 2 to 4.

[0273] In some embodiments, the linker unit comprises a first conjugation moiety for coupling with the binding molecule. In some embodiments, the first conjugation moiety is a group capable of coupling with the binding molecule. Thus, in some embodiments, the linker comprises a group formed from the coupling of the first conjugation moiety with the binding molecule.

[0274] In some embodiments, the linker unit comprises a second conjugation moiety for coupling with the payload moiety. In some embodiments, the second conjugation moiety is a group capable of coupling with the payload moiety. Thus, in some embodiments, the linker comprises a group formed from the coupling of the second conjugation moiety with the payload moiety.

[0275] In some embodiments, the linker unit comprises a peptide linker. In some embodiments, the linker unit consists essentially of a peptide linker. In some embodiments, the linker unit consists of a peptide linker. Typically, the peptide linker comprises, essentially of or consist of 1 to 10 amino acid residues. The peptide linker may comprise, consist essentially of or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the peptide linker comprises, consists essentially of or consists of 1 to 6 amino acid residues. In some embodiments, the peptide linker comprises, consists essentially of or consists of 1 to 5 amino acid residues. In some embodiments, the peptide linker comprises, consists essentially of or consists of 2 amino acid residues. In some embodiments, the peptide linker comprises, consists essentially of or consists of 3 amino acid residues. In some embodiments, the peptide linker comprises, consists essentially of or consists of 4 amino acid residues. In some embodiments, the peptide linker comprises, consists essentially of or consists of 5 amino acid residues.

[0276] In some embodiments, the linker comprises a peptide linker selected from AA1, AA1-Gly, Val-Cit, Val-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn and Gly-Gly-Phe-Gly.

[0277] In some embodiments, the amino acid residue represented by AA1is selected from

[0278]

[0279]

[0280] where position 1 is connected, optionally via a further conjugation moiety, to the portion of the molecule bearing the binding molecule and position 2 is connected, optionally via a further conjugation moiety, to the portion of the molecule bearing the payload compound.

[0281] In some embodiments, the first conjugation moiety comprises a pyrimidine-sulfone moiety. In some embodiments, the pyrimidine-sulfone moiety is linked to the peptide moiety via a C3-10 alkynoyl group.

[0282] In some embodiments, the linker unit comprises a peptide linker comprising the peptide sequence Gly-Gly-Phe-Gly.

[0283] In some embodiments, the first moiety comprises a maleimide moiety which is capable of adding to a sulfhydryl moiety on the binding molecule such that the conjugate comprises a sulfur-linked succinimidyl group. In some embodiments, the maleimide moiety is linked to the peptide moiety via a C1-10 alkanoyl group.

[0284] In some embodiments, the conjugate has the structure represented by formula I:

[0285]

[0286] wherein,

[0287] BM is the binding molecule;

[0288] q represents a connection number, and q is selected from the group consisting of integers from 1 to 16,

[0289]

[0290] each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carboncarbon double bond, and amido (preferably selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond);

[0291] Rx and Ry are each independently selected from H and C1-4 alkyl;

[0292] each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;

[0293] y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6);

[0294] each y2 is independently selected from any integer between 0 and 15 (such as 6-15); each y3 is independently selected from 1, 2, and 3;

[0295] each y4 is independently selected from 0 and 1; position 1 is attached to Tb via an S atom, and position 2 is attached to L2or L3;

[0296]

[0297] O / > H 'y2 ' / y3 ' 'ylyand •(<7^,xH

[0298]

[0299] y2°y3 y3o, y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6); each y2 is independently selected from any integer between 0 and 10 (such as 6-10); each y3 is independently selected from 1 or 2; each y4 is independently selected from 0 and 1, position 1 is attached to Li, and position 2 is attached to L3;

[0300] L3 is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from natural amino acid residues, non-natural amino acid residues, or selected from amino acid residue represented by AA1or stereoisomer thereof;

[0301]

[0302] ; in the amino acid residue represented by AA1, any one of Raand Rbis H, pm1 pm1a pm1b *e4~Rnl’ and the other is

[0303]

[0304] H,,Kand; or, Raand Rb, together with the carbon atom to which they are both attached, form a 5-6 membered heterocyclic ring, and said 5-6 membered heterocyclic ring is piperidine ring or piperazine ring; r, r1, r1aand r1bare each independently 0, 1, 2, 3, 4 or 5;

[0305] Rm1, Rn1, Rm1a, Rn1a, Rm1band Rn1bare each independently H, C1-6 alkyl or -COORx1, wherein, Rx1is C1-6 alkyl;

[0306] or, Rm1and Rn1, Rm1aand Rn1a, and Rm1band Rn1b, together with the nitrogen atom to which they are both attached, form a 5-6 membered heterocyclic ring, and of said 5-6 membered heterocyclic ring, the heteroatom is selected from 1 or 2 N atoms; the said 5-6 membered heterocyclic ring is optionally substituted with one or more R0’;

[0307] Rzis selected from C1-6 alkyl;

[0308] R° and R0’ are each independently selected from C1-6 alkyl, -NRm2Rn2or 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl; of said 5-6 membered heterocyclyl, the heteroatom is selected from 1 or 2 N atoms;

[0309] Rm2and Rn2are each independently selected from H and C1-6 alkyl;

[0310]

[0311] L4 is absent or present, when L4 is present, L4 is selected from H

[0312]

[0313] 2 is attached to W;

[0314] Ri and R2 are each independently selected from H, halogens and C1-4 alkyl; or, R1 and R2, together with the carbon atom to which they are both attached form a 5-6 membered heterocyclic ring, the heterocyclic ring contains 1, 2, or 3 of O, S, or N or any combination thereof;

[0315] R3 is selected from H and C1-4 alkyl; or R3 and X, together with the carbon atom to which they are both attached, form a 5-6 membered carbon ring;

[0316] o - W is absent or present, when W is present, W is selected from -O-, -S-, -NR4-,5o R4

[0317] and

[0318]

[0319] , position 1 is attached to X, and position 2 is attached to L4 or L3;

[0320] f ^7 R7♦— (<jk» MU X is selected from optionally substituted -(CH2)ni-,

[0321]

[0322] ”R?,, <►, position 1 is attached to the parent ring and position 2 is attached to W or L4; the substituent is selected from one or two C1-4 alkyls;

[0323] R4, Rs, and R? are each independently selected from H and C1-4 alkyl;

[0324] n, n1, n2, n3 are each independently selected from any integer between 0 and 6.

[0325] In some embodiments of formula (I), the connection number 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 of formula (I), the connection number q is an integer from 4 to 12. In some embodiments of formula (I), the connection number q is an integer from 6 to 10. In some embodiments of formula (I), the connection number q is an integer from 7 to 9. In some embodiments of formula (I), the connection numberq is 6. In some embodiments of formula (I), the connection number q is 7. In some embodiments of formula (I), the connection number q is 8. In some embodiments of formula (I), the connection number q is 9. In some embodiments of formula (I), the connection number q is 10.

[0326] In some embodiments of formula (I), the connection number q is an integer from 1 to 8. In some embodiments of formula (I), the connection number q is an integer from 4 to 8. In some embodiments of formula (I), the connection number q is 8.

[0327]

[0328] XX

[0329] In some embodiments, Li is, v 5 N A Ry °

[0330] In some embodiments, Z is selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond. In some embodiments, Z is a carbon-carbon triple bond.

[0331] In some embodiments, Rx is H or methyl. In some embodiments, Rx is H.

[0332] In some embodiments, Ry is H or methyl. In some embodiments, Ry is H.

[0333] In some embodiments, m is 2, 3 or 4. In some embodiments, m is 3.

[0334] In some embodiments, L2 is absent.

[0335] In some embodiments, L3 is selected from AA1, AA1-Gly, Val-Cit, Val-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn and Gly-Gly-Phe-Gly. In some embodiments, L3 is selected from AA1, AA1-Gly, Val-Cit, Val-AA1-Gly, AA1-Ala-Asn and Gly-Gly-Phe-Gly. In some embodiments, L3is Val-AA1-Gly.In some embodiments, the amino acid residue represented by AA1is selected from

[0336]

[0337] In some embodiments, the amino acid residue represented b

[0338]

[0339] y AA1is

[0340]

[0341] In some embodiments, L4is selected from H, and

[0342]

[0343] In some embodiments, l

[0344]

[0345] _4is H

[0346] In some embodiments, W is O.

[0347] In some embodiments, X is -(CH2)ni-. In some embodiments, n1 is 2, 3 or 4. In some embodiments, n1 is 3.

[0348] In some embodiments, the structure

[0349]

[0350] 1-1 1-2 1-3 1-4is:

[0351]

[0352] wherein position 1 is attached to the connecting atom on the binding molecule and position 2 is attached to W.In some embodiments, the structural fragment represented

[0353]

[0354]

[0355] wherein position 1 is attached to L4.

[0356]

[0357] In some embodiments, the linker-payload moiety has the structure:

[0358]

[0359] In some embodiments, the conjugate is selected from the group consisting of:

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] wherein,

[0366] q represents a connection number, and is selected from the group consisting of integers from 1 to 16, and BM is a binding molecule as defined herein.

[0367] In some embodiments, the conjugate has the structure of formula (I VC):

[0368]

[0369] wherein,

[0370] q represents a connection number, and is selected from the group consisting of integers from 1 to 16, and BM is a binding molecule as defined herein.

[0371] In some embodiments of formula (IVC), the connection number 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 of formula (IVC), the connection number q is an integer from 4 to 12. In some embodiments of formula (IVC), the connection number q is an integer from 6 to 10. In some embodiments of formula (IVC), the connection number q is an integer from 7 to 9. In some embodiments of formula (IVC), the connection number q is 6. In some embodiments of formula (IVC), the connection number q is 7. In some embodiments of formula (IVC), the connection numberq is 8. In some embodiments of formula (IVC), the connection number q is 9. In some embodiments of formula (IVC), the connection number q is 10.In some embodiments of formula (IVC), the connection number q is an integer from 1 to 8. In some embodiments of formula (IVC), the connection number q is an integer from 4 to 8. In some embodiments of formula (IVC), the connection number q is 8.

[0372] Methods

[0373] The invention provides a method for producing a BPC according to the invention, comprising contacting a binding molecule as defined herein with a suitable linker-payload compound.

[0374] Suitably, the compounds of the invention may be synthesised by the methods described in WO 2022 / 170971.

[0375] A general method for preparing a BPC may involve the coupling of the linker-payload compound to reduced, inter-chain disulphide-forming cysteine residues of an antibody. For example, the antibody may be reduced 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 may be performed at 37°C for 90 minutes.

[0376] Following reduction, a linker-payload solution, prepared at a concentration of 20 mM in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA), may be added slowly to the reduced antibody at a molar excess of 9.6. The final concentration of DMSO or DMA in the reaction mixture may be maintained at 5-10% of the total reaction volume. The reaction mixture may be incubated at room temperature (RT) for 2 hours.

[0377] The resulting antibody-drug conjugate (ADC) may be purified to remove excess unreacted linker-payload, for example using tangential flow filtration (TFF) or gel filtration, and the purified ADC may be formulated into a buffer and filtered under sterile conditions.

[0378] The linker-payload compound may be of the formula:

[0379]

[0380] wherein,

[0381] LG is a leaving group;

[0382]

[0383] each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carboncarbon double bond, and amido (preferably selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond);

[0384] Rx and Ry are each independently selected from H and C1-4 alkyl;

[0385] each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;

[0386] y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6);

[0387] each y2 is independently selected from any integer between 0 and 15 (such as 6-15);each y3 is independently selected from 1, 2, and 3;

[0388] each y4 is independently selected from 0 and 1; position 1 is attached to LG, and position 2 is attached to L2or L3;

[0389] L2is absent or present, and when L2is present, L2is selected from:

[0390]

[0391]

[0392] _

[0393]

[0394] , y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6); each y2 is independently selected from any integer between 0 and 10 (such as 6-10); each y3 is independently selected from 1 or 2; each y4 is independently selected from 0 and 1, position 1 is attached to Li, and position 2 is attached to L3;

[0395] L3is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from natural amino acid residues, non-natural amino acid residues, or selected from amino acid residue represented by AA1or stereoisomer thereof;

[0396]

[0397] ; in the amino acid residue represented by AA1, any one of Raand Rbis H, Rm1pm1a pm 1b

[0398] and the other is

[0399]

[0400] H,; or, Raand Rb, together with the carbon atom to which they are both attached, form a 5-6 membered heterocyclic ring, and said 5-6 membered heterocyclic ring is piperidine ring or piperazine ring.

[0401] r, r1, r1aand r1bare each independently 0, 1, 2, 3, 4 or 5;

[0402] Rm1, Rn1, Rm1a, Rn1a, Rm1band Rn1bare each independently H, C1-6 alkyl or -COORx1, wherein, Rx1is C1-6 alkyl;or, Rm1and Rn1, Rm1aand Rn1a, and Rm1band Rn1b, together with the nitrogen atom to which they are both attached, form a 5-6 membered heterocyclic ring, and of said 5-6 membered heterocyclic ring, the heteroatom is selected from 1 or 2 N atoms; the said 5-6 membered heterocyclic ring is optionally substituted with one or more R0’;

[0403] Rzis selected from C1-6 alkyl;

[0404] R° and R0’ are each independently selected from C1-6 alkyl, -NRm2Rn2or 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl; of said 5-6 membered heterocyclyl, the heteroatom is selected from 1 or 2 N atoms;

[0405] Rm2and Rn2are each independently selected from H and C1-6 alkyl;

[0406] L4 is absent or present, when L4 is present, L4 is selected from H

[0407]

[0408] 2 is attached to W;

[0409] R1 and R2 are each independently selected from H, halogens and C1-4 alkyl; or, R1 and R2, together with the carbon atom to which they are both attached form a 5-6 membered heterocyclic ring, the heterocyclic ring contains 1, 2, or 3 of O, S, or N or any combination thereof;

[0410] R3 is selected from H and C1-4 alkyl; or R3 and X, together with the carbon atom to which they are both attached, form a 5-6 membered carbon ring;

[0411] W is absent or present, when W is present, W is selected from -O-, -S-, -NR4-,

[0412]

[0413] and

[0414]

[0415] , position 1 is attached to X, and position 2 is attached to L4or L3;fj7 R7RHLR7X is selected from optionally substituted -

[0416]

[0417] (CH2)ni-,V R?,R?, v position 1 is attached to the parent ring and position 2 is attached to W or L4; the substituent is selected from one or two C1-4 alkyls;

[0418] R4, Rs, and R? are each independently selected from H and C1-4 alkyl; and

[0419] n, n1, n2, n3 are each independently selected from any integer between 0 and 6.

[0420] In some embodiments, LG is selected from halogen, sulfone group, a tertiary amine salt group, a diazonium salt group, -QMs, MeSCh', and CF3SO3'.

[0421] In some embodiments, LG is selected from F, Cl and MeSO2' and the tertiary amine salt group is selected from Me3N+and Et3N+.

[0422] In some embodiments, LG is selected from F and MeSO2'.

[0423] In some embodiments, the linker-payload compound is of the formula:

[0424]

[0425] wherein Rsis alkyl, preferably C1-4 alkyl, and more preferably methyl.

[0426] Nucleic acid

[0427] The present invention provides one or more nucleic acid sequence(s) encoding the binding molecule as defined herein. In other words, the present invention provides one or more nucleic acid sequence(s) capable of expressing the binding molecule as defined herein.As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other. The nucleic acid sequence(s) may be RNA or DNA sequences, or a mixture of RNA and DNA sequences.

[0428] In an embodiment, the nucleic acid sequence(s) are one or more DNA sequences, such as cDNA sequences. In an embodiment, the nucleic acid sequence is a DNA sequence, such as a cDNA sequence. In an embodiment, the nucleic acid sequence(s) are RNA sequences, such as mRNA sequences. In an embodiment, the nucleic acid sequence is an RNA sequence, such as an mRNA sequence.

[0429] The nucleic acid sequence(s) may be single-stranded or may be double-stranded. The nucleic acid sequence(s) may be, for example, genomic, recombinant, mRNA or cDNA. The nucleic acid sequence(s) may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance in vivo activity and / or stability.

[0430] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These variations in nucleic acid sequences are encompassed by the present invention. Therefore, multiple nucleic acid sequence(s) are envisaged, each of which may be different, but which still encode a binding molecule as defined herein. It is known in the art how to design and produce such nucleic acid sequences.

[0431] In some embodiments, the nucleic acid sequence(s) may be codon optimised for production in the host cell of choice. In some embodiments, the nucleic acid sequence(s) may be operably linked to further sequence(s) such as control sequence(s), e.g. promoter sequence(s), enhancer sequence(s), polyadenylation signal sequence(s) and / or other regulatory sequence(s), which control transcription and / or translation. The nucleic acid sequence(s) may be in the form of one or more expression cassettes. The nucleic acid sequences may be suitable for expression in prokaryotic cells or in eukaryotic cells, such as mammalian cells. Any promoter may be used, such as a strong promoter that is functional in prokaryotic cells or in eukaryotic cells. Suitable promoters will be known in the art. The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0432] Vector

[0433] The present invention provides a vector comprising the one or more nucleic acid sequence(s) of the invention.

[0434] Accordingly, the vector may comprise a polynucleotide comprising a nucleic acid sequence or sequences encoding the binding molecule as defined herein.The vector may be used to introduce nucleic acid sequence(s) according to the invention into a cell so that the cell expresses and / or produces the binding molecule as defined herein.

[0435] As used herein, the term “vector” may be considered interchangeable with the term “expression vector” and “expression construct”. The vector may be any vector that is suitable for introducing and / or expressing a nucleic acid sequence in a cell. The vector may comprise regulatory sequences, enhancer sequences and / or promoter sequences that promote expression of a nucleic acid sequence in a cell.

[0436] The vector according to the invention may be any agent capable of delivering nucleic acid sequence(s) according to the invention to a cell and / or expressing nucleic acid sequence(s) according to the invention in a cell. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0437] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retroviral vector or a lentiviral vector.

[0438] The vector may be capable of transfecting or transducing a cell.

[0439] Cells and related methods

[0440] The present invention provides a cell comprising one or more nucleic acid sequence(s) according to the invention, or a vector according to the invention.

[0441] The present invention provides a cell comprising a binding molecule as defined herein.

[0442] The present invention provides a cell comprising a BPC according to the invention.

[0443] The one or more nucleic acid sequence(s) or vector may, for example, be introduced into a cell by transduction or transfection in vitro or ex vivo.

[0444] As such, the present invention also provides a method for making a cell according to the invention comprising the step of introducing the one or more nucleic acid(s) according to the invention, or the vector according to the invention into said cell. In some embodiments, the nucleic acid(s) may be introduced as described herein.

[0445] In some embodiments, the cell may be capable of expressing the binding molecule as defined herein. In some embodiments, the cell may be capable of producing the binding molecule as defined herein.In some embodiments, the cell may be capable of expressing and / or producing the binding molecule as defined herein when the cell is cultured under suitable conditions.

[0446] The present invention also provides a method for producing the binding molecule as defined herein, wherein the method comprises the steps of:

[0447] (i) introducing one or more nucleic acid sequence(s) according to the invention, or a vector according to the invention into a cell; and

[0448] (ii) expressing the binding molecule thereof in the cell.

[0449] In some embodiments of the methods according to the invention, the one or more nucleic acid sequence(s) or vector may be introduced into the cell by transduction or transfection in vitro or ex vivo.

[0450] In some embodiments of the methods according to the invention, culturing the cell under suitable conditions may result in the cell expressing and / or producing the binding molecule as defined herein.

[0451] In some embodiments, the method for producing the binding molecule as defined herein thereof may further comprise step (iii) harvesting the binding molecule from the cell or cell culture supernatant of the cell.

[0452] It will be understood that the binding molecule may be harvested from the cell. It will also be understood that the binding molecule may be harvested from supernatant of the cell, for example when the binding molecule is released out of the cell into the cell culture medium that the cell is cultured in.

[0453] In some embodiments, the cell may be a prokaryotic cell or a eukaryotic cell.

[0454] In some embodiments, the cell may be a bacterial cell, a fungal cell, a yeast cell, a plant cell or an animal cell.

[0455] In some embodiments, the cell may be a mammalian cell or an insect cell. In some embodiments, the cell may be a human cell.

[0456] Composition

[0457] The present invention also provides a composition which comprises one or more BPCs according to the invention.As indicated above, the drug-antibody ratio (DAR) of the BPCs according to the invention may vary. Consequently, the composition may comprise a mixture of BPCs having a number of different DARs, and may therefore have an average DAR which is non-integral. In this specification “average DAR” and “average connection number” are synonymous.

[0458] In some embodiments, the average DAR is an integer or decimal from about 1 to about 8. In some embodiments, the average DAR is an integer or decimal from about 1 to about 16. In some embodiments, the average DAR 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, the average DAR is an integer or decimal from about 4 to about 12. In some embodiments, the average DAR is an integer or decimal from about 6 to about 10. In some embodiments, the average DAR is an integer or decimal from about 7 to about 9. In some embodiments, the average DAR is an integer or decimal from about 7.5 to about 8.5. In some embodiments, the average DAR is an integer or decimal from about 7.8 to about 8.2.

[0459] In some embodiments, the average DAR is about 6.0. In some embodiments, the average DAR is about 6.1. In some embodiments, the average DAR is about 6.2. In some embodiments, the average DAR is about 6.3. In some embodiments, the average DAR is about 6.4. In some embodiments, the average DAR is about 6.5. In some embodiments, the average DAR is about 6.6. In some embodiments, the average DAR is about 6.7. In some embodiments, the average DAR is about 6.8. In some embodiments, the average DAR is about 6.9. In some embodiments, the average DAR is about 7.0. In some embodiments, the average DAR is about 7.1. In some embodiments, the average DAR is about 7.2. In some embodiments, the average DAR is about 7.3. In some embodiments, the average DAR is about 7.4. In some embodiments, the average DAR is about 7.5. In some embodiments, the average DAR is about 7.6. In some embodiments, the average DAR is about 7.7. In some embodiments, the average DAR is about 7.8. In some embodiments, the average DAR is about 7.9. In some embodiments, the average DAR is about 8.0. In some embodiments, the average DAR is about 8.1. In some embodiments, the average DAR is about 8.2. In some embodiments, the average DAR is about 8.3. In some embodiments, the average DAR is about 8.4. In some embodiments, the average DAR is about 8.5. In some embodiments, the average DAR is about 8.6. In some embodiments, the average DAR is about 8.7. In some embodiments, the average DAR is about 8.8. In some embodiments, the average DAR is about 8.9. In some embodiments, the average DAR is about 9.0. In some embodiments, the average DAR is about 9.1. In some embodiments, the average DAR is about 9.2. In some embodiments, the average DAR is about 9.3. In some embodiments, the average DAR isabout 9.4. In some embodiments, the average DAR is about 9.5. In some embodiments, the average DAR is about 9.6. In some embodiments, the average DAR is about 9.7. In some embodiments, the average DAR is about 9.8. In some embodiments, the average DAR is about 9.9. In some embodiments, the DAR is about 10.0.

[0460] In some embodiments, the composition comprises one or more conjugates having the structure shown as formula I’:

[0461]

[0462] wherein:

[0463] BM, L1, L2, L3, L4, W, X, R1, R2 and R3 are as defined above for formula (I), either in its broadest aspect or a preferred aspect; and

[0464] q’ is an average connection number and is an integer or decimal from 1 to 16.

[0465] In some embodiments, the composition comprises one or more conjugates having the structure shown as formula (I VC‘):

[0466]

[0467] (IVC‘)

[0468] or a pharmaceutically acceptable salt thereof, wherein,q’ represents an average connection number, and is an integer or decimal from 1 to 16; and BM is a binding molecule as defined herein.

[0469] In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is an integer or decimal from about 1 to about 8. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is an integer or decimal from about 4 to about 8. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is an integer or decimal from about 1 to about 16. In some embodiments of formulae (I’) or (IVC’), the average connection number 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 of formulae (I’) or (IVC’), the average connection number q’ is an integer or decimal from about 4 to about 12. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is an integer or decimal from about 6 to about 10. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is an integer or decimal from about 7 to about 9. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is from about 7.5 to about 8.5. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is from about 7.8 to about 8.2.

[0470] In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.0. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.1. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.2. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.3. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.4. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.5. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.6. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.7. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.8. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 6.9. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.0. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.1. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.2. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.3. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.4. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.5. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.6. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.7. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about7.8. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 7.9. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.0. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.1. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.2. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.3. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.4. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.5. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.6. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.7. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.8. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 8.9. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.0. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.1. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.2. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.3. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.4. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.5. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.6. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.7. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.8. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 9.9. In some embodiments of formulae (I’) or (IVC’), the average connection number q’ is about 10.0.

[0471] Pharmaceutical Composition

[0472] The present invention also provides a composition, such as a pharmaceutical composition, which comprises the BPC according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0473] The present invention also provides a pharmaceutical composition comprising the binding molecule as defined herein, the one or more nucleic acid(s) according to the invention, or the vector according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0474] 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.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).

[0475] 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.

[0476] 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.

[0477] The term “carrier”, as used herein, may refer to a diluent, adjuvant, excipient, or vehicle.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] In some embodiments, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.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.

[0482] In some embodiments, the composition may comprise one or more vesicles, nanoparticles, lipid nanoparticle (LNPs), liposomes or polymeric mixtures.

[0483] 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.

[0484] Kit

[0485] The present invention provides a kit comprising the BPC according to the invention.

[0486] The present invention provides a kit comprising the composition according to the invention.

[0487] In some embodiments, the kit may optionally comprise instructions for using the kit to target the one or more payload(s) to a cell expressing MUC16.

[0488] Method of treatment

[0489] The present invention provides an in vitro method comprising contacting a cell with the BPC according to the invention.

[0490] The invention provides a method of treating or diagnosing a disease, comprising administering the BPC according to the invention to a subject.

[0491] The invention provides a method of treating or diagnosing a disease, comprising administering the composition according to the invention to a subject.

[0492] The invention provides the BPC according to the invention for use as a medicament.

[0493] The invention provides the composition according to the invention for use as a medicament.

[0494] The invention provides the BPC according to the invention for use in a method of therapy or a diagnostic method.

[0495] The invention provides the composition according to the invention for use in a method of therapy or a diagnostic method.In some embodiments, the disease is cancer. The medicament may be for use in the treatment of cancer. The method may be a method of treating, preventing or diagnosing cancer.

[0496] The cancer may express MUC16. In some embodiments, expression of MUC16 is increased compared to expression of MUC16 by the same non-cancerous tissue or cells.

[0497] In some embodiments, the cancer is an ovarian cancer, pancreatic cancer, breast cancer, lung cancer, oesophageal cancer, prostate cancer, bladder cancer or endometrial cancer. In some embodiments, the cancer is an ovarian cancer. In some embodiments, the cancer is pancreatic cancer.

[0498] According to the invention, the term “MUC16-positive cancer” means a cancer involving cancer cells expressing MUC16, preferably on the surface of said cancer cells.

[0499] “Cell surface” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. For example, a transmembrane protein having one or more extracellular portions is considered as being expressed on the cell surface.

[0500] MUC16 is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by MUC16-specific antibodies added to the cells which have not been disrupted.

[0501] In some embodiments, cells may express the extracellular domain of MUC16 (i.e. MUC16 ectodomain) on the surface of said cells, for example following cleavage of CA-125 from full length MUC16.

[0502] According to the invention, the term “disease” refers to any pathological state, including cancer, in particular those forms of cancer described herein. Any reference herein to cancer or particular forms of cancer also includes cancer metastasis thereof. In a preferred embodiment, a disease to be treated according to the present application involves cells expressing MUC16.

[0503] “Diseases associated with cells expressing MUC16” or similar expressions means according to the invention that MUC16 is expressed in cells of a diseased tissue or organ.

[0504] In one embodiment, expression of MUC16 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ.An increase refers to an increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is only found in a diseased tissue, while expression in a corresponding healthy tissue is repressed. For example, MUC16 is expressed in ovarian cancer tissue while expression is not detectable in non-cancerous ovarian tissue. According to the invention, diseases associated with cells expressing MUC16 include cancer diseases. Furthermore, according to the invention, cancer diseases preferably are those wherein the cancer cells express MUC16.

[0505] As used herein, a “cancer disease” or “cancer” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and / or migration. By “cancer cell” is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Preferably, a “cancer disease” is characterized by cells expressing MUC16 and a cancer cell expresses MUC16. A cell expressing MUC16 preferably is a cancer cell, preferably of the cancers described herein.

[0506] According to the invention, the term “cancer” also includes cancer metastasis of a primary tumour such as primary ovarian cancer. Thus, if reference is made, for example, to ovarian cancer, this also includes metastasis of the ovarian cancer, for example metastasis to the lung, liver and / or lymph nodes.

[0507] By “metastasis” is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumour, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumour at the target site depends on angiogenesis. Tumour metastasis often occurs even after the removal of the primary tumour because tumour cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention relates to “distant metastasis” which relates to a metastasis which is remote from the primary tumour and the regional lymph node system. In one embodiment, the term “metastasis” according to the invention relates to lymph node metastasis. One particular form of metastasis which is treatable using the therapy of the invention is metastasis originating from ovarian cancer as primary site. In preferred embodiments such ovarian cancer metastasis is metastasis into lymph nodes, metastasis into lung and / or metastasis into liver.

[0508] A refractory cancer is a malignancy for which a particular treatment is ineffective, which is either initially unresponsive to treatment, or which becomes unresponsive over time.By “treat” is meant to administer a compound or composition or a combination of compounds or compositions to a subject in order to prevent or eliminate a disease, including reducing the size of a tumour or the number of tumours in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the frequency or severity of symptoms and / or recurrences in a subject who currently has or who previously has had a disease; and / or prolong, i.e., increase the lifespan of the subject.

[0509] In particular, the term “treatment of a disease” includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

[0510] The term “patient” means according to the invention a subject for treatment, in particular a diseased subject, including human beings, nonhuman primates or other animals, in particular mammals such as cows, horses, pigs, sheep, goats, dogs, cats or rodents such as mice and rats. In a particularly preferred embodiment, a patient is a human being.

[0511] The invention will now be further described by way of examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0512] EXAMPLES

[0513] Materials

[0514] Table 1. Constructs, materials and cell lines used in the Examples

[0515] Name Comments

[0516] ADC to be tested (Formula IVC, wherein BM is

[0517] BNT-ADC-003 a binding molecule comprising SEQ ID NO: 9

[0518] and 10 and payload)

[0519] BNT-mAb-003 Naked mAb (SEQ ID NO: 9 and 10)

[0520] BNT-ADC-002-IC Isotype control ADC

[0521] BNT-payload-002 Payload

[0522] Rituximab

[0523] VHH-lgG1Fc-STR- VHH-Fc fusion protein (SEQ ID NO: 24)

[0524] fusion

[0525] MUC16 extracellular domain (SEQ ID NO: 33), MUC16-ECD

[0526]

[0527] further comprising his-tagSW1990 Target positive cell

[0528] OVCAR-3 Target positive cell

[0529] AsPc-1-Luc-GFP Target negative cell

[0530]

[0531] Binding to human MUC16 extracellular domain

[0532] Binding to the MUC16 extracellular domain (MUC16-ECD) was assessed by ELISA. Anti-his antibody (1 pg / mL, 100 pL / well) was coated at 4°C overnight prior to blocking with 2% BSA (200 pL / well) at room temperature for 1 hour. MUC16-ECD antigen (0.5 pg / ml, 100 pl / well) was incubated at room temperature for 1 hour. Antibody (4-fold dilutions from 6.25 nM in 2% BSA, 100 pl / well) was incubated at room temperature for 2 hours, prior to incubation with secondary antibody (goat anti-human IgG Fc HRP, 1:5000, 100 pl / well) at room temperature for 1 hour. TMB substrate was added and the reaction stopped with H2SO4 after 5 minutes.

[0533] BNT-mAb-003 and BNT-ADC-003 showed dose dependent binding to MUC16-ECD (Figure 1a).

[0534] In a second experiment, antibody (2, 1 or 0.5 pg / mL, 100 pL / well) was coated at 4°C overnight prior to blocking with 2% BSA (200 pL / well) at room temperature for 1 hour. MUC16-ECD antigen (400 nM, 4-fold dilution in 2% BSA, 100 pl / well) was incubated at room temperature for 2 hours, prior to incubation with secondary antibody (anti-his HRP, 1:2000, 100 pl / well) at room temperature for 1 hour. TMB substrate was added and the reaction stopped with H2SO4 after 10 minutes.

[0535] MUC16-ECD showed dose dependent binding to BNT-mAb-003 (Figure 1b).

[0536] In a third experiment, antibody (2 pg / mL, 100 pL / well) was coated at 4°C overnight prior to blocking with 2% BSA (200 pL / well) at room temperature for 1 hour. W3XX106-hPro2. ECD. AVI. His antigen (0.028, 0.084 or 0.251 nM, 50 pl / well) was incubated at room temperature for 1 hour, prior to incubation with VHH-lgG1Fc-STR-fusion (from 400 nM, 4-fold dilution in 2% BSA, 100 pl / well) at room temperature of 2 hours. Secondary antibody (anti-his HRP, 1:2000, 100 pl / well) was incubated at room temperature for 1 hour. TMB substrate was added and the reaction stopped with H2SO4 after 10 minutes.

[0537] VHH-lgG1Fc-STR-fusion showed dose dependent competition binding to BNT-mAb-003 (Figure 1c).Fc receptor binding

[0538] Surface plasmon resonance experiments for BNT-ADC-003 and Rituximab were performed using a Biacore 8K (Cytiva) machine equipped with a CM5 chip (Cytiva). Ligand was immobilised using THE™ His tag antibody (Genscript). A running buffer of 1xHBS-EP+(TEKNOVA) and regeneration buffer of 10 mM Glycine-HCI, pH 1.5 were used. A capture contact time of 30 sec and flow rate of 10 pL / min were used. Ligand and analyte concentrations were as shown in Table 2. Data were evaluated using Biacore evaluation software (Cytiva) with a 1:1 binding model or steady state affinity model.

[0539] Table 2. SPR experimental details

[0540] Working concentration

[0541] FcyRI 0.1 BNT-ADC-003: 0, 160, 320, 640, 1280, 2560, 120 600 30

[0542] 5120, 10240, 20480 and 40960 nM

[0543] Association time

[0544] Rituximab: 0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, ()sec

[0545] 10, 20 and 40 nM

[0546] FcyRlla 0.4 BNT-ADC-003: 0, 160, 320, 640, 1280, 2560, 60 90 Dissociation time 30 (H131) 5120, 10240, 20480 and 40960 nM; ()sec Rituximab: 0, 40, 80, 160, 320, 640, 1280, 2560,

[0547] 5120 and 10240 nM

[0548] Flow rate (µL / min) FcyRlla 0.4 BNT-ADC-003: 0, 160, 320, 640, 1280, 2560, 60 90 30 (R131) 5120, 10240, 20480 and 40960 nM;

[0549] Rituximab: 0, 80, 160, 320, 640, 1280, 2560,

[0550] 5120, 10240 and 20480 nM

[0551] FcyRI lb 0.4 0, 160, 320, 640, 1280, 2560, 5120, 10240, 60 90 30

[0552] 20480 and 40960 nM

[0553] FcyRllla 0.4 BNT-ADC-003: 0, 160, 320, 640, 1280, 2560, 300 300 30 (F158) 5120, 10240, 20480 and 40960 nM;

[0554] Rituximab: 0, 10, 20, 40, 80, 160, 320, 640,

[0555] 1280 and 2560 nM

[0556] FcyRllla 0.4 BNT-ADC-003: 0, 160, 320, 640, 1280, 2560, 300 300 30 (V158) 5120, 10240, 20480 and 40960 nM;

[0557] Rituximab: 0, 2.5, 5, 10, 20, 40, 80, 160, 320

[0558]

[0559] and 640 nMFcyRlllb 0.4 0, 160, 320, 640, 1280, 2560, 5120, 10240, 60 90 30 20480 and 40960 nM

[0560]

[0561] BNT-ADC-003 showed weaker binding affinity to human FcyRI, human FcyRlla (H131), human FcyRlla (R131), human FcyRlllb, human FcyRI I la (F158), human FcyRI I la (V158), and human FcyRlllb than Rituximab (Table 3 and Figures 1d, e, f, g, h, i, j).

[0562] Table 3. Fc receptor binding

[0563] Ligand Analyte ka(1 / Ms) kd(1 / s) KD(M)

[0564] BNT-ADC-003 No or weak binding

[0565] FcyRI

[0566] Rituximab 5.08E+05 3.73E-04 7.34E-10 BNT-ADC-003 No or weak binding

[0567] FcyRlla (H131)

[0568] Rituximab NA 3.51 E-06 BNT-ADC-003 No or weak binding

[0569] FcyRlla (R131)

[0570] Rituximab NA 1.36E-05 BNT-ADC-003 No or weak binding

[0571] FcyRllb

[0572] Rituximab NA 1.65E-05 BNT-ADC-003 No or weak binding

[0573] FcyRllla (F158)

[0574] Rituximab NA 1.48E-06 BNT-ADC-003 No or weak binding

[0575] FcyRI I la (V158)

[0576] Rituximab NA 2.45E-07 BNT-ADC-003 No or weak binding

[0577] FcyRlllb

[0578] Rituximab NA 5.21 E-06 BNT-ADC-003 2.61 E-06

[0579] human FcRn at NA

[0580] Rituximab pH 6.0 NA 2.43E-06 BNT-ADC-003 human FcRn at No or weak binding

[0581]

[0582] Rituximab pH 7.4 No or weak binding

[0583] Binding of BNT-ADC-003 and Rituximab to Human FcRn was assessed by SPR at pH 6.0 and 7.4 using a Biacore 8K (Cytiva) machine equipped with a CM5 chip (Cytiva). A running buffer of 1xPBST(pH 6.0) or 1xPBST(pH 7.4) and regeneration buffer of 1xPBS (pH 7.4) was used. A contact time of 60 sec and flow rate of 10 pL / min were used. Antibody and analyteconcentrations were as shown in Table 4. Data were evaluated using Biacore evaluation software (Cytiva) with a steady state affinity model.

[0584] Antibody

[0585] Table 4. FcRn SPR experimental details

[0586] Conc. (µg / mL)

[0587] Analyte

[0588] Working concentration

[0589] BNT-ADC- 003 8

[0590] FcRn 0, 23.438, 46.875, 93.75, 187.5,

[0591] 375, 750, 1500, 3000 and 6000 nM 60 90 30 Rituximab 8

[0592]

[0593] BNT-ADC-003 showed similar binding affinity to human FcRn with Rituximab at pH 6.0. BNT-ADC-003 and Rituximab both showed no or weak binding to human FcRn at pH 7.4 (Figure 1k, I and Table 3). Association time

[0594] ()sec

[0595] C1q binding

[0596] Dissociation Antibody binding to C1q was assessed by ELISA. Antibodies (3 pg / mL) were coated at 4°C overnight prior to incubation with human C1q (half-log fold serial dilution from 600 pg / ml) at 25°C for 2 hours. HRP-Anti-C1q antibody was incubated at 25°C for 1 hours, and A450 was measured.

[0597] BNT-ADC-003 showed weaker binding activity to human C1q compared with Rituximab (Figure 1m and Table 5).

[0598] Table 5. C1q binding

[0599] Sample EC50(nM)

[0600] BNT-ADC-003 No or weak binding

[0601] Rituximab 41.07

[0602]

[0603] Human lgG4 isotype control No or weak bindingBinding to MUC16-expressing cells

[0604] Target cells (OVCAR-3 and SW1990) were plated at 1E5 cells / well and antibody or ADC (starting from 400 nM, 4-fold serial dilution, 11 points) was incubated at 4°C for 1 hour. Secondary antibody (R-PE anti-human IgG Fey, 1:500) was incubated at 4°C for 0.5 hour prior to FACS analysis.

[0605] BNT-mAb-003 and BNT-ADC-003 showed dose dependent binding to OVCAR-3 with nanomolar ECso (Figure 2a, Table 6). BNT-mAb-003 and BNT-ADC-003 showed weak dose dependent binding to SW1990 (Figure 2b, Table 6).

[0606] Table 6. FACS Binding on OVCAR-3 and SW1990

[0607] Abs (nM) OVCAR-3 SW1990

[0608] EC50 Max EC50 Max

[0609] (nM) MFI (nM) MFI

[0610] BNT-mAb-003 3.0 20050 NA 536 BNT-ADC-003 2.2 16000 NA 438

[0611] NA 226 NA 172

[0612]

[0613] BNT-ADC-002-IC

[0614] Cytotoxicity assays

[0615] Cells (OVCAR-3, SW1990) were seeded overnight at 37°C. On day 0, ADCs or antibodies were added (from 400nM, 4-fold dilution, 9 doses). On day 6, assay plate was equilibrated to room temperature and CellTiter gio (CTG) was measured.

[0616] BNT-ADC-003 showed dose dependent cytotoxicity to OVCAR-3 and SW1990 cell lines (Figure 3a, b and Table 7).

[0617] Table 7. Cytotoxicity assay characterization

[0618] OVCAR-3 OVCAR-3 SW1990

[0619] EC50 Max EC50 Max EC50 Max (nM) lnh% (nM) lnh% (nM) lnh% BNT-ADC-002-IC 6.5 79.93 7.7 79.93 77 34 BNT-mAb-003 NA 20.53 NA 20.53 NA 2.6 BNT-ADC-003 2.8 85.56 3.4 85.56 27 28

[0620] ~ 0.0060 96.16 ~ 0.0060 96.16 1.8 49

[0621]

[0622] BNT-payload-002Internalisation assays

[0623] Cells (OVCAR-3, 1E5 cells / well, 100 pL / well) were seeded. Antibody was added (400 nM, 4-fold dilution in 1% BSA, 100 pL / well) and incubated at 4°C for 1 hour. Secondary antibody (goat anti human IgG Fc A647 (1:500), 100 pL / well) was added and incubated at 4°C for 0.5 hour. Internalisation was assessed at 0 hours (4°C) or 3 hours (37°C) by acid quench (100 pL glycine, pH 2.5 for -5 mins at 4°C) and FACS analysis.

[0624] BNT-ADC-003 showed dose dependent internalization on OVCAR-3. There is no acid quench step for 4°C group. As a result, this result indicates the binding signal of target. There is an acid quench step for 37°C group. As a result, this result indicates the internalization signal of target (Figure 4a, b).

[0625] CDC assays

[0626] Cells (5E4 / well) were seeded and antibody or ADC (400 nM, 4-fold dilution, 11 doses, 40 pl / well) was added. Complement was added to a final concentration of 10%. Cells were stained with PI and analysed by FACS.

[0627] BNT-ADC-003 did not induce dose dependent complement cytotoxicity on OVCAR-3 or SW1990 cell line (Figure 5a-c), consistent with presence of Fc-silencing mutations.

[0628] ADCC assays

[0629] Cells were loaded with EuTDA and seeded at 5000 cells / well. PBMC (stimulated with IL-2) was added (E: T of 50:1 or 75:1). Antibody or ADC (from 100 nM, 4-fold dilution, 10 doses, 50 pl / well) was added and incubated for at 37°C for 2 hours. Europium solution was added (RT, 15 mins) and cells were detected using Envision.

[0630] BNT-ADC-003 did not induce dose dependent ADCC effect on OVCAR-3 or SW1990 cell lines (Figure 6a-d), consistent with presence of Fc-silencing mutations.

[0631] In vivo efficacy studies in OVCAR-3 CDX model

[0632] CB-17 SCID mice were injected with OVCAR-3 (1E7 cells / 0.2 mL DPBS with 50% Matrigel). Mice (10 / group; -184 mm3tumour volume) received 3 weekly intravenous dose administrations of antibody (1 mg / kg), ADC isotype control (1 mg / kg) or ADC (0.3, 1, 3, or 8 mg / kg). Tumour volumes and body weights were measured throughout the study and analysed by two-way ANOVA in GraphPad.No bodyweight loss was observed in all groups. BNT-ADC-003 showed potent tumor inhibition in OVCAR-3 CDX model (Figure 7a, b, c).

[0633] CB-17 SCID mice were subcutaneously injected with OVCAR-3 (1E7 cells / 0.2 mL DPBS with 50% Matrigel). Mice (7 / group; -209 mm3tumour volume) received 3 weekly intravenous dose administrations of ADC (1, 3, or 8 (mg / kg)). Tumour volumes and body weights were measured throughout the study and analysed by two-way ANOVA in GraphPad.

[0634] No bodyweight loss was observed in all groups. BNT-ADC-003 showed potent tumor inhibition in OVCAR-3 CDX model. At the end of the study, one tumour-free mouse remained in the 1 mg / kg group, one tumour-free mouse remained in the 3mg / kg group, and five tumour-free mice remained in the 8mg / kg group. All of the mice were euthanized in PBS group when reached the humane end point (Figure 8a, b, c).

[0635] In vivo efficacy studies in SW1990 CDX model

[0636] Female CB-17 SCID mice were injected with SW1990 (5E6 cells in 100 pL PBS mixed with 100 pL Matrigel). Mice (10 / group; -100-150 mm3tumour volume) received 4 weekly intravenous dose administrations of antibody (3 mg / kg), ADC isotype control (3 mg / kg) or ADC (1, 3, or 8 mg / kg). Tumour volumes and body weights were measured throughout the study and analysed by two-way ANOVA in GraphPad.

[0637] No bodyweight loss was observed in all groups. BNT-ADC-003 showed potent tumor inhibition in SW1990 CDX model (Figure 9a, b, c).

[0638] In vivo mouse pharmacokinetic study

[0639] Female CB-17 SCID mice (33 / group) were intravenously administered a single dose of BNT-ADC-003 (1 or 8 mg / kg).

[0640] Terminal blood samples were collected at 0.5, 4 hours, 1, 3, 5, 7, 10, 14, 21 and 28 days postdose, followed by about 200-300 pl plasma collection, two aliquots (100-150 pl per vial) harvested for bioanalysis.

[0641] Total ADC and total Ab concentration in plasma as detected by ELISA methods and PK parameters including C0, T½, VdSS, Cl, ALICo-t, AUCo-inf, MRTo-t and MRTo-inf were determined.

[0642] Fc+Fc ELISA protocol was as follows. Plates were coated with goat anti-human IgG (1 pg / mL), and sample diluted in 2% BSA added. Biotin labelled goat anti-human IgG (0.0625 pg. ml) wasadded and plates were developed with SA-HRP and TMB, reading 450-540 nm, and analysed using SoftMax & WinNonlin.

[0643] For Fc+anti-payload ELISA, plates were coated with goat anti-human IgG (1 pg / mL), and sample diluted in 2% BSA added. BNT-payload antibody (0.5 pg. ml) was added and plates were developed with goat anti-mouse IgG-Fc Fragment HRP Conjugated and TMB, reading 450-540 nm, and analysed using SoftMax.

[0644] BNT-ADC-003 showed good half-life and clearance properties (Figure 10a, b and Table 8).

[0645] Table 8: Mouse PK summary

[0646] Compound BNT-ADC-003

[0647] Method Fc + Fc Fc + Anti payload

[0648] Dose G1: 1 mg / kg, IV G2: 8 mg / kg, IV G1: 1 mg / kg, IV G2: 8 mg / kg, IV

[0649] t % (h) 235 235 219 174

[0650] Cmax (Mg / mL) 15.9 139 10.0 179

[0651] AUC 0-t 2350 22097 1979 22158

[0652] (h* pg / mL)

[0653] Cl_obs 8.85 7.58 10.6 7.98 (mL / day / kg)

[0654] MRTINF_obs (h) 328 334 320 269

[0655] Vss_obs(ml / kg) 121 106 141 89.4

[0656]

[0657] Tolerability studies in mice

[0658] Male mice (6 / group) were intravenously administered a single dose of PBS or BNT-ADC-003 (20, 60 or 180 mg / kg) and were monitored for one week following administration of ADC. Evaluations included clinical observations and body weight measurements.

[0659] All mice were well tolerable to BNT-ADC-003 in 20 mg / kg and 60 mg / kg dose. Body weight loss were observed in BNT-ADC-003 180 mg / kg group (Figure 11a, b).NUMBERED EMBODIMENTS

[0660] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered embodiments.

[0661] 1. A binding molecule-payload conjugate (BPC) comprising a binding molecule and one or more payload moieties; wherein:

[0662] a) the binding molecule is an antibody or a fragment thereof and comprises one or more of heavy chain complementarity determining regions (HCDRs) 1-3 and one or more of light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein:

[0663] HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3, LCDR1 comprises an amino acid sequence according to SEQ ID NO: 4, LCDR2 comprises an amino acid sequence according to SEQ ID NO: 5, and LCDR3 comprises an amino acid sequence according to SEQ ID NO: 6, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences; and

[0664] b) the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers.

[0665] 2. The BPC according to embodiment 1, wherein the binding molecule comprises all of the HCDRs 1-3 and all of the LCDRs 1-3.

[0666] 3. The BPC according to embodiment 1 or embodiment 2, wherein the binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 7, or a variant having at least 80% sequence identity thereto.

[0667] 4. The BPC according to any one of embodiments 1 to 3, wherein the binding molecule comprises a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% sequence identity thereto.

[0668] 5. The BPC according to any preceding embodiment, which is an antibody drug conjugate (ADC), wherein the binding molecule comprises a heavy chain and a light chain.

[0669] 6. The BPC according to embodiment 5, wherein the heavy chain comprises an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% sequence identity thereto.7. The BPC according to embodiment 5 or embodiment 6, wherein the light chain comprises an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% sequence identity thereto.

[0670] 8. A binding molecule-payload conjugate (BPC) comprising a binding molecule and one or more payload moieties; wherein:

[0671] a) the binding molecule comprises a VHH domain comprising one or more of complementarity determining regions (CDRs) 1-3 according to the IMGT numbering scheme, wherein:

[0672] CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences; and

[0673] b) the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers.

[0674] 9. The BPC according to embodiment 8, wherein the binding molecule comprises all of the CDRs 1-3.

[0675] 10. The BPC according to embodiment 8 or 9, wherein the binding molecule comprises an amino acid sequence according to SEQ ID NO: 23, or a variant having at least 80% sequence identity thereto.

[0676] 11. The BPC according to any one of embodiments 8 to 10, wherein the binding molecule comprises a Fc region; optionally a modified Fc region; optionally wherein the binding molecule comprises an amino acid sequence according to SEQ ID NO: 29 and / or an amino acid sequence according to SEQ ID NO: 30.

[0677] 12. The BPC according to any one of embodiments 8 to 11, wherein the binding molecule comprises an amino acid sequence according to SEQ ID NO: 24, or a variant having at least 80% sequence identity thereto.

[0678] 13. The BPC according to any preceding embodiment, wherein the binding molecule specifically binds to MUC16, preferably wherein the binding molecule binds to an epitope within SEQ ID NO: 33.

[0679] 14. The BPC according to any one of the preceding embodiments, wherein the one or more payload moieties are covalently linked to the binding molecule via a linker.15. The BPC according to any one of the preceding embodiments, wherein the linker comprises or is a peptide linker.

[0680] 16. The BPC according to any one of the preceding embodiments, wherein the linker comprises a first conjugation moiety for coupling with the binding molecule.

[0681] 17. The BPC according to embodiment 16, wherein the linker comprises a second conjugation moiety for coupling with the payload moiety.

[0682] 18. The BPC according to any one of the preceding embodiments, wherein the linker comprises a peptide linker selected from AA1, AA1-Gly, Val-Cit, Val-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn and Gly-Gly-Phe-Gly.

[0683] 19. The BPC according to embodiment 18, wherein the amino acid residue represented

[0684]

[0685] 20. The BPC according to any one of embodiments 16 to 19, wherein the first conjugation moiety comprises a pyrimidine-sulfone moiety.

[0686] 21. The BPC according to embodiment 20, wherein the pyrimidine-sulfone moiety is linked to the peptide moiety via a C3-10 alkynoyl group.

[0687] 22. The BPC according to embodiment 18, wherein the linker comprises a peptide linker comprising the peptide sequence Gly-Gly-Phe-Gly.23. The BPC according to any one of embodiments 16 to 19, wherein the first conjugation moiety comprises a maleimide moiety which is capable of adding to a sulfhydryl moiety on the binding molecule such that the conjugate comprises a sulfur-linked succinimidyl group.

[0688] 24. The BPC according to embodiment 23, wherein the maleimide moiety is linked to the peptide moiety via a C1-10 alkanoyl group.

[0689] 25. The BPC according to any preceding embodiment, wherein the BPC has a drugantibody ratio (DAR) which is an integer between 1 to 16, preferably between 1 to 8.

[0690] 26. The BPC according to any preceding embodiment, wherein the BPC comprises more than one payload moiety, optionally wherein the payload moieties are different.

[0691] 27. The BPC according to any preceding embodiment, wherein the one or more payload moieties are independently selected from a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a colored agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumor cell growth, promotes tumor cell apoptosis or necrosis.

[0692] 28. The BPC according to embodiment 27, wherein the payload moiety is a drug.

[0693] 29. The BPC according to embodiment 28, wherein the drug is a cytotoxic drug, immune modulator, or a STING inhibitor.

[0694] 30. The BPC according to embodiment 29, wherein the cytotoxic drug is a tubulin inhibitor, a DNA damaging agent, a type I topoisomerase (TOPO1) inhibitor, an auristatin, a maytansinoid, or a calicheamicin.

[0695] 31. The BPC according to embodiment 30, wherein the cytotoxic drug is a TOPO1 inhibitor.

[0696] 32. The BPC according to embodiment 31, wherein the TOPO1 inhibitor is a camptothecin or an exatecan.

[0697] 33. The BPC according to any one of embodiments 1 to 32, wherein the conjugate has the structure represented by formula I:

[0698]

[0699] or a tautomer, a mesomer, a racemate, an enantiomer or a diastereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein,

[0700] BM is the binding molecule;

[0701]

[0702] each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon- carbon double bond, and amido (preferably selected from a direct bond, a carboncarbon triple bond, and a carbon-carbon double bond);

[0703] Rx and Ry are each independently selected from H and C1-4 alkyl;

[0704] each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;

[0705] y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6);

[0706] each y2 is independently selected from any integer between 0 and 15 (such as 6-15); each y3 is independently selected from 1, 2, and 3;each y4 is independently selected from 0 and 1; position 1 is attached to BM via an S atom, and position 2 is attached to L2 or L3;

[0707]

[0708] and 6 (such as 4, 5, and 6); each y2 is independently selected from any integer between 0 and 10 (such as 6-10); each y3 is independently selected from 1 or 2; each y4 is independently selected from 0 and 1, position 1 is attached to Li, and position 2 is attached to L3;

[0709] L3 is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from natural amino acid residues, nonnatural amino acid residues, or selected from amino acid residue represented by AA1or stereoisomer thereof;

[0710] o

[0711]

[0712] ; in the amino acid residue represented by AA1, any one of Raand Rbis H,

[0713] and the other is

[0714]

[0715] H, and Rb, together with the carbon atom to which they are both attached, form a 5-6 membered heterocyclic ring, and said 5-6 membered heterocyclic ring is piperidine ring or piperazine ring.

[0716] r, r1, r1aand r1bare each independently 0, 1, 2, 3, 4 or 5;

[0717] Rm1, Rn1, Rm1a, Rn1a, Rm1band Rn1bare each independently H, C1-6 alkyl or -COORx1, wherein, Rx1is C1-6 alkyl;or, Rm1and Rn1, Rm1aand Rn1a, and Rm1band Rn1b, together with the nitrogen atom to which they are both attached, form a 5-6 membered heterocyclic ring, and of said 5-6 membered heterocyclic ring, the heteroatom is selected from 1 or 2 N atoms; the said 5-6 membered heterocyclic ring is optionally substituted with one or more R0’;

[0718] Rzis selected from C1-6 alkyl;

[0719] R° and R0’ are each independently selected from C1-6 alkyl, -NRm2Rn2or 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl; of said 5-6 membered heterocyclyl, the heteroatom is selected from 1 or 2 N atoms;

[0720] Rm2and Rn2are each independently selected from H and C1-6 alkyl;

[0721]

[0722] position 2 is attached to W;

[0723] Ri and R2 are each independently selected from H, halogens and C1-4 alkyl; or, R1 and R2, together with the carbon atom to which they are both attached form a 5-6 membered heterocyclic ring, the heterocyclic ring contains 1, 2, or 3 of O, S, or N or any combination thereof;

[0724] R3 is selected from H and C1-4 alkyl; or R3 and X, together with the carbon atom to which they are both attached, form a 5-6 membered carbon ring;

[0725] W is absent or present, when W is present, W is selected from -O-, -S-, -NR4-,

[0726]

[0727]

[0728] and, position 1 is attached to X, and position 2 is attached to L4or L3;fj7 R7RHLR7X is selected from optionally substituted -

[0729]

[0730] (CH2)ni-,V R?,R?, v position 1 is attached to the parent ring and position 2 is attached to W or L4; the substituent is selected from one or two C1-4 alkyls;

[0731] R4, Rs, and R? are each independently selected from H and C1-4 alkyl;

[0732] n, n1, n2, n3 are each independently selected from any integer between 0 and 6; and q represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

[0733] 34. The BPC according to embodiment 33, wherein Li is selected from

[0734]

[0735] N

[0736] and

[0737]

[0738] 35. The BPC according to embodiment 34, wherein Li is

[0739]

[0740] 36. The BPC according to embodiment 33 or 34, wherein Z is selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond.

[0741] 37. The BPC according to embodiment 36, wherein Z is a carbon-carbon triple bond. 38. The BPC according to any one of embodiments 33 to 37, wherein Rx is H or methyl.

[0742] 39. The BPC according to embodiment 38, wherein Rx is H.

[0743] 40. The BPC according to any one of embodiments 33 to 39, wherein Ry is H or methyl.

[0744] 41. The BPC according to embodiment 40, wherein Ry is H.42. The BPC according to any one of embodiments 33 to 41, wherein m is 2, 3 or 4. 43. The BPC according to embodiment 42, wherein m is 3.

[0745] 44. The BPC according to any one of embodiments 33 to 43, wherein L2 is absent.

[0746] 45. The BPC according to any one of embodiments 33 to 44, wherein L3is selected from AA1, AA1-Gly, Val-Cit, Val-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn and Gly-Gly-Phe-Gly.

[0747] 46. The BPC according to embodiment 45, wherein L3 is selected from AA1, AA1-Gly, Val-Cit, Val-AA1-Gly, AA1-Ala-Asn and Gly-Gly-Phe-Gly.

[0748] 47. The BPC according to embodiment 46, wherein L3 is Val-AA1-Gly.

[0749] 48. The BPC according to any one of embodiments 45 to 47, wherein the amino acid

[0750]

[0751] 49. The BPC according to embodiment 48, wherein the amino acid residue represented

[0752]

[0753] 50. The BPC according to any one of embodiments 33 to 49, wherein L4 is selected from

[0754] H, and I

[0755]

[0756] 51. The BPC according to embodiment 50, wherein L4 is H

[0757] 52. The BPC according to any one of embodiments 33 to 51, wherein W is O.

[0758] 53. The BPC according to embodiment 52, wherein X is -(CH2)ni-- 54. The BPC according to any one of embodiments 33 to 53, wherein n1 is 2, 3 or 4.55. The BPC according to embodiment 54, wherein n1 is 3.

[0759] 56. The BPC according to any one of embodiments 33 to 55, wherein the structure • - L1- L2— L3— 1_4— js.

[0760]

[0761] wherein position 1 is attached to the connecting atom on the binding molecule and position 2 is attached to W.

[0762] 57. The BPC according to any one of embodiments 33 to 56, wherein the structural

[0763]

[0764] attached to L4.

[0765] 58. The BPC according to any one of embodiments 33 to 57, wherein the structural

[0766]

[0767] 59. The BPC according to any one of embodiments 33 to 58, wherein the linker-payload

[0768] comprises the structure:

[0769]

[0770] 60. The BPC according to embodiment 33, wherein the conjugate is selected from the group consisting of:

[0771]

[0772]

[0773]

[0774]

[0775]

[0776] or a pharmaceutically acceptable salt thereof wherein,

[0777] BM is the binding molecule as defined in any one of embodiments 1 to 13; and

[0778] q represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

[0779] 61. The BPC according to embodiment 28 wherein the conjugate is

[0780]

[0781] or a pharmaceutically acceptable salt thereof wherein

[0782] BM is the binding molecule as defined in any one of embodiments 1 to 13; and

[0783] q represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

[0784] 62. The BPC according to embodiment 60 or 61, wherein q is an integer from 1 to 12. 63. The BPC according to embodiment 62, wherein q is an integer from 1 to 8.

[0785] 64. The BPC according to embodiment 63, wherein q is an integer from 4 to 8.

[0786] 65. The BPC according to embodiment 64, wherein q is an integer and is 8.

[0787] 66. A method for producing a BPC according to any one of embodiments 1 to 13, comprising contacting a binding molecule as defined in any one of embodiments 1 to 13 with a suitable linker-payload compound.

[0788] 67. A method according to embodiment 66, wherein the linker-payload compound is of the formula:

[0789]

[0790] wherein,

[0791] LG is a leaving group;

[0792]

[0793] each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carboncarbon double bond, and amido (preferably selected from a direct bond, a carboncarbon triple bond, and a carbon-carbon double bond);

[0794] Rx and Ry are each independently selected from H and C1-4 alkyl;

[0795] each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;

[0796] y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6);

[0797] each y2 is independently selected from any integer between 0 and 15 (such as 6-15); each y3 is independently selected from 1, 2, and 3;

[0798] each y4 is independently selected from 0 and 1; position 1 is attached to LG, and position 2 is attached to L2or L3;

[0799]

[0800]

[0801] y 6 y y3o, y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6); each y2 is independently selected from any integer between 0 and 10 (such as 6-10); each y3 is independently selected from 1 or 2; each y4 is independently selected from 0 and 1, position 1 is attached to Li, and position 2 is attached to L3;

[0802] L3is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from natural amino acid residues, nonnatural amino acid residues, or selected from amino acid residue represented by AA1or stereoisomer thereof;

[0803]

[0804] ; in the amino acid residue represented by AA1, any one of Raand Rbis H, pm1 pm1a pm1b

[0805] and the other is

[0806]

[0807] H,,Kand; or, Raand Rb, together with the carbon atom to which they are both attached, form a 5-6 membered heterocyclic ring, and said 5-6 membered heterocyclic ring is piperidine ring or piperazine ring.

[0808] r, r1, r1aand r1bare each independently 0, 1, 2, 3, 4 or 5;

[0809] Rm1, Rn1, Rm1a, Rn1a, Rm1band Rn1bare each independently H, C1-6 alkyl or -COORx1, wherein, Rx1is C1-6 alkyl;

[0810] or, Rm1and Rn1, Rm1aand Rn1a, and Rm1band Rn1b, together with the nitrogen atom to which they are both attached, form a 5-6 membered heterocyclic ring, and of said 5-6 membered heterocyclic ring, the heteroatom is selected from 1 or 2 N atoms; the said 5-6 membered heterocyclic ring is optionally substituted with one or more R0’;

[0811] Rzis selected from C1-6 alkyl;

[0812] R° and R0’ are each independently selected from C1-6 alkyl, -NRm2Rn2or 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl; of said 5-6 membered heterocyclyl, the heteroatom is selected from 1 or 2 N atoms;

[0813] Rm2and Rn2are each independently selected from H and C1-6 alkyl;

[0814]

[0815] position 2 is attached to W;

[0816] Ri and R2are each independently selected from H, halogens and C1-4 alkyl; or, Ri and R2, together with the carbon atom to which they are both attached form a 5-6 membered heterocyclic ring, the heterocyclic ring contains 1, 2, or 3 of O, S, or N or any combination thereof;

[0817] R3 is selected from H and C1-4 alkyl; or R3 and X, together with the carbon atom to which they are both attached, form a 5-6 membered carbon ring;

[0818] o

[0819] W is absent or present, when W is present, W is selected from -O-, -S-, -NR4-,

[0820]

[0821]

[0822] and, position 1 is attached to X, and position 2 is attached to L4 or L3;

[0823] fj7 R7

[0824] ('jh* MU, X is selected from optionally substituted -

[0825]

[0826] (CH2)ni-,v R?,R7, v position 1 is attached to the parent ring and position 2 is attached to W or L4; the substituent is selected from one or two C1-4 alkyls;

[0827] R4, Rs, and R? are each independently selected from H and C1-4 alkyl;

[0828] n, n1, n2, n3 are each independently selected from any integer between 0 and 6.

[0829] 68. A method according to embodiment 67, wherein LG is selected from halogen, sulfone group, a tertiary amine salt group diazonium salt group, -OMs, MeSO2-, and CF3SO3-.69. A method according to embodiment 68, wherein LG is selected from F, Cl and MeSO2- and the tertiary amine salt group is selected from Me3N+ and Et3N+.

[0830] 70. A method according to embodiment 68, wherein LG is selected from F and MeSO2- 71. A method according to embodiment 67, wherein the linker-payload compound is of the formula:

[0831]

[0832] 72. One or more nucleic acid sequence(s) encoding a binding molecule as defined in any one of embodiments 1 to 13; optionally wherein the one or more nucleic acid sequence(s) is an RNA sequence.

[0833] 73. A vector comprising the one or more nucleic acid sequences(s) according to embodiment 72.

[0834] 74. A cell comprising the one or more nucleic acid sequence(s) according to embodiment 72, the vector according to embodiment 73, the binding molecule as defined in any one of embodiments 1 to 13, or the BPC according to any one of embodiments 1 to 65; optionally, wherein the cell is capable of expressing the binding molecule as defined in any one of embodiments 1 to 13.

[0835] 75. A composition comprising the BPC according to any one of embodiments 1 to 65, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0836] 76. An in vitro method comprising contacting a cell with the BPC according to any one of embodiments 1 to 65.

[0837] 77. A method of treating or diagnosing a disease, comprising administering the BPC according to any one of embodiments 1 to 65 or the composition according to embodiment 75 to a subject.

[0838] 78. The BPC according to any one of embodiments 1 to 65 or the composition according to embodiment 75 for use as a medicament.79. The BPC according to any one of embodiments 1 to 65 or the composition according to embodiment 75 for use in a method of therapy or a diagnostic method.

[0839] 80. The BPC or composition for use according to embodiment 79 wherein the method is a method of treating, preventing or diagnosing cancer.

[0840] 81. The BPC or composition for use according to embodiment 80, wherein the cancer expresses MUC16; optionally wherein the expression of MUC16 is increased compared to the expression of MUC16 by the same non-cancerous tissue or cells.

[0841] 82. The BPC or composition for use according to any one of embodiments 80 to 81, wherein the cancer is ovarian cancer, pancreatic cancer, breast cancer, lung cancer, oesophageal cancer, prostate cancer, bladder cancer or endometrial cancer.

[0842] 83. A kit comprising a BPC according to any one of embodiments 1 to 65 or a composition according to embodiment 75.

[0843] 84. An ADC comprising a binding molecule and one or more payload-linker moieties; wherein:

[0844] a) the binding molecule is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence according to SEQ ID NO: 9 and the light chain comprises an amino acid sequence according to SEQ ID NO: 10; and

[0845] b) the one or more payload-linker moieties have the structure:

[0846]

[0847] 85. A BPC comprising a binding molecule and one or more payload moieties; wherein: a) the binding molecule is a VHH-Fc fusion protein comprising an amino acid sequence according to SEQ ID NO: 24; and

[0848] b) the one or more payload-linker moieties have the structure:

[0849]

Claims

CLAIMS1. A binding molecule-payload conjugate (BPC) comprising a binding molecule and one or more payload moieties; wherein:a) the binding molecule is an antibody or a fragment thereof and comprises one or more of heavy chain complementarity determining regions (HCDRs) 1-3 and one or more of light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein:HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3, LCDR1 comprises an amino acid sequence according to SEQ ID NO: 4, LCDR2 comprises an amino acid sequence according to SEQ ID NO: 5, and LCDR3 comprises an amino acid sequence according to SEQ ID NO: 6, optionally wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences; andb) the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers.

2. The BPC according to claim 1, wherein the binding molecule comprises all of the HCDRs 1-3 and all of the LCDRs 1-3.

3. The BPC according to claim 1 or claim 2, wherein the binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 7, or a variant having at least 80% sequence identity thereto, and / or a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% sequence identity thereto.

4. The BPC according to any preceding claim, which is an antibody drug conjugate (ADC), wherein the binding molecule comprises a heavy chain and a light chain.

5. The BPC according to claim 4, wherein the heavy chain comprises an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% sequence identity thereto, and / or the light chain comprises an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% sequence identity thereto.

6. A binding molecule-payload conjugate (BPC) comprising a binding molecule and one or more payload moieties; wherein:a) the binding molecule comprises a VHH domain comprising one or more of complementarity determining regions (CDRs) 1-3 according to the IMGT numbering scheme, wherein:CDR1 comprises an amino acid sequence according to SEQ ID NO: 17, CDR2 comprises an amino acid sequence according to SEQ ID NO: 18, and CDR3 comprises an amino acid sequence according to SEQ ID NO: 19, optionally wherein one or more of the CDRs comprise one, two or three amino acid mutations relative to the recited sequences; andb) the one or more payload moieties is covalently linked to the binding molecule, optionally via one or more linkers.

7. The BPC according to embodiment 6, wherein the binding molecule comprises all of the CDRs 1-3.

8. The BPC according to embodiment 6 or 7, wherein the binding molecule comprises an amino acid sequence according to SEQ ID NO: 23, or a variant having at least 80% sequence identity thereto.

9. The BPC according to any one of embodiments 6 to 8, wherein the binding molecule comprises a Fc region; optionally a modified Fc region; optionally wherein the binding molecule comprises an amino acid sequence according to SEQ ID NO: 29 and / or an amino acid sequence according to SEQ ID NO: 30.

10. The BPC according to any one of embodiments 6 to 9, wherein the binding molecule comprises an amino acid sequence according to SEQ ID NO: 24, or a variant having at least 80% sequence identity thereto.

11. The BPC according to any one of the preceding claims, wherein the one or more payload moieties are covalently linked to the binding molecule via a linker, optionally wherein the linker comprises or is a peptide linker, optionally wherein the peptide linker is selected from AA1, AA1-Gly, Val-Cit, Val-Ala, Val-AA1, Val-AA1-Gly, AA1-Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn and Gly-Gly-Phe-Gly.

12. The BPC according to any preceding claim, wherein the one or more payload moieties are independently selected from a drug, a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a colored agent, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand, another active agent that inhibits tumor cell growth, promotes tumor cell apoptosis ornecrosis, optionally wherein the drug is a cytotoxic drug, immune modulator, or a STING inhibitor.

13. The BPC according to claim 12, wherein the cytotoxic drug is a tubulin inhibitor, a DNA damaging agent, a type I topoisomerase (TOPO1) inhibitor, an auristatin, a maytansinoid, or a calicheamicin.

14. The BPC according to any one of claims 1 to 13, wherein the conjugate has the structure represented by formula I:or a tautomer, a mesomer, a racemate, an enantiomer or a diastereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein,BM is the binding molecule;each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carboncarbon double bond, and amido (preferably selected from a direct bond, a carboncarbon triple bond, and a carbon-carbon double bond);Rx and Ry are each independently selected from H and C1-4 alkyl;each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6);each y2 is independently selected from any integer between 0 and 15 (such as 6-15); each y3 is independently selected from 1, 2, and 3;each y4 is independently selected from 0 and 1; position 1 is attached to BM via an S atom, and position 2 is attached to L2 or L3;y 0 y y3o, y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6); each y2 is independently selected from any integer between 0 and 10 (such as 6-10); each y3 is independently selected from 1 or 2; each y4 is independently selected from 0 and 1, position 1 is attached to Li, and position 2 is attached to L3;L3 is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from natural amino acid residues, nonnatural amino acid residues, or selected from amino acid residue represented by AA1or stereoisomer thereof;o; in the amino acid residue represented by AA1, any one of Raand Rbis H,and the other isH, and Rb, together with the carbon atom to which they are both attached, form a 5-6membered heterocyclic ring, and said 5-6 membered heterocyclic ring is piperidine ring or piperazine ring.r, r1, r1aand r1bare each independently 0, 1, 2, 3, 4 or 5;Rm1, Rn1, Rm1a, Rn1a, Rm1band Rn1bare each independently H, C1-6 alkyl or -COORx1, wherein, Rx1is C1-6 alkyl;or, Rm1and Rn1, Rm1aand Rn1a, and Rm1band Rn1b, together with the nitrogen atom to which they are both attached, form a 5-6 membered heterocyclic ring, and of said 5-6 membered heterocyclic ring, the heteroatom is selected from 1 or 2 N atoms; the said 5-6 membered heterocyclic ring is optionally substituted with one or more R0’;Rzis selected from C1-6 alkyl;R° and R0’ are each independently selected from C1-6 alkyl, -NRm2Rn2or 5-6 membered heterocyclyl optionally substituted with C1-6 alkyl; of said 5-6 membered heterocyclyl, the heteroatom is selected from 1 or 2 N atoms;Rm2and Rn2are each independently selected from H and C1-6 alkyl;position 2 is attached to W;Ri and R2are each independently selected from H, halogens and C1-4 alkyl; or, Ri and R2, together with the carbon atom to which they are both attached form a 5-6 membered heterocyclic ring, the heterocyclic ring contains 1, 2, or 3 of O, S, or N or any combination thereof;Rs is selected from H and C1-4 alkyl; or R3 and X, together with the carbon atom to which they are both attached, form a 5-6 membered carbon ring;oW is absent or present, when W is present, W is selected from -O-, -S-, -NR4-,*5o R4and *4*5, position 1 is attached to X, and position 2 is attached to L4or L3;R7fX is selected from optionally substituted -(CH2)ni-,v R?,R7,, position 1 is attached to the parent ring and position 2 is attached to W or L4; the substituent is selected from one or two C1-4 alkyls;R4, R5, and R7are each independently selected from H and C1-4 alkyl;n, n1, n2, n3 are each independently selected from any integer between 0 and 6; and q represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

15. The BPC according to claim 14, wherein the linker-payload comprises the structure:

16. The BPC according to claim 15, wherein the conjugate isor a pharmaceutically acceptable salt thereof whereinBM is the binding molecule as defined in any one of claims 1 to 10; andq represents a connection number, and q is selected from the group consisting of integers from 1 to 16.

17. The BPC according to any one of claims 14 to 16, wherein q is selected from the group consisting of integers 1 to 8, preferably wherein q is selected from the group consisting of integers 4 to 8.

18. A method for producing a BPC according to any one of claims 1 to 17, comprising contacting a binding molecule as defined in any one of claims 1 to 10 with a suitable linkerpayload compound.

19. A composition comprising the BPC according to any one of claims 1 to 17, together with a pharmaceutically acceptable carrier, diluent or excipient.

20. A method of treating or diagnosing a disease, comprising administering the BPC according to any one of claims 1 to 17, or the composition according to claim 19, to a subject.

21. The BPC according to any one of claims 1 to 17, or the composition according to claim 19, for use in a method of therapy or a diagnostic method, optionally wherein the method is a method of treating, preventing or diagnosing cancer, optionally wherein the cancer expresses MUC16; optionally wherein the expression of MUC16 is increased compared to the expression of MUC16 by the same non-cancerous tissue or cells, optionally wherein the cancer is ovarian cancer, pancreatic cancer, breast cancer, lung cancer, oesophageal cancer, prostate cancer, bladder cancer or endometrial cancer.

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