Camptothecin analogs conjugated to a glutamine residue in a protein, and their use

Abstract

Described herein are protein-drug conjugates and compositions thereof that are useful, for example, for target-specific delivery of therapeutic moieties, e.g., camptothecin analogs and / or derivatives. In certain embodiments, provided are specific and efficient methods for producing protein-drug constructs (e.g., antibody-drug conjugates) utilizing a combination of transglutaminase and 1,3-cycloaddition techniques. Camptothecin analogs, antibody-drug conjugates, and compositions which comprise glutaminyl-modified antibodies and camptothecin analog payloads and are provided.

Description

CAMPTOTHECIN ANALOGS CONJUGATED TO A GLUTAMINE RESIDUE IN A PROTEIN, AND THEIR USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial Nos. 63 / 051,172, filed on July 13, 2020, and 63 / 154,531, filed on February 26, 2021, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates), pharmaceutical compositions, and methods of treating disease therewith. Also provided are specific and efficient methods for producing protein-drug constructs utilizing a combination of transglutaminase and 1,3-cycloaddition techniques. More specifically, the present disclosure relates to protein-drug conjugates (e.g., antibody-drug conjugates) comprising camptothecin analogs and derivatives. SEQUENCE LISTING

[0003] An official copy of the sequence listing is submitted concurrently with the specification via EFS-Web as a paper copy of an ASCI| formatted sequence listing with a file name of 250298_000244_SL.txt, and a size of about 996 kilobytes, and created on July 12, 2021. The sequence listing contained in this paper copy of the ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND OF THE DISCLOSURE

[0004] Proliferative diseases are characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can result in death. Abnormal proliferation, for example, cancer, is caused by both external factors (e.g., tobacco, chemicals, radiation and infectious organisms) and internal factors (inherited mutations, immune system conditions, the mutations that occur from metabolism). These causal factors may act together or in sequence to initiate or promote abnormal proliferation. Cancer is treated by surgery, radiation, chemotherapy, hormones and immunotherapy. However, there is a need for more effective anti-proliferation drugs.

[0005] The ideal anti-proliferation therapy would enable targeted delivery of highly cytotoxic agents to tumor cells and would leave normal cells unaffected. Conventional chemotherapeutic treatment is limited because of the toxic side-effects that arise from effects of the drug on non-cancerous cells. Various approaches to targeted drug delivery have been tried, including the use of conjugates of tumor targeted probes (such as antibodies or growth factors) with toxins such as pseudomonas or diphtheria toxins, which arrest the synthesis of proteins and cells. However, the side effects include reaction of the immune system due to non-human components of the conjugates. Further, the half-life of the drug conjugates was limited due to elimination from the circulation through renal filtration, and schematic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-targeted organs and tissues.

[0006] Another approach uses passive drug carriers such as polymers, liposomes, and polymeric micelles to take advantage of the hyper-permeability of vascular endothelia of tumor tissue. Polymeric drugs and macromolecules accumulate within solid tumors due to an enhanced permeability and retention mechanism. However, barriers of using such targeted deliveries include fast clearance of foreign particles from the blood, and technological hindrances in obtaining highly standardized, pharmaceutically acceptable drug delivery systems with the necessary specificity and selectivity for binding tumor cells.

[0007] Protein conjugates, such as antibody conjugates, utilize the selective binding of a binding agent to deliver a payload to targets within tissues of subjects. The payload can be a therapeutic moiety that is capable of taking action at the target.

[0008] Several techniques for conjugating linkers and payloads to antibodies are available. Many conjugates are prepared by non-selective covalent linkage to cysteine or lysine residues in the antibody. This non-selective technique can result in a heterogeneous mixture of products with conjugations at different sites and with different numbers of conjugations per antibody. Thus, there is a need in the art for methods and techniques that provide site-selective antibody conjugation.

[0009] There is a need in the art for additional safe and effective anti-tumor targeting agents that can bind to various antigens to provide enhanced the treatment of diseases such as cancer for use in monotherapy and combination therapies. In certain embodiments, the present disclosure meets the needs and provides other advantages.

[0010] The foregoing discussion is presented solely to provide a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application. SUMMARY OF THE DISCLOSURE

[0011] Various non-limiting aspects and embodiments of the disclosure are described below.

[0012] In one aspect, the present disclosure provides a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)}k)n (A), wherein: BA is an antibody or an antigen-binding fragment thereof: Gln is a glutamine residue; L1 is absent or a first linker: Bis a branching unit comprising at least one adduct of group B’ and group B”, wherein N=N 4 { N—N . one of the groups B’ and B” is selected from -Na and N-N : and the other of the A Ga oN | lL a TR Nl, 2 ' 4 groups B’ and B” is selected from —f=g Har cand \— / where Qis CorN; L2 is a second linker covalently attached to the branching unit B via the at least one group B”; M is absent or a moiety having the structure 3 , where R, R’, and R” are independently at each occurrence hydrogen or a C4-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Wg PEN oll y ZT O- , A 4 a 0 ®); k is an integer from 1 to 12; mis an integer from 1 to 30, and nis an integer from 1 to 30

[0013] In another aspect, the present disclosure provides a compound having a structure according to Formula (I): BA-(GIn-NH-L1-B-(-L2-M-Dxd)}n (I), wherein: BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is absent or a first linker: B is a branching unit comprising at least one adduct of group B’, where the group B’ is x Nes, EY N=N - gmt £7 TR aa selected from -Na, —$"=="5, ow N-N ;and \..” / ,whereQisCorN; L2 is a second linker covalently attached to the branching unit B via at least one group B”, wherein the group B’ and the group B” form the at least one adduct; AK M is absent or a moiety having the structure ke , where R, R’, and R” are independently at each occurrence hydrogen or a C1-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Wm O° xz CN Say, 2 AX *P) k is an integer from 1 to 12, and nis an integer from 1 to 30.

[0014] In one embodiment, the BA is an antibody or an antigen-binding fragment thereof.

[0015] In one embodiment, the BA is an anti-HER2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-MET / MET bispecific antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti- PSMA antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, or an antigen-binding fragment thereof.

[0016] In one embodiment, the BA is an anti-STEAP2 antibody comprising an HCVR / LCVR amino acid sequence pair as set forth in Table 1.

[0017] In one embodiment, the BA is an anti-HER2 / HER2 bispecific antibody. In one embodiment, the BA binds two separate epitopes of HER2 protein.

[0018] In one embodiment, the anti-HER2 / HER2 bispecific antibody comprises: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human HER2; and wherein D2 specifically binds a second epitope of human HER2.

[0019] In one embodiment, the D1 and D2 do not compete with one another for binding to human HER2.

[0020] In one embodiment, the BA is an anti-MET / MET bispecific antibody. In one embodiment, the BA binds two separate epitopes of MET protein.

[0021] In one embodiment, the anti- MET / MET bispecific antibody comprises: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human MET; and wherein D2 specifically binds a second epitope of human MET.

[0022] In one embodiment, the D1 and D2 do not compete with one another for binding to human MET.

[0023] In one embodiment, the BA is an anti-MET / MET bispecific antibody comprising an HCVR / LCVR amino acid sequence pair as set forth in Table 3.

[0024] In one embodiment, the BA is an anti-MET / MET bispecific antibody comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen binding domain comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR 1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096-2098, and wherein the D2 antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2038-2040-2042-2094-2096-2098.

[0025] In one embodiment, the BA is an anti-MET / MET bispecific antibody H4H14639D, which comprises a D1 derived from H4H13306P2 and a D2 derived from H4H13312P2.

[0026] In one embodiment, the anti-STEAP2 comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 58; 74 / 58; 82 / 58; 90 / 58; 98 / 58; 106 / 114; 122 / 130; 138 / 146; 154 / 162; 170 / 178; 186 / 194; 202 / 210; 218 / 226; 234 / 242; 250 / 258; 266 / 274; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

[0027] In one embodiment, the glutamine residue Gin is naturally present ina CH2 or CH3 domain of the BA.

[0028] In one embodiment, the glutamine residue Gin is introduced to the BA by modifying one or more amino acids.

[0029] In one embodiment, the Gin is Q295 or N297Q.

[0030] In one embodiment, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0031] In one embodiment, L1 comprises C1. alkyl, phenyl, -NH-, -C(O)-, -(CH2),-NH- C(0)-, -(CH2)u-C(0)-NH-, -(CH2-CH2-O)w~, -(CH2)u-(O-CH2-CH2)\-C(O)-NH-, a peptide unit comprising from 2 to 4 amino acids, or combinations thereof, each of which may be optionally substituted with one or more of -S-, -S(Oz)-, -C(0)-, -C(O2)-; and COH, wherein subscripts u and v are independently an integer from 1 to 8.

[0032] In one embodiment, L1 is selected from the group consisting of: HO, 0 " o op 9 RY o. bse ~ “oN ren LE Fg Akt ya Yh 5 A Yel YY sa Ta AR ni Sg Hh Na NO) ~ poser fon OE Re LoD oe * and H EA 1 © * , wherein Rais a group comprising an alkyne, an s-cyclooctene, a maleimide, an amine, a ketone, an aldehyde, a azide, a tetrazine, a trans-cyclooctene, a maleimide, an amine, a ketone, an aldehyde, a carboxylic acid, an ester, a thiol, a sulfonic acid, a tosylate, a halide, a silane, a cyano group, a carbohydrate group, a biotin group, a lipid residue and wherein subscripts x, n, p and q are independently an integer from 0 to 12, and combinations thereof.

[0033] In one embodiment, B comprises one adduct of group B'.

[0034] In one embodiment, -NH-L1-B’ is selected from the group consisting of: Ay AOA gy AOD (8) H os o. a. ©) EN Po nO NPENY wg Onn y Ag A H J SN NNN: NP EN NNN NEN) h ~ ~ ; Ay ET Zon A. 07 Ong A RT H i B " N Wa Wo GN No AO, 07 {8 } FN pm H X - 2 Tv gr 8) H Ashi Ha a © 9 ot . od FL J = H ry J Su no A where the Fi is the amino point of attachment to the glutamine residue of the BA.

[0035] In one embodiment, the group B’ is an azide (-Ns), and the adduct of the group B’ comprises a triazole.

[0036] In one embodiment, B comprises two adducts of group B'.

[0037] In one embodiment, B comprises three adducts of group B'.

[0038] In one embodiment, B comprises at least four adducts of group B'.

[0039] In one embodiment, B comprises a group selected from the group consisting of: pe Sg I eT Rega ET me pe he go £2 Was a i Hoy Ae 0 en or Be % oe a3 ¢ & of 5 21 N a Al 1 AB de yr 5 2 No " Tow Se 8) Noxd ! Go 7 A BY a. “ H fr 2 o Lote Ze re % LOA , Pa % CHB) ST } OFFA ) oH [ AA ue H oh w soe Ney e , PLA Re OA GAB Ap spe fl Sse Ny O55 ~E) 1 " - : , 0 y d , and 008) where (B') comprises points of attachment of the adduct of the group B’.

[0040] In one embodiment, B is selected from the group consisting of: x Nr hy © AO On, i sy GPO HL Ong % Po ~~ on Jor NO y y oO sii: “E A ~Y Mp On ors a o XK SN gers Dns, dy N ®) H “O_o. 4 - Nye 8) & pS oN grr BE 6 trfmnpbonons } 5 0 GPA Og Nm) Bem oP" fz i torn A £3 Foe? uel ® Sse 2 In EY 25 \ 8) 3 c oo t ®) su ~~ 5 hs W SE il hil dl LE | NOB) one embodiment, -NH-L1-B is selected from the group ct 1 (8) Lm A 3 @) i gr YN 2 0 Aron, gn H i AN go Om gon Re oO PN On y gry i o. o. Roe wo oO Litre ® doy o / - Or A YI * , L Amon Tg y og H 0 i “ ~ Org o a PO Yr AS AC id H ’ H Meg K Nya g® o ou Mem Fa : N nr pyar On, 2 . or Fyfon i : 0 ed 0 © ayo, -s Bin AB FE B Ae ® Pz Li} Ta & Ayo 5 & E oo ; y co $e bo wl << a BE @) y or \, 5 — iB) i ’ ] 7 ¢ rE ra CPR ” TR \ oe (OOo AB FyOoSAy H yr a LL van n No Oo) A where the >, is the amino point of attachment to the glutamine residue ¢

[0042] In one embodiment, the group B' is an azide (-Na), and the adduct of the group B’ comprises a triazole.

[0043] In one embodiment, the adduct of the group B’ and the group B” has a structure selected from the group consisting of: STN TN Ud t CO Ee \ OD 2 A N | 7 wail Ar xT { IB a , and Rs wherein Q is C or N.

[0044] In one embodiment, M is absent. EAE

[0045] In one embodiment, M is k ,where R, R’, and R” are hydrogens 9 u st A at each occurrence, i.e, Mis AK , where R is hydrogen and R’ and

[0046] In one embodiment, M is si AT 1 R” together form a 5-membered ring, i.e. M is Yeeiid

[0047] In one embodiment, n is 2. In one embodiment, n is 4. In one embodiment, n is 8. In one embodiment, nis 12. In one embodiment, n is 16. In one embodiment, n is 24.

[0048] In one embodiment, L2 has a structure according to Formula (L2): B’-SP1-B2-(-SP2-AA-SP3), (L2), wherein: B” is a group capable of covalently attaching to the group B’; SP1 is absent or a first spacer unit; B2 is absent or a branching unit; SP2 is absent or a second spacer unit; AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit covalently attached to the Dxd, and p is an integer from 1 to 12.

[0049] In one embodiment, the at least one group B’ is selected from the group consisting N=N, Lp NN e@slay i May “2 TN 77 i ii pt y of -Na, N=N | —f=— fg for Ne... , and combinations thereof.

[0050] In one embodiment, SP1 is absent or selected from the group consisting of © a & [I NP and Le

[0051] In one embodiment, the B2 is absent or selected from the group consisting of Ox GQ 1 o 54 4 NAY " = 2. % Vn om = > rE >, #3 Ad ~ Non 0 APO AN. 9% he A Forno? Le Lo wos he Ye ° cg ’ -d 7 and AF

[0052] In one embodiment, SP2 is absent or selected from the group consisting of a C16 alkyl, -(CH2-CHz-O)v~, -NH-, -C(O)-, -NH-C(O}-, -NH-(CHz)u-, -NH-(CH2),-C(O)-, -NH-(CH2-CHz- O)v, -NH-(CH2-CH2-O)-C(QO)-, -NH-(CH2-CH2-0)~(CHz)u-, -NH-(CH2-CH2-0)~(CH2)u-C(0)-, - (CHz2):-NH-C(O)-, -NH-(CHz),-NH-C(O)-, -NH-(CHz),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8.

[0053] In one embodiment, AA is a peptide unit comprising from 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

[0054] In one embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0055] In one embodiment, AA is selected from the group consisting of glycine-glycine- glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine- phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)), L- glutamic acid-valine-citrulline (-EVC), and D-glutamic acid-valine-citrulline (°EVC). jc je ” ng i Z Ny Hh NA ~p LA aA N a <& GC I$ * Amide 4 , and combinations thereof, wherein R. is independently at each HO, HO© HO, Mo HO, No HONE Hora occurrence absent or a group selected from OH and OH

[0057] In one embodiment, the M-Dxd has a structure selected from the group consisting of 0 Ay No? Qo pl Ao bh pet and OH, o HoT nl 0 H ir Ap Ie eS H SR Ry o 1 LM F NN 2 Ne OHO, and WNBERIY EY Ta 2 Ire ra In Ar al Bik wherein R is a hydrogen or a C-C4 alkyl, and where « represents the point of attachment to L2.

[0058] In one embodiment, the compound has a structure: a o Aa \ a 7 - ohn ion dy, Wo {oN 0 Yd rR Ap + el LL & at S ew Y BAL, By Pym Kg LT TETAS ~ LL Y os, y a on - Fo

[0059] In one embodiment, the compound has a structure: a Qa ~Amdoa dy, Fm 0 4 Sy A oN Qphismmmmn Sd &C iY & a A i I fo VF WNT a o A i I“ — spodnmion ly, 2 =o Haden IF II YY SE / <o oY a Ay T , nw F 4

[0060] In one embodiment, the compound has a structure: 3 fonts , SNL e ord Yo Ponds Ty YR SO — * ™ Ls, / .

[0061] In another aspect, the present disclosure provides a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)i)n (1), wherein: BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is absent or a first linker; B is a branching unit comprising aa Nd | On Te ! \ \. N N= Fa t 0 Ny a xT Ny \ on I 2” N J "De HD 0, VN Now \ { . i” = ~~ ~~ } . Os d , M TH+ f } : 3! 1 is absent or a moiety having the structure & , where R, R’, and R” are independently at each occurrence hydrogen or a C1-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Byreps® Fe = TY A a F \ Ie = "4 ®; k is an integer from 1 to 12, and nis an integer from 1 to 30.

[0062] In another aspect, the present disclosure provides a compound according to Formula (L2-P), (L2'-P), or (L2"-P): B”-SP1-B2-(-SP2-AA-SP3-M-Dxd)p (L2-P), HzN-SP1-B2-(-SP2-AA-SP3-M-Dxd), (L2'-P), maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd), (L2"-P), wherein: N=N 44 px NeN i.e = — B” is selected from the group consisting of -Na, N-N | ~fr=—ti f= QTD LOGON ne and Sn and Se Q SP1 is absent or a first spacer unit selected from the group consisting of and a ee SP2 is absent or a second spacer unit selected from the group consisting of a C1.s alkyl, - (CH2-CH2-O)v-, -NH-, -C(O)-, -NH-C(0O)-, -NH-(CH2)-, -NH-(CH2)u-C(O)-, -NH-(CH2-CH2- O)v-, -NH-(CH2-CH2-0)y-C(O)-, -NH-(CH2-CH2-0),-(CHz)u-, -NH-(CH2-CH2-O)\~(CHz)u-C(O)-, -(CH2)y-NH-C(Q)-, -NH-(CHz2)u-NH-C(O)-, -NH-(CHz),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8; AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit selected from the group consisting of, ro se S AL i +4 a 4 i NE *h NA pe Ae AA Y } oO o oO , wherein R. is independently at each occurrence absent or HO. HO How A HO, re ° LPN a group selected from OH and OH e ! o Nb 2 ¥ « M is absent or & , where R, R’, and R” are independently at each occurrence hydrogen or a C+-Cs alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): Bynagt® ; SN r 1 TJ AH > —, (P), and p is an integer from 1 to 12.

[0063] In one embodiment, AA is a peptide unit comprising from 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, alanine and citrulline, and combinations thereof.

[0064] In one embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0065] In one embodiment, AA is selected from the group consisting of glycine-glycine- glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine- phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)) and glutamic acid-valine-citrulline (EVC).

[0066] In one embodiment, the compound has a structure selected from the group consisting of: PS | 3 o Adon ITs LITT vy ol 2 JF NY Ux 1 Sy NH, Y S . 0 ypolnpion dy, Tommi AL LO 0s FE ° 0 Ng " Sf ¥ a N\ o H 4 H Tes k Grid J BH IN oy i y d u d Hou Ay ng prgomady As Ad 2 HON NT EY . oH le CZ “SN J A i - 7 CNH EF Nery wi aH TN . [4] a i MH oly rtd LI 5 ~~ HO" Y oy CH HN y ~N\ J A NH P Nay Ade Aided = TI Clary ot & 7 eto x ZN, ¢ J 2 Thon, \ i : = 07 N 4 | =o yy RL o = / QF oP OH Hos AA Co OH LN H I oO o oo 0 o H ’ f | ps NOR a. =A YY y | oO Jon Y HOA Aa OH 7 oan HOY ¥° y 0G I nS ai Sek, AAY° a oS AO os wi © , 0 oa OQ —, LT F 9 0 Ky 2 0. XE ; ~~ Co enero dy 1 o | Chirpbonimmnmonnyig LE ALT YY Seg + 5 o FL LR, ~N § Ig woke Sy AY © o % =e) ~~ tet in i | Qn mmmmmingid 20 NY LO £ | 1 YY x wel LL, J 5 Ny or a pharmaceutically acceptable salt thereof.

[0067] In one embodiment, the compound has a structure selected from the group consisting of: > ° ONT ey Soyo Oro L : d 7 “7 & wo A Noo Wh Noo Wh i 5 "a 3 arm onn JL t Ae orn 2 bs ee Re | berm I ry EH NB he Noo Syn H or a pharmaceutically acceptable salt thereof.

[0068] In one embodiment, the compound has a structure according to Formula (L2-P), (L2'-P) or (L2"-P) selected from the group consisting of: , NS SNS PL | 3 en ym JY oy XB, ~ Nr x Pa “ #: " O Na k _ A Ko Yo Chongming Idina d,, $7 © 5 #7 | oO o oN Lo NNN ool, As a rey LI O 9 SNH, H ~ XK Neo ° She Q ¢ H WH . . Ory H a Ay 3 t a N i C0 Se % 2 . tel JA LO MS Gas PY a s & CL, 7 | ¥ C1 aH WA Sy NH, I 0: NH nL guilt Leo HN. N 20 o ! A fl ok N 0 9 9 CNY Tw onrngligh LAA o J 0 « a wa ” 7 vue % 2 oS ou - <Q Sy pom e % : x oH D Xa OY : y CH, 2 Ay - 3 4 3 5 &) 2 Pe WH Q o H Khoo, . SY y " i 1 i or = f # : A HL, A Q } Ou mnAg ‘ Omg " Yr : A SNR § 0 9 ™ i i nn LCL ~ Hoo CY Ferg p ST CN il ~ op a HN - ~o HN Sg Ong . OF "NH t I O. g fondo t,, i o OY r Ls 1 0 5 HN, ) bomen Ay I a4 EB 1 5 4g a Q 2 o Lg Hook, nL ! SORT 6 NT, Cr hn a wl IT Pop ~ tJ a i g oN o. oO! A Op, oy 2 oN Pay 5° 2 5 3 HX y™ 0 Aaa, food, 2 =o monk LE OTT Sead ~ 0 Sy an Ppa, ate ror ht Oy = L3- I RB SSAA) IF ann Sng Ne ; A i gy Son I oon ou Re oo pe q - J oo ay $8 VS " o , com fo My, 2 de Mngt TY XE SGLEA & “OH oN NH Y Nak, ua 1 J jg ~~ nh I ez Hn ~ PEE a oY wo Oo ah on pw he OA pAymmron / v 3 H (po 1, . 3 M3 HN [NN Ay 0 \, BOINSCALE Ras Jur oY ry q : & oe HO | NA, > o y a Q ery o : o Ags Fo 0, Xn 1 Oy mO. He 2 5 ; oie FA I 5 , ~ ~ 9 2 Li 1 Cob ~ 9 Sg H a \, I NT NH, H ? GTN 0 PN No, : I TY TW w Ogy NH \ Hal” On J i : . arn OOK Syn o) NN Spo 3 HN, 1 4 i 4 oO fel hp A ~ 3 AAT [3 H ] ° : ~ oN , Pon 5 1 < NN Y -: 9 H 8 gh Li A Cry as Hak, fas AS © A FEY Co : ~ ~ ad he 1 va he aon ~ a Sy, y oN Pon ~ Sy = . WL 8 ’ oo gph = : gy fA a. * rs yr SR Sod H Nr T So | J: bY & } NT UNHy 4 Po = Linn lod Lh 2 : Hedy {, X or ’ : a Aer pd T 0H 3 : pe RS % N NH 1 Ay a I) Ro 3 room Ao dy 3 rnp A wy SG YIVTCY X AE tg A LL, A So rctmgmin i t / No ea Ne [ 4. "ommnded LY Sn TALC oh BN LA, ¥ J Hus H I oo ge Ke ; a TE ° Se ’ x SUSTES d8Soaas ans Jue ty YEN roel at Et Sond 0. A, N < : y J PAYY & : rtrd, ~ "ar FI ytd a i H i ¥ TTT 5 omni 207 x a, ‘ i In ’ YE ry “3 & pr. oe Sows aa ) 6 Lo LA Jb A Se Xx o oon NH ~ Sutalyh Nr Le y el x rrp lr ® RA A S ny : \ ¥ a Ea Sw oy . . Jel, | NY y pte dh LL ~y x Nn DY C3 Dd Pom ~ Q Sr p— Sow on A A ’ ELT 8 LT, X Q é Pon Sr edd } } J Hoo = Le Ln LLY Front HL ? Pon Se, Sy ~ 2 ww Aor She ee Yr 7 Be 0 A 1 Y T. y ° rey ™i C1 YP SA Pro uy 1, ) XL { + | 5 Eo T or oe 9 o i Oe WIT 0 ene 4 2 Q gS Cr pe o YA $f oh ox Nw ~ rp A adage? on ~ og Sw H, Pon 5 a DR Sf eh ror 4 3 0 o é Pom “on 3 POA Oy 5 ~~ 1 i f NOY 8 er TLL AL Y 7 Aon Shoes Sy ° Q Qt LY 8 Ory J oyiel lt 4 = 8 | ~ ~ 3 T Pa Svs A i pm ™~ LA, AT A CY Ay G8 ’ wet dt oA a L ° Hon Se - RUN St ° ° ° oS Lh 9 ss Oey SIA yy 2 i] i Eo I omg Sern fh 5 fe C y ae efron AIR LIS NY x 5 a Fond 3 y ce ¥ ! 1 o o Qo < i i 4 5 2 Se Lh iN A 8,1 Yr ~My NY { of ( Y i re LL, Xr and dom to Ad, 3 am SIL ALT 5 f AEE GE Or JULIETTE X ~ wor Se rd © “ Qa re FL LA oP gon dl, oF ee II Imm dfn np ATA OT LIE J NX SERA GA [ 5 wr Lh ya Li & ~ pond 3 1 Lo I TE GE MI mr SS “YY { Lh ws Yh A

[0069] In another aspect, the present disclosure provides an antibody-drug conjugate according to formula (ll): Ab-(GIn-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd)i)p)n (lI), wherein Ab is an antibody; Gln is a glutamine residue; L1 is absent or a first linker; B is a branching unit comprising at least one adduct of group B’ and group B”, where 200 LID ays (OFF the group B' is selected from -Na, Rn “foo N-N and at least one group B”, wherein B’-SP1-B2-(-SP2-AA-SP3-M-Dxd)p is the compound according to formula (L2-P) according to any of the preceding embodiments, and wherein the compound of formula (L2-P) is covalently attached to the antibody via the adduct of the group B’ and the group B”; k is an integer from 1 to 12, p is an integer from 1 to 30, and n is an integer from 1 to 30.

[0070] In one embodiment, the present disclosure provides an antibody-drug conjugate comprising an antibody and a linker-payload, wherein the linker-payload comprises the structure: SF o H o ty o o I OY redone Ay o Nod © Ng Z ~ 0 Fon o F , or o i o A Q WE 9 ao Ayo Ny ={_o A o ~~ A Hog 2 ba ad Og hy A Hl / NY ANN SoS =, BM No a where «nn represents the point of attachment to the antibody, directly or through a second linker, or a pharmaceutically acceptable salt thereof.

[0071] In another aspect, the present disclosure provides a composition comprising a population of compounds according to any of the preceding embodiments, having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.

[0072] In one embodiment, the composition according to the disclosure has a DAR of about 1.0 to about 2.5. In one embodiment, the composition according to the disclosure has a DAR of about 2.

[0073] In one embodiment, the composition according to the disclosure has a DAR of about 3.0 to about 4.5. In one embodiment, the composition according to the disclosure has a DAR of about 4.

[0074] In one embodiment, the composition according to the disclosure has a DAR of about 8.5 to about 8.5. In one embodiment, the composition according to the disclosure has a DAR of about 8.

[0075] In one embodiment, the composition according to the disclosure has a DAR of about 10 to about 14. In one embodiment, the composition according to the disclosure has a DAR of about 12.

[0076] In one embodiment, the composition according to the disclosure has a DAR of about 14 to about 18. In one embodiment, the composition according to the disclosure has a DAR of about 16.

[0077] In one embodiment, the composition according to the disclosure has a DAR of about 22 to about 24.5. In one embodiment, the composition according to the disclosure has a DAR of about 24.

[0078] In another aspect, the present disclosure provides a compound having a structure according to Formula (P-I): ing to Formula (P-I): oO Ry pan Gino Ri Rp oa oNH ree pF V( \ SHS (PH), wherein BR, R- RR. and R. are inde wherein R41, Rz, Ra, and Ry are independently a hydrogen or a C1-C4 alkyl, or wherein R2 and Rs form a 5-membered or a 6-membered ring, or a pharmaceutically acceptable salt thereof.

[0079] In one embodiment, the compound is Is] Ayo? H NH Fo” ~ Nd or a pharmaceutically acceptable salt thereof.

[0080] An antibody-drug conjugate comprising an antibody, a linker, and a payload, o of IL o Ha Ney ri wherein the payload is yop oe be Ny o Pes FONT) H wo or a pharmaceutically acceptable salt thereof.

[0081] An antibody-drug conjugate comprising an antibody, a linker, and a payload, q Ad A N oP A NH

[0082] In one embodiment, the compound has a structure according to Formula (P2): oN 0 yg] NOY lH Se Se 0 | = oH N F NTN , No wherein the payload is OMG, or a pharmaceutically acceptable salt thereof, where «a represents the point of attachment to the linker.

[0082] In one embodiment, the compound has a structure according to Formula (P2): JN | In one embodiment, the compound h ha Nop 0 NH rN LO 2 N- F NN pe ~ SHO (P2), wherein R is 2 hvdroaen or a C+-C4 alkyl wherein R is a hydrogen or a C+-C4 alkyl, or a pharmaceutically acceptable salt thereof.

[0083] In one embodiment, the compound is LVVoy) 11 OTe SPOUTS, Ue COITPpOUridd ls wml 0 NH C RN Lo FT, “ef CH 0 | or a pharmaceutically acceptable salt thereof.

[0084] In one aspect, the present invention provides a pharmaceutical composition comprising the compound according to any of the preceding embodiments, and a diluent, a carrier, and / or an excipient.

[0085] In one aspect, the present invention provides a method of treating a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the compound according to any of the preceding embodiments, or the composition of any one of the preceding embodiments.

[0086] In one embodiment, the condition is cancer.

[0087] In one embodiment, the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

[0088] In one embodiment, the condition is HER2+ breast cancer.

[0089] In one aspect, the present invention provides a method of selectively delivering a compound into a cell, wherein the compound is according to any one of the preceding embodiments.

[0090] In one aspect, the present invention provides a method of selectively targeting an antigen on a surface of a cell with a compound, wherein the compound is according to any one of the preceding embodiments.

[0091] In one embodiment, the cell is a mammalian cell.

[0092] In one embodiment, the cell is a human cell.

[0093] In one embodiment, the cell is a cancer cell.

[0094] In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0095] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)i)n (A), wherein BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is a first linker; B is a branching unit comprising at least one adduct of a group B’ and a group B”; L2 is a second linker covalently attached to the branching unit B via at least one group B”; SAK M is absent or b , where R, R’, and R” are independently at each occurrence hydrogen or a C4-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent comprising a structure according to Formula (P): pS pe. wh A ~~ ~ < P), k and m are independently an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NH,) with a compound L1-B, wherein the branching unit B comprises the at least one group B’, b) contacting the product of step a) with k or more equivalents of a compound L2-(-M-Dxd)m, wherein the linker L2 comprises at least one group B”, N= 0 “4 i wherein one of the groups B’ and B” is selected from -N3 and N-N ; and the other ~ OY = LIC Tx of the groups B’ and B” is selected from f= Ao ; and Nr where Q is C or N; and ¢) isolating the produced compound of Formula (1).

[0096] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)x)n (A), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one adduct of a group B’ and a group B” as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above; M is ? l R 3A KK ror absent or fe , where R, R’, and R” are as described above; Dxd is an anti- tumor agent comprising a structure according to Formula (P): Ws, 0 TY rT TT NN Pp ] Ne Fa Pa o 4 -\ , 3 — H 6 (P), k and m are independently an integer from 1 to 12, and nis an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least one group B’, with k or more equivalents of a compound L2-(-M-Dxd)m, wherein the linker L2 comprises at least one group B” capable of covalently binding with the group B’, N=N 4 5 { N-N - wherein one of the groups B’ and B” is selected from -Nj and N-N : and the other i nd ~ “ia, . OT ees” of the groups B’ and B” is selected from =f “Po ; and oe where Q is C or N; thereby producting L1-B-(-L2-(-M-Dxd)m); b) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NH,) with the L1-B-{-L.2-(-M-Dxd)m)k product of step a), and ¢) isolating the produced compound of Formula (1).

[0097] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)i)a (0, wherein BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is a first linker; B is a branching unit comprising at least one adduct of a group B’ and a group B”; L2 is a second linker covalently attached to the branching unit B via at least one group B”: EAE M is absent or ke , where R, R’, and R” are independently at each occurrence hydrogen or a C1-Cy4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent comprising a structure according to Formula (P): Wor” Apr F TT ARE ~AX ®) k is an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NH.) with a compound L1-B, wherein the branching unit B comprises the at least one group B’, b) contacting the product of step a) with k or more equivalents of a compound L2- M-Dxd, wherein the linker L2 comprises the at least one group B” capable of covalently attaching to the group B’, N=N HH wherein one of the groups B’ and B” is selected from -N3 and N~N and the other mY Sr aC AF TN of the groups B’ and B” is selected from gm oe ; and Neer where Q is C or N; and ¢) isolating the produced compound of Formula (1).

[0098] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)x)a (1), wherein BA is an antibody or an antigen-binding fragment thereof, Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one adduct of a group B’ and a group B” as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above; M is BAK absent or & , where R, R’, and R” are as described above; Dxd is an anti- tumor agent comprising a structure according to Formula (P): Kon? dle ge 1 1 Nr 0 re 7 < 5 A (P), kis an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least one group B’ with k or more equivalents of a compound L2-M-Dxd, wherein the linker L2 comprises at least one group B”, thereby producting L1-B-(-L2-M-Dxd)x, wherein one of {hs { NN the groups B’ and B’ is selected from -N; and ~~ N~N : and the other of the groups B’ and B” SAAS where QisCorN: Re Ngee” Tr 1 is selected from —5===§~ “oe ; and Nes b) contacting, in the presence of a transglutaminase, the binding agent BA comprising at least one glutamine residue Gin (BA-GIn-NHz) with the L1-B-(-L2-M-Dxd)« product of step a), and ¢) isolating the produced compound of Formula (1).

[0099] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (l11): BA-(GIn-NH-L2"-P)), (111), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L2-P is H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p as described above, and n is an integer from 1 to 30; Q . . . a. soe SP1 is absent or a first spacer unit selected from the group consisting of o and ~ and © B2 is absent or a branching unit; SP2 is absent or a second spacer unit selected from the group consisting of a C1.¢ alkyl, -(CH2-CH2-O)v-, -NH-, -C(QO)-, -NH-C(O)-, -NH-(CH2)u-, -NH-(CH2)u-C(O)-, -NH-(CHa- CH2-O)-, -NH-(CHz-CH2z-O),-C(O)-, -NH-(CHz-CH2-O),-(CHz)u~, -NH-(CH2-CH2-O) (CH2)u-C(0)-, -(CH2)u-NH-C(O)-, -NH-(CH2)u,-NH-C(O)-, -NH-(CH2)u-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8: AA is absent or a peptide unit comprising from 2 to 4 amino acids; Fo Fe 0 # 0. } Cl i I oo pe % L pe A AA To ZC Pm or i ) , wherein Rc is independently at each occ HO Ho. A HOO HO, 0 Lor AN or a group selected from OH and OH AK M is absent or 4 , where R, R’, and R” are independently at each occurrence hydrogen or a C4-Cs4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): pS pe. wh A ~~ ~ < P), p is an integer from 1 to 30; wherein the method comprises the steps of: b) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gln (BA-GIn-NH2) with the L2'-P, and ¢) isolating the produced compound of Formula (III).

[0100] In one embodiment, the glutamine residue GIn is naturally present ina CH2 or CH3 domain of the BA.

[0101] In one embodiment, wherein the glutamine residue Gin is introduced to the BA by modifying one or more amino acids.

[0102] In one embodiment, wherein the Gin is Q295 or N297Q.

[0103] In one embodiment, wherein the transglutaminase is microbial transglutaminase (MTG).

[0104] In one embodiment, M is absent, or the M-Dxd has a structure selected from the group consisting of 2 2 Ng 0 Fr Ym, “a and > os . up consisting of 11 NA 0 x N oN Ay NH H SRR LAA F NTN o Nef OHO, and whores Bie o Warrnnan ore So: oik wherein R is a hydrogen or a C4-C4 alkyl, and where « represents the point of attachment to L2.

[0105] In one embodiment, the compound L2-Dxd has a structure selected from the group consisting of: / 2 foody | 0 NY WN Gn Orme bp 0 I SF { Ae. 0 ryredryeady Stren 0” Y oF “] ° Ss Ug N Clan, ; ON [A o H 4 H tne k Grid J 8H TN ” x ~ too, as I Cr SH Ho jo N Nahe I aH TN HO, ) i Ko mdm ioo RY i. Ue X \ 1 or Jr Neto fot S Arte Ido d,, ~ © © Py Nt Clary ot & 7 eto x ZN Cd BA fon. i Noro A rgd Ron = Ey or 7 Q Pa - [a oN rosy, San —— rx @ © y § Arpt diye dy, A WOH , Ax OH Ho oH Hoe eS Ww 9 pid i Io A I~ He a3 & 5 & os Sf y a Q 0, N a _— Hood, Co | i § Me on 8 TY ¥ Sd Srirphongbonges ve sdend 5 ox ub oho Shae JF 2 ted 3.78, ~~ Ro 0A Ras | > i 5, 0 162, Xx i or YY ~ Hy o®s 4 | C ob mgr igh tty A Hof Y Y z "a So IN Tr HOO Sh J F 3 | A H # | or a pharmaceutically acceptable salt thereof.

[0106] In one embodiment, the compound L2-Dxd has a structure selected from the group consisting of: —_ — Yop ror wren J HY 5 “ J ow A he ~ Syn EH, ~ Syn To —o T% ~~ SY Le pion A he a LL d 3 A re J ow, pe 1 ° 9 9 ¢ yo rs { Arete RA AL 9 A= Sa TY 1 0 a 7° Nr ~ 0 Seb, H LT No Sb or a pharmaceutically acceptable salt thereof.

[0107] In one embodiment, the compound L2-Dxd has a structure selected from the group consisting of: ih E oy Io 2 reyes 8 Ayheamman ed YY ne SOA ~ Tx Sp, S / Je n £ wa od ww 8 Xa Sen, ~~ Pa . $ 0 o a A rompiamoningrin At Ae eb TY mo 5 “> & ost wa Y 9 0 @, Ny 0 oN Hoo Ay, EAST 9 Shaky H STN Ao RH F Ny 0 _A_ / 0 oy, 5 = ~NA Ho | N ; iw So Pan Ao Noy bY 2 O 3 «© 3 Hl pu ood, . Say . ANITA PR 5 0 Sy . oC fol 1 : : : yeh A Jol oy ' | N M.., ’ | 5 HL A YX ” “x i Q ¥ S89 : oY OQ OOH 2 Sy i | © Oy NH ~eAS go? aR Hh 2 o 9 A Hoo hl 2 9 NY ononglpk Li JI a J Q “ Q - & -n Yt J - on So, HH pom e % : x oH D Xa OY : y CH, 2 Ay - 3 4 3 5 &) 2 Pe WH Q o H Khoo, . SY y " i 1 i or = f # : A HL, A Q } Ou mnAg ‘ Omg " Yr : A SNR § 0 9 ™ i i nn LCL ~ Hoo CY Ferg p ST CN il ~ op a HN - ~o HN Sg Ong . OF "NH t I O. g fondo t,, i o OY r Ls 1 0 5 HN, ) bomen Ay I a4 EB 1 5 4g a Q 2 o Lg Hook, nL ! SORT 6 NT, Cr hn a wl IT Pop ~ tJ a i g oN o. oO! A Op, oy 2 oN Pay 5° 2 5 3 HX y™ 0 Aaa, food, 2 =o monk LE OTT Sead ~ 0 Sy an Ppa, ate ror ht Oy = L3- I RB SSAA) IF ann Sng Ne ; A i gy Son I oon ou Re oo pe q - J oo ay $8 VS " o , com fo My, 2 de Mngt TY XE SGLEA & “OH oN NH Y Nak, ua 1 J jg ~~ nh I ez Hn ~ PEE a oY wo Oo ah on pw he OA pAymmron / v 3 H (po 1, . 3 M3 HN [NN Ay 0 \, BOINSCALE Ras Jur oY ry q : & oe HO | NA, > o y a Q ery o : o Ags Fo 0, Xn 1 Oy mO. He 2 5 ; oie FA I 5 , ~ ~ 9 2 Li 1 Cob ~ 9 Sg H a \, I NT NH, H ? GTN 0 PN No, : I TY TW w Ogy NH \ Hal” On J i : . arn OOK Syn o) NN Spo 3 HN, 1 4 i 4 oO fel hp A ~ 3 AAT [3 H ] ° : ~ oN , Pon 5 1 < NN Y -: 9 H 8 gh Li A Cry as Hak, fas AS © A FEY Co : ~ ~ ad he 1 va he aon ~ a Sy, y oN Pon ~ Sy = . WL 8 ’ oo gph = : gy fA a. * rs yr SR Sod H Nr T So | J: bY & } NT UNHy 4 Po = Linn lod Lh 2 : Hedy {, X or ’ : a Aer pd T 0H 3 : pe RS % N NH 1 Ay a I) Ro 3 room Ao dy 3 rnp A wy SG YIVTCY X AE tg A LL, A So rctmgmin i t / No ea Ne [ 4. "ommnded LY Sn TALC oh BN LA, ¥ J goed LN oA © i YOY Ce Sdn wo 9 Aor Sh ¢ Ho oo y Vomongled LA | Saag ed re Te h an, or Et Sond 0. A, N < : y J PAYY & : rtrd, ~ "ar FI ytd a i H i ¥ TTT 5 omni 207 x a, ‘ i In ’ YE ry “3 & pr. oe Sows aa ) 6 Lo LA Jb A Se Xx o oon NH ~ Sutalyh Nr Le y el x rrp lr ® RA A S ny : \ ¥ a Ea Sw oy . . Jel, | NY y pte dh LL ~y x Nn DY C3 Dd Pom ~ Q Sr p— Sow on A A ’ ELT 8 LT, X Q é Pon Sr edd } } J Hoo = Le Ln LLY Front HL ? Pon Se, Sy ~ 2 ww Aor She ee Yr 7 Be 0 A 1 Y T. y ° rey ™i C1 YP SA Pro uy 1, ) XL { + | 5 Eo T or oe 9 o i Oe WIT 0 ene 4 2 Q gS Cr pe o YA $f oh ox Nw ~ rp A adage? on ~ og Sw H, Pon 5 a DR Sf eh ror 4 3 0 o é Pom “on 3 POA Oy 5 ~~ 1 i f NOY 8 er TLL AL Y 7 Aon Shoes Sy ° Q Qt LY 8 Ory J oyiel lt 4 = 8 | ~ ~ 3 T Pa Svs A i pm ™~ LA, AT A CY Ay G8 ’ wet dt oA a L ° Hon Se - RUN St ° ° ° oS Lh 9 nb ATH : ST Xx 3, I ! Lo o (7, : SI Wo y XJ ! oe uP AEA Ahn A 1 i hep dy 2 / i v oh mpfr dol 8.1 Oy $i 08 rw YN s LL 7 : EYE 6 Pd EN wy Wh, or ; i wy 4, $ Wg Ft Ses NETS CEE acs age Jed “YY yy rye HG, 5 , and ~ Jad, 3 png deh LI Ay (Jf SOY rh Z woe San Egat oA wf Qa tigger Fogg A LY a Ou { Ye o YY 1 crm A Y 5s vac oh re Lik 2 5 wp ES 1 AY pg OEY Soni A LR 2 I WY ! hry ER ELE C 5 L oy Sym, ol T

[0108] These and other aspects of the present disclosure will become apparent to those skilled in the art after a reading of the following detailed description of the disclosure, including the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0110] Figure 1 is a schematic demonstrating two-step site-specific generation of Dxd- ADCs according to an embodiment of the disclosure. The first step is conjugation of one or more first linkers (L1-B’) with a glutamine residue on an antibody via a transglutaminase (e.g., MTG)- mediated conjugation reaction. The second step is conjugation of antibody-L1-B to one or more Linker 2-Payloads (L2P).

[0111] Figures 2A and 2B are schematics demonstrating specific non-limiting embodiments of the disclosure. Figure 2A is a schematic of a two-step site-specific generation of Dxd-ADCs with glutamine residues at position 295 having a DAR of 2 times n times m according to an embodiment of the present disclosure. Figure 2B is a schematic of a two-step site- specific generation of Dxd-ADCs with a glutamine residue at positions 295 and 297 having a DAR of 4 times n times m according to an embodiment of the present disclosure.

[0112] Figure 3A is a schematic demonstrating two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the disclosure. The first step is to conjugate a linear first linker 1 (L1-B’) comprising one azide moiety (-Ns) to glutamine residues at positions 295 and 297 of an antibody via an MTG-mediated conjugation reaction, generating an antibody having 4 azide-comprising linkers attached to it (Ab-(N3)4). The second step is to attach Ab-(Na)s to a specific Linker2-Payload (L2P) via the azide-cycloalkyne 1,3 cycloaddition reaction, generating a Dxd-ADC with a DAR of 4. Figure 3B depicts schematics of ADCs and exemplary amino azido linkers having a DAR of 2 or 4 suitable for use in an embodiment of the present disclosure depicted in Figure 3A.

[0113] Figure 4A is a schematic demonstrating two-step site-specific generation of one specific embodiment of a Dxd-ADC according to the disclosure. The first step is to conjugate a branched first linker 1 (L1-B’) comprising two azide moieties (-N3) to glutamine residues at positions 295 and 297 of an antibody via an MTG-mediated conjugation reaction, generating an antibody having 8 azide-comprising linkers attached to it (Ab-(Na)e). The second step is to attach Ab-(Na)s to a specific Linker2-Payload (L2P) via the azide-cycloalkyne 1,3 cycloaddition reaction, generating a Dxd-ADC with a DAR of 8. Figure 4B depicts schematics of ADCs and exemplary branched alkyl azide amine linkers suitable for use in an embodiment of the present disclosure depicted in Figure 4A.

[0114] Figure 5 is a schematic demonstrating three approaches to producing site-specific ADCs with DAR4 to DAR24 on Antibody-Q295 / 297 according to an embodiment of the disclosure.

[0115] Figure 6 is a schematic demonstrating three approaches to producing site-specific ADCs with DAR2 to DAR 12 on Antibody-Q295 according to an embodiment of the disclosure.

[0116] Figure 7A is a schematic demonstrating two-step Approach | for making an exemplary 8DAR Branch-Linker-Payload ADC (Ab-AL1-LP39). Figure 7B is a schematic demonstrating two-step Approach Il for making an exemplary 8DAR Branch-Linker-Payload ADC (Ab-BL2-LP22). Figure 7C is a schematic demonstrating one-step Approach Ill for making 4DAR ADC with Linear-Linker-P and 8DAR ADC with Branch-Linker-Payload.

[0117] Figure 8A (SNU16, FGFR2-amplified gastic cancer) and Figure 8B (SNU16 tumor bearing mice) show tumor volume vs. days after treatment for anti-FGFR2b Dxd ADCs (DARS) according to the disclosure. These ADCs demonstrated significant anti-tumor efficacy against SNU-16 human gastric cancer xenografts.

[0118] Figure 9A (SNU16, FGFR2-amplified gastic cancer) and Figure 9B (SNU16 tumor bearing mice) show tumor volume vs. days after treatment for anti-FGFR2b Dxd ADCs (DAR4) according to the disclosure. These ADCs demonstrated significant anti-tumor efficacy against SNU-16 human gastric cancer xenografts.

[0119] Figure 10 is a matrix illustrating the components of 272 exemplary MET x MET bispecific antibodies disclosed herein. Each numbered cell of the matrix identifies a unique bispecific antibody comprising a “D1” antigen binding domain and a “D2” antigen binding domain, wherein the D1 antigen binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDRs from the corresponding anti-MET antibody listed along the Y-axis, and wherein the D2 antigen binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDRs from the corresponding anti- MET antibody listed along the X-axis. DETAILED DESCRIPTION

[0120] Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the disclosure that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the disclosure is intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present disclosure. Definitions

[0121] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0122] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0123] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician. In some embodiments, treatment comprises methods wherein cells are ablated in such manner where disease is indirectly impacted. In certain embodiments, treatment comprises depleting immune cells as a hematopoietic conditioning regimen prior to therapy.

[0124] A “subject” or “patient” or “individual” or “animal”, as used herein, refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0125] As used herein the term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0126] The phrase “pharmaceutically acceptable salt’, as used in connection with compositions of the disclosure, refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those disclosed in Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66:1, incorporated herein by reference. Examples of salts include, but are not limited to, acid derived, base derived, organic, inorganic, amine, and alkali or alkaline earth metal salts, including but not limited to calcium salts, magnesium salts, potassium salts, sodium salts, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, p toluene sulfonic acid, salicylic acid, and the like. In some examples, a payload described herein (e.qg., a rifamycin analog described herein) comprises a tertiary amine, where the nitrogen atom in the tertiary amine is the atom through which the payload is bonded to a linker or a linker-spacer. In such instances, bonding to the tertiary amine of the payload yields a quaternary amine in the linker-payload molecule. The positive charge on the quaternary amine can be balanced by a counter ion (e.g., chloro, bromo, iodo, or any other suitably charged moiety such as those described herein).

[0127] Ranges can be expressed herein as from “about” or “approximately” one particular value and / or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value.

[0128] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if the other such compounds, material, particles, or method steps have the same function as what is named.

[0129] Compounds of the present disclosure include those described generally herein, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0130] As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C+—Cxz for straight chain, C2—Cz for branched chain), and alternatively, about 1-10 carbon atoms, or about 1 to 6 carbon atoms. In some embodiments, a cycloalkyl ring has from about 3-10 carbon atoms in their ring structure where such rings are monocyclic or bicyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C1—Cs for straight chain lower alkyls).

[0131] As used herein, the term “alkenyl” refers to an alkyl group, as defined herein, having one or more double bonds.

[0132] As used herein, the term “alkynyl” refers to an alkyl group, as defined herein, having one or more triple bonds.

[0133] The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring.

[0134] The term “halogen” means F, Cl, Br, or |; the term “halide” refers to a halogen radical or substituent, namely -F, -Cl, -Br, or -I.

[0135] The term ‘adduct’, e.g., “an adduct of group B” of the present disclosure encompasses any moiety comprising the product of an addition reaction, e.g., an addition reaction of group B’, independent of the synthetic steps taken to produce the moiety.

[0136] The term “covalent attachment’ means formation of a covalent bond, i.e., a chemical bond that involves sharing of one or more electron pairs between two atoms. Covalent bonding may include different interactions, including but not limited to o-bonding, m-bonding, metal-to-metal bonding, agostic interactions, bent bonds, and three-center two-electron bonds. When a first group is said to be “capable of covalently attaching” to a second group, this means that the first group is capable of forming a covalent bond with the second group, directly or indirectly, e.g., through the use of a catalyst or under specific reaction conditions. Non-limiting examples of groups capable of covalently attaching to each other may include, e.g., an amine and a carboxylic acid (forming an amide bond), a diene and a dienophile (via a Diels-Alder reaction), and an azide and an alkyne (forming a triazole via a 1,3-cycloaddition reaction).

[0137] As described herein, compounds of the disclosure may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0138] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.

[0139] Unless otherwise stated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure.

[0140] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 'C- or '3C- or 14C -enriched carbon are within the scope of this disclosure.

[0141] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0142] Unless otherwise stated, all crystalline forms of the compounds of the disclosure and salts thereof are also within the scope of the disclosure. The compounds of the disclosure may be isolated in various amorphous and crystalline forms, including without limitation forms which are anhydrous, hydrated, non-solvated, or solvated. Example hydrates include hemihydrates, monohydrates, dihydrates, and the like. In some embodiments, the compounds of the disclosure are anhydrous and non-solvated. By "anhydrous" is meant that the crystalline form of the compound contains essentially no bound water in the crystal lattice structure, i.e., the compound does not form a crystalline hydrate.

[0143] As used herein, "crystalline form” is meant to refer to a certain lattice configuration of a crystalline substance. Different crystalline forms of the same substance typically have different crystalline lattices (e.g., unit cells) which are attributed to different physical properties that are characteristic of each of the crystalline forms. In some instances, different lattice configurations have different water or solvent content. The different crystalline lattices can be identified by solid state characterization methods such as by X-ray powder diffraction (PXRD). Other characterization methods such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), solid state NMR, and the like further help identify the crystalline form as well as help determine stability and solvent / water content.

[0144] Crystalline forms of a substance include both solvated (e.g., hydrated) and non- solvated (e.g., anhydrous) forms. A hydrated form is a crystalline form that includes water in the crystalline lattice. Hydrated forms can be stoichiometric hydrates, where the water is present in the lattice in a certain water / molecule ratio such as for hemihydrates, monohydrates, dihydrates, etc. Hydrated forms can also be non-stoichiometric, where the water content is variable and dependent on external conditions such as humidity.

[0145] In some embodiments, the compounds of the disclosure are substantially isolated. By "substantially isolated" is meant that a particular compound is at least partially isolated from impurities. For example, in some embodiments a compound of the disclosure comprises less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities. Impurities generally include anything that is not the substantially isolated compound including, for example, other crystalline forms and other substances.

[0146] Certain groups, moieties, substituents, and atoms are depicted with a wavy line. The wavy line can intersect or cap a bond or bonds. The wavy line indicates the atom through which the groups, moieties, substituents, or atoms are bonded. For example, a phenyl group that SH, Cs Ho a is substituted with a propyl group depicted as: » 2 has the following structure: CH, n1471 The evnreccinn “HER?” ar

[0147] The expression “HER2” or “human epidermal growth factor receptor 2” refers to a member of the human epidermal growth factor receptor family. The protein is also known as NEU; NGL; HER2; TKR1; CD340; HER-2; MLN 19; HER-2 / neu. HER2 can refer to the amino acid sequence as set forth in NCBI accession No. NP_004439.2. Amplification or over- expression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years the protein has become an important biomarker and target of therapy for approximately 30% of breast cancer patient. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression "HER2" means human HERZ unless specified as being from a non-human species, e.g., "mouse HER2," "monkey HER2," etc.

[0148] The phrase "an antibody that binds HER2" or an "anti-HER2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize HER2.

[0149] The phrase an “anti-HER2 / HER2” antibody, e.g., an “anti-HER2 / HER2 bispecific antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize two different HER2 epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise a first antigen-binding domain (D1) which specifically binds a first epitope of human HER2 and a second antigen-binding domain (D2) which specifically binds a second epitope of human HER2.

[0150] The expression “STEAP2,” as used herein, refers to six-transmembrane epithelial antigen of prostate 2. STEAP2 is an integral, six-transmembrane-spanning protein that is highly expressed in prostate epithelial cells and is a cell-surface marker for prostate cancer, for example STEAP2 was found to be expressed in significant levels on an LNCaP prostate cell line (Porkka, et al. Lab Invest 2002, 82:1573-1582). STEAP2 (UniProtkKB / Swiss-Prot: Q8NFT2.3) is a 490- amino acid protein encoded by STEAP2 gene located at the chromosomal region 7g21 in humans, see e.g., the amino acid sequence of human STEAP2 as set forth in Tables 1 and 2.

[0151] As used herein, "an antibody that binds STEAP2" or an "anti-STEAP2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize STEAP2.

[0152] The phrase "an antibody that binds MET" or an "anti-MET antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize MET. The expressions “MET,” “c-Met,” and the like, as used herein, refer to the human membrane spanning receptor tyrosine kinase.

[0153] The phrase an “anti-MET / MET” antibody, e.g., an “ant-MET / MET bispecific antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize two different MET epitopes. In some embodiments, bispecific antibodies and antigen-binding fragments thereof comprise a first antigen-binding domain (D1) which specifically binds a first epitope of human MET and a second antigen-binding domain (D2) which specifically binds a second epitope of human MET.

[0154] All amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. § 1.822 (B)(J)

[0155] The term “protein” means any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. As used herein, “protein” includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives like CHO- K1 cells).

[0156] All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression "STEAP2" means human STEAP2 unless specified as being from a non-human species, e.g., "mouse STEAP2," "monkey STEAP2," etc.

[0157] The amino acid sequence of an antibody can be numbered using any known numbering schemes, including those described by Kabat et al., ("Kabat" numbering scheme); Al- Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al, 1996, J. Mol. Biol. 262:732-745 ("Contact’ numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, selection of a numbering scheme is not intended to imply differences in sequences where they do not exist, and one of skill in the art can readily confirm a sequence position by examining the amino acid sequence of one or more antibodies. Unless stated otherwise, the "EU numbering scheme" is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra).

[0158] The term “glutaminyl-modified antibody" refers to an antibody with at least one covalent linkage from a glutamine side chain to a primary amine compound of the present disclosure. In particular embodiments, the primary amine compound is linked through an amide linkage on the glutamine side chain. In certain embodiments, the glutamine is an endogenous glutamine. In other embodiments, the glutamine is an endogenous glutamine made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). In additional embodiments, the glutamine is polypeptide engineered with an acyl donor glutamine-containing tag (e.g., glutamine-containing peptide tags, Q- tags or TGase recognition tag).

[0159] The term "TGase recognition tag” refers to a sequence of amino acids comprising an acceptor glutamine residue and that when incorporated into (e.g., appended to) a polypeptide sequence, under suitable conditions, is recognized by a TGase and leads to cross-linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reaction partner. The recognition tag may be a peptide sequence that is not naturally present in the polypeptide comprising the TGase recognition tag. In some embodiments, the TGase recognition tag comprises at least one Gin. In some embodiments, the TGase recognition tag comprises an amino acid sequence XXQX (SEQ ID NO: 1935), wherein X is any amino acid (e.g., conventional amino acid Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, lle, Met, Pro, Thr, Lys, or Trp or nonconventional amino acid). In some embodiments, the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO:1936), LLQG (SEQ ID N0O:1937), LSLSQG (SEQ ID NO:1938), gGGLLQGG (SEQ ID NO:1939), gLLQG (SEQ ID NO:1940), LLQ, gSPLAQSHGG (SEQ ID NO:1941), gLLQGGG (SEQ ID NO:1942), gLLQGG (SEQ ID NO:1943), gLLQ (SEQ ID NO:1944), LLQLLQGA (SEQ ID NO:1945), LLQGA (SEQ ID NO:1946), LLQYQGA (SEQ ID NO:1947), LLQGSG (SEQ ID NO:1948), LLQYQG (SEQ ID NO:1949), LLQLLQG (SEQ ID NO:1950), SLLQG (SEQ ID NO:1951), LLQLQ (SEQ ID NO:1952), LLQLLQ (SEQ ID NO:1953), and LLQGR (SEQ ID NO:1954). See for example, W02012059882, the entire contents of which are incorporated herein.

[0160] The term “antibody,” as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), 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. In different embodiments, the FRs of the antibody (or antigen-binding portion thereof) can be identical to the human germline sequences, or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0161] The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0162] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDRS peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobedies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.

[0163] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL or VL- VL dimers.

[0164] Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0165] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that can be found within an antigen- binding fragment of an antibody of the present description include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2: (V) VH-CH1-CH2- CH3: (vi) VH-CH2-CH3: (vii) VH-CL; (viii) VL-CH1: (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed herein, the variable and constant domains can be either directly linked to one another or can be linked by a full or partial hinge or linker region. A hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0166] Moreover, an antigen-binding fragment of an antibody of the present description can comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed herein in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0167] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present description using routine techniques available in the art.

[0168] The antibodies of the present description can function through complement- dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity” (CDC) refers to lysis of antigen-expressing cells by an antibody of the description in the presence of complement. “Antibody-dependent cell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.

[01689] In certain embodiments, the antibodies of the description, e.g., ant-HER2 antibodies, or anti-HER2 / HER2 bispecific antibodies, or anti-MET antibodies, or anti-MET / MET bispecific antibodies, or anti-STEAP2 antibodies, are human antibodies. The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the description can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0170] The antibodies can, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (See, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in Vivo.

[0171] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification. The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using a human IgG1 hinge. The instant description encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which can be desirable, for example, in production, to improve the yield of the desired antibody form.

[0172] The antibodies of the description can be isolated or purified antibodies. An “isolated antibody” or “purified antibody,” as used herein, means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody” for purposes of the present description. For example, an antibody that has been purified from at least one component of a reaction or reaction sequence, is a “purified antibody” or results from purifying the antibody. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody or purified antibody can be substantially free of other cellular material and / or chemicals.

[0173] The antibodies disclosed herein can comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present description includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).

[0174] Furthermore, the antibodies of the present description can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, improved drug-to-antibody ratio (DAR) for antibody-drug conjugates, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present description.

[0175] The term “aglycosylated antibody” refers to an antibody that does not comprise a glycosylation sequence that might interfere with a transglutamination reaction, for instance an antibody that does not have saccharide group at N297 on one or more heavy chains. In particular embodiments, an antibody heavy chain has an N297 mutation. In other words, the antibody is mutated to no longer have an asparagine residue at position 297 according to the EU numbering system as disclosed by Kabat et al. In particular embodiments, an antibody heavy chain has an N297Q or an N297D mutation. Such an antibody can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at site apart from any interfering glycosylation site or any other interfering structure. Such an antibody also can be isolated from natural or artificial sources. Aglycosylated antibodies also include antibodies comprising a T299 or S298P or other mutations, or combinations of mutations that result in a lack of glycosylation.

[0176] The term “deglycosylated antibody” refers to an antibody in which a saccharide group at is removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of saccharide groups is accomplished enzymatically, included but not limited to via PNGase.

[0177] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope can include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0178] The terms “conjugated protein” or “conjugated antibody” as used herein refers to a protein or an antibody covalently linked to one or more chemical moieties. The chemical moiety can include an amine compound of the present disclosure. Linkers (L) and payloads (D) suitable for use with the present disclosure are described in detail herein. In particular embodiments, a conjugated antibody comprising a therapeutic moiety is an antibody-drug conjugate (ADC), also referred to as an antibody-payload conjugate, or an antibody-linker-payload conjugate.

[0179] The term “Drug-to-Antibody Ratio” or (DAR) is the average number of therapeutic moieties, e.g., drugs, conjugated to a binding agent of the present disclosure.

[0180] The term “Linker Antibody Ratio” or (LAR), also denoted as the lower case | in some embodiments, is the average number of reactive primary amine compounds conjugated to a binding agent of the present disclosure. Such binding agents, e.g., antibodies, can be conjugated with primary amine compounds comprising, e.g., a suitable azide or alkyne. The resulting binding agent, which is functionalized with an azide or an alkyne can subsequently react with a therapeutic moiety comprising the corresponding azide or alkyne via the 1,3-cycloaddition reaction.

[0181] The phrase “pharmaceutically acceptable amount” refers to an amount effective or sufficient in treating, reducing, alleviating, or modulating the effects or symptoms of at least one health problem in a subject in need thereof. For example, a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an amount effective for modulating a biological target using the antibody or antibody-drug-conjugates provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, from about 0.001% up to about 10%, and any amount in between, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of an antibody or antibody-drug-conjugate provided herein.

[0182] The phrase “reaction pH” refers to the pH of a reaction after all reaction components or reactants have been added.

[0183] The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0184] As applied to polypeptides, the term “substantial similarity” or “substantially similar’ means that two peptide sequences, when optimally aligned, such as by the programs gAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (8) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. In some embodiments, conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0185] Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256. 1443-1445. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0186] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, gCG software contains programs such as gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., gCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in gCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another particular algorithm when comparing a sequence of the description to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402. Protein-Drug Conjugate Compounds

[0187] According to the foregoing objective and others, the present disclosure provides protein-drug conjugate compounds, e.g., antibody-drug conjugate compounds, and precursors and intermediates thereof, pharmaceutical compositions, and methods for treating certain diseases in a subject in need of such treatment. According to the disclosure, the protein-drug conjugate compounds provided herein comprise a glutaminyl-modified binding agent conjugated with a primary amine compound linked to a therapeutic moiety, e.g., camptothecin analog moiety, as described herein.

[0188] In one aspect, the present disclosure provides compounds comprising a binding agent according to the present disclosure, (e.g., an antibody or a fragment thereof), having one or more glutamine residues conjugated to one or more camptothecin analogs, e.g., Dxd, via an optional first linker, a branching unit comprising at least one adduct, and an optional second linker. Illustrative non-limiting examples include Formula (I) and Formula (Il) described herein. In specific embodiments of a protein-drug conjugate according to the disclosure, wherein the binding agent is an antibody, (e.g., a monoclonal antibody), the term “antibody drug conjugate” or ADC is optionally used.

[0189] In one aspect, the present disclosure provides a compound having a structure according to Formula (C): BA-GIn-NH-L1-B-(-L2-(-M-Camp)m)n wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is absent or a first linker; B is a branching unit comprising at least one adduct of group B’ and group B”, where the group B’ is a first component, e.g., a first cycloaddition component; L2 is a second linker covalently attached to the branching unit B via the at least one group B”, wherein B” is a second component, e.g., a second cycloaddition component, and wherein the group B’ and the group B” form the at least one adduct; M is absent or a moiety having the structure 5° y ®t be , where R, R’, and R” are independently at each occurrence hydrogen or a C+-C20 alkyl, or wherein R’ and R” together form a ring; Camp is a camptothecin analog, and m and n are independently an integer from 1 to 30.

[0190] In one aspect, the present disclosure provides a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m))n (A), wherein: BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is absent or a first linker; B is a branching unit comprising at least one adduct of group B’ and group B”, wherein Ne=N 4 one of the groups B’ and B” is selected from -Na and NN : and the other of the Qe 1 ¥e groups B’ and B” is selected from f= Pe ; and Ne , where Q is CorN: L2 is a second linker covalently attached to the branching unit B via the at least one group B”: SAK M is absent or a moiety having the structure i ,whereR, R’, and R” are independently at each occurrence hydrogen or a C4-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): pS pe. wh A ~~ ~ < P); k is an integer from 1 to 12; m is an integer from 1 to 30, and nis an integer from 1 to 30

[0191] In one aspect, the present disclosure provides a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd))a (I), wherein BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is absent or a first linker; B is a branching unit comprising at least one adduct of group B’, Nx ad Nee QAO =i LT NIT where the group B’ is selected from -N3, woman ES N-N ; and oe where Q is C or N; L2 is a second linker covalently attached to the branching unit B via at least one group B”, wherein the group B’ and the group B” form the at least one adduct; M is absent EAE or a moiety having the structure k , where R, R’, and R” are independently at each occurrences hydrogen or a C1-Cs alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Wr 0 OX er ANN rh, <5 — ne (P), and k and n are independently an integer from 1 to 30. Linker L1

[0192] In certain embodiments, linker L1 is absent.

[0193] In certain embodiments, linker L1 is present and is covalently attached to the amine of a glutamine residue of the binding agent BA.

[0194] In certain embodiments, linker L1 comprises an alkyl (e.g., a C120 alkyl, or a C1.12 alkyl, or a C1.¢ alkyl), -NH-, -C(0)-, -(CH2)y-NH-C(O)-, -(CH2)y-C(0O)-NH-, -(CH2-CH2-O)v-, -(CHz).- (O-CHz-CH2)\-C(O)-NH-, a peptide unit comprising from 2 to 4 amino acids, or combinations thereof, each of which may be optionally substituted with one or more of -S-, -S(O2)-, -C(O)-, - C(O2)-; or -CO2H, wherein subscripts u and v are independently an integer from 1 to 8.

[0195] In certain embodiments, the free (unconjugated) linker L1 comprises a primary amine for attachment to the glutamine residue via a transglutamination reaction.

[0196] In one embodiment, linker L1 comprises one or more polyethylene glycol (PEG) units. In one embodiment, L1 comprises 2, or 3, or 4, or 5, or 8, or 7, or 8, or 9, or 10 PEG units.

[0197] In one embodiment, linker L1 comprises a disulfide (-S-S-) bond.

[0198] In one embodiment, linker L1 comprises a -S(O,)- moiety.

[0199] In one embodiment, one or more carbons on linker L1 is substituted with -COzH.

[0200] In one embodiment, linker L1 comprises a peptide unit comprising from 2 to 4 amino acids, or a peptide unit comprising 2 amino acids, a peptide unit comprising 3 amino acids, or a peptide unit comprising 4 amino acids.

[0201] In one embodiment, linker L1 comprises a peptide unit comprising 2 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, and citrulline, and combinations thereof. In one particular embodiment, linker L1 comprises a valine-citrulline unit.

[0202] In one embodiment, linker L1 is selected from the group consisting of: “, COS Acro 5 Boe HO. 20 " ap 9 8H 0 Se % eon Tptadx addlpton bhose(d Li el OY wed AK r hk Sp lx A ~X (LOM ¥ I = w 1 , and hls Y T wherein Ra is a group comprising an alkyne, an azide, atetrazine, a trans-cyclooctene, a maleimide, an amine, a ketone, an aldehyde, a carboxylic acid, an ester, a thiol, a sulfonic acid, a tosylate, a halide, a silane, a cyanogroup, a carbohydrate group, a biotin group, a lipid residue, and wherein subscripts x, n, p and q are independently an integer from 0 to 12, and combinations thereof. Branching unit B

[0203] Branching unit B comprises at least one adduct ofgroup B'. In certain embodiments, B comprises one adduct of group B’. In certain embodiments, B comprises two adducts of group B'. In certain embodiments, B comprises three adducts of group B'.

[0204] In certain embodiments, B comprises at least four adducts of group B’. In certain embodiments, B comprises four adducts of group B'. In certain embodiments, B comprises five adducts of group B'. In certain embodiments, B comprises six adducts of group B’.

[0205] Generally, an adduct of group B’ according to the present disclosure encompasses any moiety comprising the product of an addition reaction of group B’, independent of the synthetic steps taken to produce the moiety.

[0206] In some embodiments, the adduct of group B' may be a product of a substituted maleimide and, e.g., a thiol, or a substituted trans-cyclooctene, e.g.: Hale A I ON fo Ty ¥ where n is an integer from 0 to 12 and, e.g., a tetrazine.

[0207] In some embodiments, the adduct of group B' may be the product of a 1,3- cycloaddition reaction between an azide and an alkyne moiety. Without wishing to be bound by theory, the azide-alkyne cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole. NN

[0208] More specifically, an adduct ofgroup B’ selected from -Na, =i ] ’ N=N Ne LO 4 IY y N-N and where Q is C or N mav encompass a 1 3- : and \ for N-N and © where Q is C or N, may encompass a 1,3- cycloaddition adduct of the group B' and the group B’ selected from -Ns, f= 0 2M Ne wl Yor Tv mr where OO is C or N wherein the aroun B” is ; and “Ho N-N and where Q is C or N, wherein the group B” is complementary to the group B’ to form a 1,3-cycloaddition adduct.

[0209] By way of a non-limiting example, group B' may be an azide (-N3), and group B” =~ ee may be an alkyne-containing group, €.g., es oe ; or N

[0210] By way of another non-limiting example, group B’ may be an alkyne- AA - ia, IVE Need containing group, e.g., Sef * ;or N\ and group B” may be an azide.

[0211] In one embodiment, the adduct of the group B’' and the group B’ comprises a triazole moiety. In one particular embodiment, the adduct of the group B’ and the group B” has a structure selected from the group consisting of: TN and © whereinQis CorN. ON #, d 0 , wh

[0212] As stated above, in one embodiment, B comprises one adduct of the group B'.

[0213] In specific embodiments, L1-B is selected from the group consisting of: Ay Le NN) N 0 Pg FO 2 a AO - NN ng Sr On ye 7 wr Crag) Foe Ong On rn Oe ng a Ons BY H HOO Ay wy CN a ee] Ay 2 i EE PNT Br rnd arr (EY Ul em rm) Ame o H Q 0 $ a MX 6 - Hrs Sug Mn, MN, Carr BY) § = ( ld " HO Ya a aw 8 or “ig 1 NA A LX Ay - H o 2 Sy ng i MY be ei —— oH where the EN is the amino point of attachment to the glutamine residue of the BA, and (B') is the adduct of the group B’.

[0214] In one embodiment, the group B' is an azide (-Ns), and the adduct of the group B’ comprises a triazole.

[0215] According to one embodiment of the present disclosure, linkers L1-B may be azide amine linkers (AL), which comprise an amine group which directly attaches to the antibody, a PEG-containing base structure, and an azide functional group B’ (n=1).

[0216] The basic component structures of non-limiting exemplary azide amine linkers are listed shown in Figure 3B. Specific structures synthesized as examples are provided below.

[0217] In one embodiment, B comprises at least two adducts of the group B'. In specific embodiments, B comprises: 9 i hcl Low Hf a : lt H CC He ome 3 iS - = Ao AB) ) “ A + Ag ” 9 IA ye ye * Ya ’ HR L 4 : wn Sg ARE 7 BY fd Yo. a "he So ® ou Fa of ned at Ou HEY ST of. (8) 5 IKE : nt a ~ AE Le 1 ’ 3 Ne [7 RT Oa HE OA gE aE CT O MOSS Ne OFA) x 1 o. # } ! & 4 , and oo" where (B') comprises points of attachment of the adduct of the group B’.

[0218] In specific embodiments, B is selected from the group consisting of: 9 IS AE) go © a iB) { oo i gr | ~s YO SONU NPN i Oe 8.0 x oO (a) Ou gy Te § 0 AAO gE) dC N k 2 HOM y Sonphongao AN: )] Oo 9 gi ASE H He H eg ns Ny Ne & oyfngrm b n Agar Ors Nor gor 8) N . 1 Ay N ~~ Tn ~ Og Ne mB) Sen Pg Feds So Yo : So P See @), y N ©) 5 A Clr ®) oxo Ny ~ ha ® (ONO B Li] OB specific embodiments, L1-B is selected from the group ct eNO: & ol Bg pd Py rt®s y 2 “ A yi ahora [Hen On gl Be Pg - Re Nang O © Jmgromgnon AL > o. of 7 ~ 8) NPN To 0 AA ARAN ON FR el ¥ ¥ B® H Neg, i a oO a ‘ a I a a a He le u Semper i Ox Hamp | oP . AO gn Ogg Bo Pg N Ne ¥ 2 oh ny } i Agen EI N’ NH i A oF (BY H Fe Pr = Zl Aon ? ms -_ Ayman mg Hynes Ke 3 ®) 0 ¢ Seong od Br @ 3 r FE pd o 7 oT Som ~ -Q 1 GrA-0 ed — ®) (RNA I a (ONO E) FO H Uo be oo Choa Kooga BY) where the IN is the amino point of attachment to the glutamine residue of the BA.

[0220] According to another embodiment of the present disclosure, linkers L1-B may be branched-alkyl azide amine linkers (BL) comprising an amine group which directly attaches to the BA (e.g., an antibody), a branched-alkyl PEG containing base structure, and 2 to 6 azide functional groups B’' (n=2-6).

[0221] The basic component structures of exemplary non-limiting branched-alkyl azide amine linkers are listed in Figure 4B. Specific structures synthesized as examples are provided below. Linker L2

[0222] In certain embodiments of the present disclosure, L2 has a structure according to Formula (L2): B”-SP1-B2-(-SP2-AA-SP3), (L2),wherein: B” is a group capable of covalently attaching to the group B’; SP1 is absent or a first spacer unit; B2 is absent or a branching unit; SP2 is absent or a second spacer unit; AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit, and p is an integer from 1 to 12.

[0223] In certain embodiments of the present disclosure, L2 has a structure according to Formula (L2’): HzN-SP1-B2-(-SP2-AA-SP3), (L2") wherein: SP1 is absent or a first spacer unit; B2 is absent or a branching unit; SP2 is absent or a second spacer unit; AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit, and p is an integer from 1 to 12.

[0224] In certain embodiments of the present disclosure, L2 has a structure according to Formula (L2%): Maleimide-N-SP1-B2-(-SP2-AA-SP3), (L2"), wherein: SP1 is absent or a first spacer unit; B2 is absent or a branching unit; SP2 is absent or a second spacer unit; AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit, and p is an integer from 1 to 12.

[0225] In some embodiments, the linker L2 comprises a group B” capable of covalently attaching to the group B’ as described above. SNe

[0226] In certain embodiments, the group B” is selected from -Ns, ~fm=g— LO LN OF — N-N and \ where QisCorN , where Qis CorN. i and Nes ;

[0227] By way of a non-limiting example, the group B’ may be an alkyne- Nx 0 TE ON ll REE = containing group, e.g., fmf = por Ne

[0228] By way of another non-limiting example, group B” may be an azide.

[0229] In one embodiment, the adduct of the group B’ and the group B’ comprises a triazole moiety. In one particular embodiment, the adduct of the group B’ and the group B” has a structure selected from the group consisting of: ITN and Nu ONY and “=”, wherein Q is C or N, or a regioisomer thereof.

[0230] In one embodiment, the first spacer SP1 is absent. Cc 0.

[0231] In another embodiment, SP1 is selected from the group consisting of % A Q Bh il and Are

[0232] In one embodiment, the branching unit B2 is absent.

[0233] In one embodiment, the branching unit B2 according to the disclosure has the structure of one of B1-B5 depicted below. Branching Unit B2 B1 B2 B3 B4 BS 4 = ai | Os, “sr. a Y NY 5 wx | | A NU 3 : FOr Oy A 3 g | Structure | Sol | 7 ; On or L , ha Ath | ¢ } oy y ~~ ) } LAN —_— PF

[0234] In one embodiment, the second spacer SP2 is absent.

[0235] In another embodiment, SP2 is selected from the group consisting of an alkyl (e.g., a Ci.20 alkyl, or a Cy.12 alkyl, or a C1.10 alkyl, or a C1. alkyl, or a C1. alkyl), -(CH2-CH2-O)v-, -NH-, -C(0)-, -NH-C(0)-, -NH-(CHz)s-, -NH-(CH2),-C(O)-, -NH-(CH2-CH2-O)y-, -NH-(CH2-CHz-O)\~ C(0)-, -NH-(CH2-CHz-O)\-(CHz2)u-, -NH-(CH2z-CH2-0)~(CH2)u-C(0)-, -(CH2)»-NH-C(O)-, -NH- (CH2)y-NH-C(O)-, -NH-(CHa).,-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8.

[0236] In certain embodiments, AA is a peptide unit comprising from 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

[0237] In one embodiment, AA is a peptide unit comprising 2 amino acids. In one embodiment, AA is a peptide unit comprising 3 amino acids. In one embodiment, AA is a peptide unit comprising 4 amino acids.

[0238] In one particular embodiment, AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

[0239] In another particular embodiment, AA is selected from the group consisting of glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF), and glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)) and glutamic acid-valine-citrulline (EVC).

[0240] In one embodiment, the third spacer SP3 is absent.

[0241] In another embodiment, SP3 is selected from the group consisting of bodiment, SP3 is s sh (PS Re Re Lr hE sh (VPS re A 4 8 c ! Bd 4 , and combinations thereof, wherein R. is independently at each HO HO How Ag HO, No I 7 TIT HOP NOK HOP NO occurrence absent or a group selected from QOH and OH

[0242] In one embodiment, the spacer SP3 is covalently attached to the camptothecin analog, e.g., Dxd or M-Dxd.

[0243] In one embodiment, the linker L2 comprises from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or from about 1 to about 8, or from about 1 to about 4, or from about 1 to about 2 (SP2-AA-SP3) moieties, and the linker-payload L2-Dxd comprises from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or from about 1 to about 6, or from about 1 to about 4, or from about 1 to about 2 Dxd payload molecules. Moiety M

[0244] In certain embodiments, moiety M is absent. f ! se AS Ke J ¥ x

[0245] In certain embodiments, M is present and has a structure & where R, R’, and R” are independently at each occurrence hydrogen or alkyl, or wherein R’ and R” together form a ring, e.g., a 3-membered to an 8-membered ring. . J si

[0246] In certain embodiments, M is present and has a structure & where R, R’, and R” are independently at each occurrence hydrogen or a C+-Cs alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring.

[0247] In one embodiment, R is a hydrogen.

[0248] In one embodiment, R’ is a hydrogen. In one embodiment, R’ is a C1-Cs alkyl.

[0249] In one embodiment, R” is a hydrogen. In one embodiment, R” is a C1-Cs alkyl.

[0250] In one embodiment, R’ and R” together form a 5-membered ring. In one embodiment, R’ and R” together are —(CHz)s-.

[0251] In one embodiment, R’ and R” together form a 6-membered ring. In one embodiment, R’ and R” together are —(CHz)s-.

[0252] In one embodiment, R, R’, and R” are hydrogens at each occurrence, i.e. M is si A 2

[0253] In another embodiment, R is hydrogen and R’ and R” together form a 5-membered ring, SNP e.g., R’ and R” together are —(CHz)s- and M is — Payloads

[0254] In certain embodiments, the payloads of the present disclosure are camptothecin analogs and / or derivatives. Terils, UE payivals Ul LTE pPIest RS O NS UT SNF Et Ne al ND HO © Camptothecin

[0255] Camptothecin (CPT), shown above, is a topoisomerase poison. It was discovered in 1966 by M. E. Wall and M. C. Wani in systematic screening of natural products for anticancer drugs. It was isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China used as a cancer treatment in Traditional Chinese Medicine. Camptothecin showed remarkable anticancer activity in preliminary clinical trials. However, it has low solubility, so synthetic and medicinal chemists have developed numerous syntheses of camptothecin and various derivatives to increase the benefits of the chemical, with good results. Four camptothecin analogs have been approved and are used in cancer chemotherapy today: topotecan, irinotecan, belotecan, and deruxtecan (Dxd).

[0256] Trastuzumab deruxtecan (T-Dxd) is an antibody-drug conjugate that includes a human epidermal growth factor receptor 2 (HER2)-directed antibody trastuzumab and a topoisomerase | inhibitor conjugate deruxtecan (Dxd, a derivative of exatecan). It was approved for use in the United States in December 2019. Exatecan, shown below, is a camptothecin analog. oH hie coq SAR 0 Ly Tov F N \ / . A D 0 ~~ o “eb Ne—~( HC © OHO Exatecan, left, and deruxtecan (Dxd), right

[0257] In one embodiment, the payload of the present disclosure is deruxtecan (Dxd).

[0258] In certain embodiments, the payload of the present disclosure is a compound having the structure P-I: oR 7 R. HN KAA 0 Pl Hy op Ry Rp NH be RS ak oO FONT 7 4 © Se HS (PH), R. are indanandantlv a hvdranan ar an wherein R+, Rz, and Rs, and R4 are independently a hydrogen or an alkyl, e.g., a C4-C12 alkyl, or a Cq-Cs alkyl, or a C4-Cs alkyl, or a C4-C4 alkyl, or wherein Rz and Rj together form a 5-membered or a 6-membered ring, or a pharmaceutically acceptable salt thereof.

[0259] In one embodiment, Ry is a hydrogen.

[0260] In one embodiment, Rz is a hydrogen. In one embodiment, Rz is a C1-Cs alkyl.

[0261] In one embodiment, Ra is a hydrogen. In one embodiment, Ra is a C+-Cs alkyl.

[0262] In one embodiment, Rq4 is a hydrogen. In one embodiment, Rq is a C1-Cs alkyl.

[0263] In one embodiment, Ri, Rz, and Rs, and R4 are a hydrogen in each occurrence. In one embodiment, the compound of the present disclosure is d of the present disclosure is c HN A on 0 NT FN -. oy, No OHO, or a pharmaceutically acceptable salt thereof.

[0264] In one embodiment, Rz and Rs together form a 5-membered ring. In one embodiment, Rz and Rj together are —(CHy)a-.

[0265] In one embodiment, Rz and Rs together form a 6-membered ring. In one embodiment, Rz and Rj together are —(CH2)4-.

[0266] In one embodiment, R1 is a hydrogen, and Rz and Rs together form a 5-membered ring.

[0267] In one embodiment, the compound of the present disclosure has a structure according to Formula (P-11): Ny Hef. A v HY f Yd A NA A 0 Yor © A oh Ry Rs Q AAA ” FL o Sep OHO wherein R is a hydrogen or an alkyl, e.g., a C+-C12 alkyl, or a C4-Ce alkyl, or a C4-Cs alkyl, or a C1-C4 alkyl, or a pharmaceutically acceptable salt thereof.

[0268] It should be understood by one of skill in the art that the compound P-II depicted above is also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure. For example, the R and S configurations for each asymmetric center, are within the scope of the present disclosure. By way of an example, the two isomers depicted below are within the scope of the present disclosure: isomers depicted below are within the scope of the present di 0 0 HN i 3 oN ENT Nop? o NH QC oNH NYY 0 SS © [ i N—f = © © Ne) ) im and ing TETHER ERY Fra amin pm ben Irv ill vy onl lin pd dtd 4 Eg bt a de i i

[0269] In one embodiment, the compound of the present disclosure is compound of the present disclosur Hy org © ~ oNH RPE oO F “N Ln Nd $C Tomb ; or a pharmaceutically acceptable salt thereof.

[0270] In one embodiment, the payload according to the disclosure is conjugated to form a protein-drug conjugate (e.g., an antibody-drug conjugate). In one embodiment, the payload is covalently attached to a moiety M. In one embodiment, the payload is M-Dxd. In one embodiment, M-Dxd has a structure selected from the group consisting of 0 Ay Noe? o pl Ao b Nw and OHO, a Ho Na A ng ngz© H “Y Ay AN N MS B je) AGN F N 4 Yam o Ne ~ OHO | and n Rie a hudranan ar a C..0, alley] and wit wherein R is a hydrogen or a C4-C4 alkyl, and where «a represents the point of attachment to 2.

[0271] The present disclosure also relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound as described above or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0272] In one embodiment, the compound according to the disclosure has the following structure: / ec. 9 33 oF roy oo - = 7 $7 gE LJ Hol . = 1 {yom Ais oH 4 ~ XN er [ A " . Ogpthar? . 5 . "Ny . wiv rere of 5! = | sey Ney a § Tn 2 10 T AL Ei i - I aKa 0. ory eS 3 7 rr ~ Se ] 5 no F / , wherein BA is a binding agent (e.g., an antibody or an antigen-binding fragment thereof).

[0273] In one embodiment, the compound according to the disclosure has the following structure: No Ng Sy oo . ord dioac dy, 3 So) C a od YY XE \ uf wat ye A Me cys grotto He dmonmmrn n A I AST La oe atl | St A Xr mn 3 / , wherein BA is a binding agent (e.g., an antibody or an antigen-binding fragment thereof).

[0274] In one embodiment, the compound according to the disclosure has the following structure: ~~ TR wT 3 . LA, xX / . Hr o a & i i TORII 3 TT ks 5.2 — ry sa gbly ; Lh 4 wherein BA is a binding agent (e.g., an antibody or an antigen-binding fragment thereof).

[0275] In one embodiment, the compound according to the disclosure has the following structure: ~~ i il . LA, Xr / , 7 Q \ homo Snty 0 y ey Roo, 0 p= I Hadron IJ LOTT XL | CT wr Y Lh, / , wherein BA is a binding agent (e.g., an antibody or an antigen-binding fragment thereof).

[0276] The present disclosure also relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound as described above or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients. Linker-Payloads (L2-P)

[0277] In another aspect, the present disclosure provides a compound according to Formula (L2-P): B"-SP1-B2-(-SP2-AA-SP3-M-Dxd)p (L2-P),wherein: N=N, Sp NN ] i . a, TN HT B” is selected from the group consisting of -Na, N-N rfid ff ANE i and OU Q 0. SP1 is absent or a first spacer unit selected from the group consisting of % Ae and 9 He ° B2 is absent or a branching unit; SP2 is absent or a second spacer unit selected from the group consisting of a C1.s alkyl, -(CHz- CH2-O)-, -NH-, -C(O)-, -NH-C(0O)-, -NH-(CH2).-, -NH-(CH2).,-C(QO)-, -NH-(CH2-CH2-O)~, -NH- (CH2-CH2-0)+-C(0)-, -NH-(CHz-CH2-0)~(CHz)u-, -NH-(CH2-CH5-0)y-(CH2).-C(O)-, -(CH2),-NH- C(0)-, -NH-(CHz),-NH-C(0)-, -NH-(CH2),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8; AA is absent or a peptide unit comprising from 2 to 4 amino acids; AA IS absent or a pepuae unit comprising from < to 4 amino acids, £9 TC # ~* SP3 is absent or a third spacer unit selected from the group consisting of, & % AL Tow ) I Tn STN £9. NP of LA ~~ Y , wherein SP3 is absent or a thir se E_ To NANA T HOS, HO... No rg HOP NO R. is independently at each occurrence absent or a group selected from OH and HO© HO, A 5 ses OM PN M is absent or ke , where R, R’, and R” are independently at each occurrence hydrogen or a C+-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): ¥W, ~ AN H ry YO Qo | i - ANNE 9 — =< (P), and p is an integer from 1 to 12.

[0278] In another aspect, the present disclosure provides a compound according to Formula (L2'-P): H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p (L2-P), wherein: N=N SL 5 CC N-N ef TN #7 B” is selected from the group consisting of -Na, N-N | ~fm—H, f= i and NS oO i ; i iat seo sg SP1 is absent or a first spacer unit selected from the group consisting of ~ and He ¢ B2 is absent or a branching unit; SP2 is absent or a second spacer unit selected from the group consisting of a C+.6 alkyl, -(CH2- CH2-O)v-, -NH-, -C(O)-, -NH-C(0)-, -NH-(CH2).-, -NH-(CH2),-C(O)-, -NH-(CHz-CH2-O)~, -NH- (CH2-CH2-0)\-C(0)-, -NH-(CH2-CH2-0)\~(CHz)u-, -NH-(CH2-CH2-0)\-(CH2)«-C(O)-, -(CH2)u-NH- C(0)-, -NH-(CHz),-NH-C(O)-, -NH-(CH2),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8; AA is absent or a peptide unit comprising from 2 to 4 amino acids; ae A 4 A “ SP3 is absent or a third spacer unit selected from the group consisting of, 4 , Wherein SP3 is absent or a third spacer unit selected from the group consisting of, oN % a a 8 ¥ , HOS, HO, Mo Ho NG R. is independently at each occurrence absent or a group selected from OH and HOp© HO. Ag NP. OH TN M is absent or b , where R, R’, and R” are independently at each occurrence hydrogen or a C+-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): EH p@ rf NN x . oO AN {) - — ry (P), and p is an integer from 1 to 12.

[0279] In another aspect, the present disclosure provides a compound according to Formula (L2"-P): Maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p (L2"-P), wherein: N=N 2 pe CO N=N cy Tx #7 B” is selected from the group consisting of -Na, N=-N | ~§m—h f= and OU Q 0. SP1 is absent or a first spacer unit selected from the group consisting of % Ae and ° Ae C B2 is absent or a branching unit; SP2 is absent or a second spacer unit selected from the group consisting of a C+.s alkyl, -(CH2- CHz-O)v-, -NH-, -C(O)-, -NH-C(0)-, -NH-(CH2).-, -NH-(CH2),-C(O)-, -NH-(CHz-CH2-O)~, -NH- (CH2-CH2-0)\-C(0)-, -NH-(CH2-CH2-0)\~(CHz)u-, -NH-(CH2-CH2-0)\-(CH2)«-C(QO)-, -(CH2)u-NH- C(0)-, -NH-(CHz),-NH-C(0)-, -NH-(CH2),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8; AA is absent or a peptide unit comprising from 2 to 4 amino acids; AAS absent or a pepliae unit comprising rom £ 0 4 amino acids; £9 L & ~~ SP3 is absent or a third spacer unit selected from the group consisting of, 8 , wherein TEA Rohoaid a LSS AH HO. HO. Ag | SE. 1 HOOK Rc is independently at each occurrence absent or a group selected from OH and HO° HO.,. 0 EN OH oR M is absent or b , where R, R’, and R” are independently at each occurrence hydrogen or a C+-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): Roping AN H © CY De AN 9 sgl nd (P), and p is an integer from 1 to 12.

[0280] In certain embodiments, the linker-payload L2-P, L2-P, L2"-P according to the disclosure has a structure selected from the group consisting of: Nol “a a. 9% a J HoX ede an AIA LITT NY $C « Te NT b Cla # F g Stats, 3S 1 ONY ON a ey ory 3 Th 7 Hl H AA " foo, eh EE a Hog Hog y 2 3 Grigg | i SK 2m Toe 18.3 =o HE or OF no SF \ J A Nha Bn ZN 5 9a, 0 wo, dy mom Mo, OY HOY NOT or OF ng vw py J ia ox { | Ne KH Y / bi iL = vy i oA ° ¢ ¥ By NS Qs x { Arron amaondid oA, 5 Fe ? Tt —_ Ardent ) ve rf a o © Nama ty mn Sih YN NYT oH oy Ho. : Po i o OH Zy forse Ton Bs Sertomy Cr ry ENP o A rN oO HOw An Ay OH Go HO I PN Af Ia Adda dy, - Bs 0 0 oO Pun “No ft —_ —_ ogy OA A LAT ’ La o: % oA No LES ~ a Sebo Pe TX ~ oa Sb, = To Ta 3 ; ory AS ANON Ee # é A 1 ~ 0 7 rt CA J rn > — be pig Zyptoot av, YY A 2k. LO 2 nA] 1, - fmm 2 EA § Og No o ng tiaL, H il POISAARAG 3 ema ITI LY SE Oo Te XJ LK, A a ” No —N 4 R=) ~ > Y 2X Na A A 5 = 145 Halo mgr On Yd N ih AL ° Y 0 4 LE, HONGO Sy ¥ F 1 1.3 3d | ~ nd reed ” { =o | adorplompmtangmisngh ehh Pie ° AA Wy g TIVE, WH Ho No Shu, A : £ 1 a u . 5 oe ore yoy Hmong Bm Aid LAY ~ fo ~ © C AEN A oy GAA Ay HO" Ye NN TY a °c Q ru oo 1 HoT 3 a Oo y dak ory 2 ~ | ery orange fel UR A I LL > a EAE ER & woo Sa, A or a pharmaceutically acceptable salt thereof.

[0281] Branched Linker2-Payloads (BL2P)

[0282] In another aspect, the present disclosure provides L2-P which comprise one or more branching units. Exemplary branching units B1-B5 according to the disclosure are depicted below. B1 B2 B3 B4 B5 5 b r~ he ) Bi " we 2] >; CC Sid AJ Fpirmgrtns “ 3 / 2) “1 oi | | Structure [ 5 2

[0283] Structures for exemplary branched linker2-payloads (BL2P) according to the disclosure are provided below. Structures mo Kn gy Aegid | Cyr wssaraniy a Yo | Go - . Wa wy (PAY ! N y Jf - w TE No LJ , H Fi CS Morgatangmtio od om AE Hon i IS © 3 . propping 3 Do oh r LO ry 25 at bh, NF NA ¥ : Shy om n W ‘ 5 Q UV oe amededialdy, | Le ™ ~ 9 Sy “NH, bit 2k. Ce TAA OT SS ale Y Cx on, A Yq reread bo Ao Gh J ! i LHe LT, NH Pil gor on hen, FEN ON 1 & ‘ g oH a 3 0 Hi vw 8 .o eyo = | mm & O A x a oH oon Se XJ oN oa An 3 eo J pt NFAT Ho Ao he 5 = o mo &- WTA Seedy Ty ¥ adeeb, CS fmm A 1 Highs 3 3 : ha, Sr ’ o W a qa ono ado dead, =o | rn IA ITY fof, ie ~N 7 HO | ne ol 9 PN - So Sl A J Nm Ong ey w ht oF 3 5 W oi Q CG Q 3 C gy , Moody, 0 =| w ono ory we va $ NK NF “3 bomen dS, ; Ss 8: ° Na iQ! Lo Ay 5 ————— - ra Te 8 a anes mond, 5 | rp § o x wT < {pb SC F p= y ET Ya oe ~ men Ro A, = Lng fp OE EE | & y ox o [NN Fon gle, pe Ce rAd, 33 | Tr + aod d gs SRT te 357 | ~ . AE Brn RAV & : — hye a RS Leal npn 33 3 nthe & jog Kp 5 o | ol Au Nh % | a A f parry. 3% | o, op 2 Yas, crhegaty HC Seo | O “My A pom ga or UR, I “ 2 iw Gi " nd . ~ H 3 pi SET Fegndy 0 | x2 "OH Sb ’ opie LO Ayn , rey pe 3 9 © Pou ~ a Sy - bg Se NT ie ot Sy Hon Sy HN SON Og : Sp 8 i q 3 "™ tad Xk 8 OY Of ° | ronpled Lt A o A= ws] a FURST, A N oon Sys Y ha a fom Tr. 1 3 | SN Lo SO SL h a Sn, u SLA a UN, dh Ms on A Ah pl A grt hy Apt © ory POE WA ol. Osp on Sebo, Ay ~~ 0. APO * oy Y Rony 30 | oN Sy Ot SO (PENSE R REN oA Mao Fels ° NTS NT, IN a. Fun Soh, : I) - = e , oe Q a cd Ay os by PS LY oy Sy ° | wo ah 3 WL oF Speman ~ Lo " nT ” Stat Sl % Lengthy £ & CY XE oi hu 7 HE | eet CR Hey Sd ~ Lh, POY Se ~ I wl oy Hon Stns, hy dumsraiund E ; Cs rg T REE oS rep Hp Beye Sy al RAN Aedes] > Ht hg ‘ or OG o Myth ® 2 aN, Amlioo Ly, 0 mo | ott Med Jk T HPS er ~Y Tey SO i I { i ¢ Hon Se 5 y & Nooo No N a hon Sif a ~ "a 0 g "4 Xorg fp bY SER Pon ho hat Ty a. Foto X,, oC bod g [oe LS (4 img lS wy <1 . i > pl on Sire, 7 ¢ ~N Sh ’ 8 FL" 8 nT 5 ~ nei od Ao Nhe FT Tron ; x o ~ Q “Ry x R. if 3 - 7S HN, i Bak pl dk & 1 On rhe WY SEE | \ ° f ' a Gk 3 pref Sg | yan hd Ae hh . ) Entel AAC epee 2 Che No Xr en | J ~ 9 Sythe ody, 3 Hr 0 3 Ox pnd oe 5 ha r Dag Null wn Na, Ag AZ C0 ANAT wb YET ox a a b on Sythe Xr yr i £ . . pono paradpepiendy 2 | Hon Sw A Sy -. pred # 2 r , i | or HO K ~ LA ’ 2 NTS OS TS o oy ty awa spt A Lo J edn A TTF x / ™ 5 . o Be oy COC Ory S| Pri ry wl Ao Sh pe erred h i £7 Ponty S34 Jk LL, <r - ; 4 ap ! Poa 1 £ X : \. oy orga BRED: yy 7 1 ) fore HO TTT 9 | oN a Aon, Fo Sony do Li Ey YY ro © L v2 J oo Sg pe pop TE . rere 3% p= FET A 2% “4 ok Her Sh Hn rg Mey Ax vr IT SY Smial, I Bphrerdompiorni258] be Lh xr hu we Ne -Y L : {goad 3S sys rye Sent A AO a ? ws Lh A

[0284] In one embodiment, the compound (i.e., the linker-payload) according to the disclosure has the structure: o FP § ° Adon ITA LITT HY L ; Wd] pg CN LL, : or a pharmaceutically acceptable salt thereof.

[0285] In one embodiment, the compound (i.e., the linker-payload) according to the disclosure has the structure: Noy x toed, o HT dyn dL AO ROR Jeon — 5% Y Lh, y a Ho F , or U Q Axo o Lo oye N id eI monannL, A ° nd fd N LI 1 I Sy NH H F or a pharmaceutically acceptable salt thereof.

[0286] In another aspect, the present disclosure provides an antibody-drug conjugate according to Formula (II): Ab-(GIn-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd))p)n (10, wherein Ab is an antibody; Gin is a glutamine residue; L1 is absent or a first linker as described above; B is a branching unit as described above comprising at least one adduct of group B’ and A A i A N=N, ANNE SL group B”, where the group B’ is selected from -N3, gw “he N-N and at least one group B”, wherein B”-SP1-B2-(-SP2-AA-SP3-M-Dxd)p is the compound according to formula (L2-P) as described above, and wherein the compound of formula (L2-P) is covalently attached to the antibody via the adduct of the group B’' and the group B”, k is an integer from 1 to 12, and p and n are independently an integer from 1 to 30.

[0287] In one embodiment, the antibody-drug conjugate according to the present disclosure comprises an antibody and a linker-payload, wherein the linker-payload comprises the structure: o Q Mey ny tet \ =o WS nd simp x Ory ros, A AS N T Noi No LL Nos LI F , or o MX x foe k ° edn ommmnn I La ALOT NY x 2 WJ A g % 1 A a Y or a pharmaceutically acceptable salt thereof, where «a represents the point of attachment to the binding agent (e.g., an antibody), directly or through a second linker. Binding Agents

[0288] In one embodiment, the effectiveness of the protein-drug conjugate embodiments described herein depend on the selectivity of the binding agent to bind its binding partner. In one embodiment of the present disclosure, the binding agent is any molecule capable of binding with some specificity to a given binding partner. In one embodiment, the binding agent is within a mammal where the interaction can result in a therapeutic use. In an alternative embodiment, the binding agent is in vitro where the interaction can result in a diagnostic use. In some aspects, the binding agent is capable of binding to a cell or cell population.

[0289] Suitable binding agents of the present disclosure include proteins that bind to a binding partner, wherein the binding agent comprises one or more glutamine residues. Suitable binding agents include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell binding or peptide binding molecules or substances.

[0290] In one embodiment the binding agent is an antibody. In certain embodiments, the antibody is selected from monoclonal antibodies, polyclonal antibodies, antibody fragments (Fab, Fab’, and F(ab)2, minibodies, diabodies, triabodies, and the like). Antibodies herein can be humanized using methods described in US Patent No. 6,596,541 and US Publication No. 2012 / 0096572, each incorporated by reference in their entirety. In certain embodiments of the protein-drug conjugate compounds of the present disclosure, BA is a humanized monoclonal antibody. For example, BA can be a monoclonal antibody that binds HER2, MET, or STEAP2. In certain embodiments of the protein-drug conjugate compounds of the present disclosure, BA is a bispecific antibody, e.g., an anti-HER2 / HER2 bispecific antibody, or an anti-MET / MET bispecific antibody.

[0291] In the present disclosure, the antibody can be any antibody deemed suitable to the practitioner of skill. In some embodiments, the antibody comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody comprises one or more gin295 residues. In certain embodiments, the antibody comprises two heavy chain polypeptides, each with one gIn295 residue. In further embodiments, the antibody comprises one or more glutamine residues at a site other than a heavy chain 295. Such antibodies can be isolated from natural sources or engineered to comprise one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain are within the skill of the practitioners in the art. In certain embodiments, the antibody is aglycosylated.

[0292] The antibody can be in any form known to those of skill in the art. In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a kappa light chain. In certain embodiments, the light chain is a lambda light chain.

[0293] In certain embodiments, the antibody comprises a heavy chain. In some aspects, the heavy chain is an IgA. In some aspects, the heavy chain is an IgD. In some aspects, the heavy chain is an IgE. In some aspects, the heavy chain is an IgG. In some aspects, the heavy chain is an IgM. In some aspects, the heavy chain is an IgG1. In some aspects, the heavy chain is an 1gG2. In some aspects, the heavy chain is an 1gG3. In some aspects, the heavy chain is an IgG4. In some aspects, the heavy chain is an IgA1. In some aspects, the heavy chain is an IgA2.

[0294] In some embodiments, the antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is a F(ab’)2 fragment. In some aspects, the antibody fragment is a Fab’ fragment. In some aspects, the antibody fragment is an scFv (sFv) fragment. In some aspects, the antibody fragment is an scFv-Fc fragment.

[0295] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0296] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody.

[0297] The antibody can have binding specificity for any antigen deemed suitable to those of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., receptor) or a growth factor. Exemplary antigens include, but are not limited to, molecules such as renin; agrowth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alphai-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor vme, factor 1X, tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-l-alpha); a serum albumin, such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as betalactamase; DNase; 19E; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA- 4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-B; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF- B1, TGF-B2, TGF- 3, TGF-R4, or TGF- BS; insulin-like growth factor-I and -2 (IGF-I and IGF-2); des(I-3)-IGF-l (brain IGF-I), insulin-like growth factor binding proteins, EpCAM, gD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, IRTAI, IRTA2, IRTA3, IRTA4, IRTAS; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CDII, CDI4, CDI9, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CDI52, or an antibody which binds to one or more tumor- associated antigens or cell-surface receptors disclosed in US Publication No. 2008 / 0171040 or US Publication No. 2008 / 0305044 and incorporated in their entirety by reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon, such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, gM-CSF, and g-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the HIV envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, such as CDlla, CDllb, CDllc, CDI8, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as AFP, ALK, B7H4, BAGE proteins, B-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase 1X), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRVIII, endoglin, Epcam, EphA2, ErbB2 / HER2, ErbB3 / HER3, ErbB4 / HER4, ETV6-AML, Fra-1, FOLR1, gAGE proteins (e.g., gAGE-1, 2), gD2, gD3, globoH, glypican-3, gM3, gp100, HER2, HLA / B-raf, HLA / EBNA1, HLA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, mL-IAP, Muc1, Muc16 (CA-125), MET, MUM1, NA17, NGEP, NY- BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-a, PDGFR-B, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, STn, survivin, TAG-72, TGF-B, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, and fragments of any of the herein-listed polypeptides.

[0298] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, B7H4, gPNMB, UPK3A, and LGRS5. Exemplary antigens also include, but are not limited to, MUC16, PSMA, STEAP2, and HER2.

[0299] In some embodiments, antigens also include, but are not limited to, hematologic targets, e.g., CD22, CD30, CD33, CD79a, and CD79b.

[0300] Some embodiments herein are target specific for therapeutic or diagnostic use. In one embodiment, binding agents are prepared to interact with and bind to antigens defined as tumor antigens, which include antigens specific for a type of tumor or antigens that are shared, overexpressed or modified on a particular type of tumor. Examples include: alpha-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloid leukemia, B- RAF, CLPP or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma as well as the following shared tumor-specific antigens, for example: BAGE- 1, GAGE, gnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2. In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antibody binds STEAP2, MUC16, EGFR, EGFRVIII, FGR2, or PRLR.

[0301] In some embodiments, the antigens include HER2. In some embodiments, the antigens include STEAP2. In some embodiments, the antigens include MET. In some embodiments, the antigens include EGFRVIII. In some embodiments, the antigens include MUC16. In some embodiments, the antigens include PRLR. In some embodiments, the antigens include PSMA. In some embodiments, the antigens include FGFR2.

[0302] In some embodiments, the BA is an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti- PRLR antibody, an anti-PSMA antibody, or an anti-FGFR2 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an anti-FOLR1 antibody, or an antigen-binding fragment thereof.

[0303] In some embodiments, the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. Anti-HER2 Antibodies Suitable for Protein-Drug Conjugates

[0304] In some embodiments, the antibody is an anti HER2 antibody. In some embodiments, the antibody is trastuzumab, pertuzumab (2C4) or margetuximab (MGAH22). In some embodiment, the antibody is frastuzumab. According to certain embodiments, protein-drug conjugates, e.g., ADCs, according to the disclosure comprise anti-HER2 antibody. In some embodiment, the anti-HER2 antibody may include those described in WO 2019 / 212965 A1.

[0305] In some embodiments, the antibody is an anti-HER2 / HER?2 bispecific antibody, which comprises a first antigen-binding domain (D1) which specifically binds a first epitope of human HER2 and a second antigen-binding domain (D2) which specifically binds a second epitope of human HER2.

[0306] In certain embodiments, D1 and D2 domains of an anti-HER2 / HER2 bispecific antibody are non-competitive with one another. Non-competition between D1 and D2 for binding to HER2 means that, the respective monospecific antigen binding proteins from which D1 and D2 were derived do not compete with one another for binding to human HER2. Exemplary antigen- binding protein competition assays are known in the art.

[0307] In certain embodiments, D1 and D2 bind to different (e.g., non-overlapping, or partially overlapping) epitopes on HER2.

[0308] In one non-limiting embodiment, the present disclosure provides protein-drug conjugates comprising a bispecific antigen-binding molecule comprising: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human HER2; and wherein D2 specifically binds a second epitope of human HER2.

[0309] Anti-HER2 / HER2 bispecific antibodies may be constructed using the antigen- binding domains of two separate monospecific anti-HER2 antibodies. For example, a collection of monoclonal monospecific anti-HER2 antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise against one another for cross-competition to a HER2 protein. If two different anti-HER2 antibodies are able to bind to HERZ at the same time (i.e., do not compete with one another), then the antigen-binding domain from the first anti-HER2 antibody and the antigen-binding domain from the second, non- competitive anti-HER2 antibody can be engineered into a single anti-HER2 / HER2 bispecific antibody in accordance with the present disclosure.

[0310] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. As will be made evident by the present disclosure, any antigen binding construct which has the ability to simultaneously bind two separate, non-identical epitopes of the HER2 molecule is regarded as a bispecific antigen-binding molecule. Any of the bispecific antigen-binding molecules described herein, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology) as will be known to a person of ordinary skill in the art.

[0311] In another aspect, the disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof which specifically binds HER2 and a pharmaceutically acceptable carrier. In one non-limiting embodiment, the antibody may bind two separate epitopes on the HERZ protein, i.e., the antibody is a HER2 / HER2 bispecific antibody. In a related aspect, the disclosure features a composition which is a combination of an anti- HER2 / HER2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-HER2 / HER2 antibody. Additional combination therapies and co-formulations involving the anti-HER2 / HER2 bispecific antibodies of the present disclosure are disclosed elsewhere herein.

[0312] In another aspect, the disclosure provides therapeutic methods for targeting / killing tumor cells expressing HER2 using an anti-HER2 / HER2 bispecific antibody of the disclosure, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-HER2 / HER2 antibody of the disclosure to a subject in need thereof. In some cases, the anti-HER2 / HER2 antibodies (or antigen-binding fragments thereof) can be used for treating breast cancer, or may be modified to be more cytotoxic by methods, including but not limited to, modified Fc domains to increase ADCC (see e.g., Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods for increasing the efficiency of tumor ablation.

[0313] The present disclosure also includes the use of an anti-HER2 antibody of the disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by HER2-expressing cells. In one aspect, the disclosure relates to a compound comprising an anti-HER2 antibody or antigen-binding fragment, or a HER2 / HER2 bispecific antibody, as disclosed herein, for use in medicine. In one aspect, the disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine.

[0314] In yet another aspect, the disclosure provides bispecific ant-HER2 / HER2 antibodies for diagnostic applications, such as, e.g., imaging reagents. Anti-STEAP2 Antibodies Suitable for Protein-Drug Conjugates

[0315] In some embodiments, the antibody is an anti-six-transmembrane epithelial antigen of prostate 2 (STEAP2), i.e., an anti-STEAP2 antibody. STEAP2, which works as a shuttle between the Golgi complex and the plasma membrane, is a metalloreductase which reduces iron and copper, facilitating their import into the cell. STEAP2 is mainly localized to epithelial cells of the prostate. STEAP2 is also expressed in normal heart, brain, pancreas, ovary, skeletal muscle, mammary gland, testis, uterus, kidney, lung, trachea, colon, and liver. STEAP2 is over-expressed in cancerous tissues, including prostate, bladder, cervix, lung, colon, kidney, breast, pancreatic, stomach, uterus, and ovarian tumors (Gomes, |.M. et al., 2012, Mol. Cancer Res. 10:573-587; Challita-Eid- P.M., et al., 2003, WO 03 / 087308; Emtage, P.C.R., 2005, WO 2005 / 079490).

[0316] In one aspect, suitable anti-STEAP antibodies are those disclosed in US2018 / 0104357. Exemplary anti-STEAP2 antibodies according to the present disclosure are listed in Tables 1 and 2 herein. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs) and light chain variable regions (LCVRs), as well as heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDRS3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti- STEAP2 antibodies. Table 2 sets forth the sequence identifiers of the nucleic acid molecules encoding the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-STEAP2 antibodies.

[0317] The present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0318] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0319] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-STEAP2 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N).

[0320] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR 1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0321] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0322] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 85%, at least 98% or at least 99% sequence identity.

[0323] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR 1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 85%, at least 98% or at least 99% sequence identity.

[0324] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 85%, at least 98% or at least 99% sequence identity.

[0325] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 85%, at least 98% or at least 99% sequence identity.

[0326] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3 / LCDRS3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-STEAP2 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 256 / 264 (e.g., H2M11162N).

[0327] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-STEAP2 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR 1-LCDR2-LCDR3 amino acid sequences set is selected from the group consisting of SEQ ID NOs: 252-254-256-260-262-264 (e.g. H2M11162N).

[0328] In a related embodiment, the present disclosure provides antibodies, or antigen- binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDRS3) contained within an HCVR / LCVR amino acid sequence pair as defined by any of the exemplary anti-STEAP2 antibodies listed in Table 1. For example, the present disclosure includes antibodies, or antigen-binding fragments thereof, comprising the HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 250 / 258 (e.g., H2M11162N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al- Lazikani et al., J. Mol. Biol. 273:927-848 (1987); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

[0329] The present disclosure also provides nucleic acid molecules encoding anti- STEAP2 antibodies or portions thereof. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0330] The present disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0331] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0332] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0333] The present disclosure also provides nucleic acid molecules encoding any of the HCDRS3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0334] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0335] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0336] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0337] The present disclosure also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDRS3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti- STEAP2 antibodies listed in Table 1.

[0338] The present disclosure also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti- STEAP2 antibodies listed in Table 1.

[0339] The present disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 85%, at least 98% or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-STEAP2 antibody listed in Table 1.

[0340] The present disclosure also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-STEAP2 antibody. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences as set forth in Table 1. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0341] The present disclosure includes anti-STEAP2 antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[0342] In another aspect, the disclosure provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof which specifically binds STEAP2 and a pharmaceutically acceptable carrier. In a related aspect, the disclosure features a composition which is a combination of an anti-STEAP2 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-STEAP2 antibody. Additional combination therapies and co-formulations involving the anti-STEAP2 antibodies of the present disclosure are disclosed elsewhere herein.

[0343] In another aspect, the disclosure provides therapeutic methods for targeting / killing tumor cells expressing STEAP2 using an anti-STEAP2 antibody of the disclosure, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-STEAP2 antibody of the disclosure to a subject in need thereof. In some cases, the anti-STEAP2 antibodies (or antigen-binding fragments thereof) can be used for treating prostate cancer, or may be modified to be more cytotoxic by methods, including but not limited to, modified Fe domains to increase ADCC (see e.g., Shield et al. (2002) JBC 277:26733), radioimmunotherapy, antibody-drug conjugates, or other methods for increasing the efficiency of tumor ablation.

[0344] The present disclosure also includes the use of an anti-STEAP2 antibody of the disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by STEAP2-expressing cells. In one aspect, the disclosure relates to a compound comprising an anti-STEAP2 antibody or antigen-binding fragment, or a STEAP2xCD3 bispecific antibody, as disclosed herein, for use in medicine. In one aspect, the disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine.

[0345] In yet another aspect, the disclosure provides monospecific anti-STEAP2 antibodies for diagnostic applications, such as, e.g., imaging reagents.

[0346] In yet another aspect, the disclosure provides therapeutic methods for stimulating T cell activation using an anti-CD3 antibody or antigen-binding portion of an antibody of the disclosure, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody

[0347] In another aspect, the present disclosure provides an isolated antibody or antigen- binding fragment thereof that binds STEAP2-expressing C4-2 cells with an EC50 of less than 50 nM as measured by FACS analysis. In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds and is internalized by STEAP2-expressing C4-2 cells.

[0348] The disclosure further provides an antibody or antigen-binding fragment that competes for binding to human STEAP2 with a reference antibody comprising an HCVR / LCVR amino acid sequence pair as set forth in Table 1. In another aspect, the disclosure provides an antibody or antigen-binding fragment that competes for binding to human STEAP2 with a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 58; 74 / 58; 82 / 58; 90 / 58; 98 / 58; 106 / 114; 122 / 130; 138 / 146; 154 / 162; 170 / 178; 186 / 194; 202 / 210; 218 / 226; 234 / 242; 250 / 258; 266 / 274; 282 / 290; 298 / 306; 314 / 322; 330 / 338: 346 / 354; 362 / 370; and 378 / 386.

[0349] The disclosure furthermore provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human STEAP2 as a reference antibody comprising an HCVR / LCVR amino acid sequence pair as set forth in Table 1. In another aspect, the antibody or antigen-binding fragment binds to the same epitope on human STEAP2 as a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 58; 74 / 58; 82 / 58; 90 / 58; 98 / 58; 106 / 114; 122 / 130; 138 / 146; 154 / 162; 170 / 178; 186 / 194; 202 / 210; 218 / 226; 234 / 242; 250 / 258; 266 / 274; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

[0350] The disclosure further provides an isolated antibody or antigen-binding fragment thereof that binds human STEAP2, wherein the antibody or antigen-binding fragment comprises: the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and the CDRs of a light chain variable region (LCVR) having an amino acid sequence as set forth in Table 1. In another aspect, the isolated antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs:2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 58; 74 / 58; 82 / 58; 90 / 58; 98 / 58; 106 / 114; 122 / 130; 138 / 146; 154 / 162; 170 / 178; 186 / 194; 202 / 210; 218 / 226; 234 / 242; 250 / 258; 266 / 274; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386. In yet another aspect, the isolated antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected from the group consisting of: SEQ ID NOs:4-8-8-12-14-16; 20-22-24-28-30-32; 36-38-40-44-46- 48; 52-54-56-60-62-64; 68-70-72-60-62-64; 76-78-80-60-62-64; 84-86-88-60-62-64; 92-94-96- 60-62-64; 100-102-104-60-62-64; 108-110-112-116-118-120; 124-126-128-132-134-136; 140- 142-144-148-150-152; 156-158-160-164-166-168; 172-174-176-180-182-184; 188-190-192-196- 198-200; 204-206-208-212-214-216; 220-222-224-228-230-232; 236-238-240-244-246-248; 252-254-256-260-262-264, 268-270-272-276-278-280; 284-286-288-292-294-296; 300-302-304- 308-310-312; 316-318-320-324-326-328,; 332-334-336-340-342-344; 348-350-352-356-358-360; 364-366-368-372-374-376; and 380-382-384-388-390-392.

[0351] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds human STEAP2, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 74, 82, 90, 98, 1086, 122, 138, 154, 170, 1886, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, and 378; and (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10; 26; 42; 58 114; 130; 1486; 162; 178; 194; 210; 226, 242; 258; 274; 290; 3086; 322; 338; 354; 370; and 386. In a further aspect, the isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs:2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 58; 74 / 58; 82 / 58; 90 / 58; 98 / 58; 106 / 114; 122 / 130; 138 / 146; 154 / 162; 170 / 178; 186 / 194; 202 / 210; 218 / 226; 234 / 242; 250 / 258; 266 / 274; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

[0352] According to another aspect, the present disclosure provides antibody-drug conjugates comprising an anti-STEAP2 antibody or antigen-binding fragment thereof as described above and a therapeutic agent (e.g., an anti-tumor agent, e.g., a camptothecin analog, e.g., Dxd). In some embodiments, the antibody or antigen-binding fragment and the anti-tumor agent are covalently attached via a linker, as discussed above. In various embodiments, the anti- STEAP2 antibody or antigen-binding fragment can be any of the anti-STEAP 2 antibodies or fragments described herein.

[0353] Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences of anti-STEAP?2 antibodies

[0354] Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-STEAP2 antibodies according to the disclosure. The corresponding nucleic acid sequence identifiers are set forth in Table 2. Table 1: Amino Acid Sequence Identifiers of anti-STEAP2 antibodies SEQID NOs: Antibody 18 20 29 24 26 28 30 32 34 236 38 40 42 44 46 48 50 52 54 56 58 680 62 64 66 B68 70 72 58 B80 B82 B84 74 76 78 80 58 680 62 64 82 84 86 88 58 B60 B62 B84 90 92 04 8 58 B60 B62 B84 o8 100 102 104 58 680 62 64 H1H11897P2 108 110 112 114 116 118 120 H1H7968P | 122 124 126 128 130 | _132 134 136 H1H7969P | 138 140 142 144 146 | _148 150 152 H1H7970P | _154 156 158 160 162 | _164 166 168 H1H7971P_|_170 172 174 176 178 | _180 182 184 H1H7972P | 186 188 190 192 194 | 196 198 200 HTM7804N_|_202 204 206 208 210 | 212 214 216 HIM7814N | 218 | 220 222 224 226 | 228 230 232 HI1M7832N | 234 | 238 238 240 242 | 244 246 248 260 262 264 276 278 280 H2M11164N 284 286 288 290 | 292 294 296 H2M7806N_| 298 | 300 302 304 306 | 308 310 312 H2M7807N_| 314 | 316 318 320 322 | 324 326 328 H2M780SN_| 330 | 332 334 336338 | 340 342 344 H2M7810N_| 346 | 348 350 352 354 | 356 358 360 H2M7811N_| 362 | 364 366 368 370 | 372 374 376 HoM7812N | 378 | 380 380 384 386 | 388 390 392 Antibody ">Y | HCVR | HCDR1 | HCDR2 | HCDRS | LCVR | LCDR1 | LCDR? | LCDRS | | Designation 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 58 60 62 64 74 76 78 80 58 60 62 64 82 84 86 88 58 60 62 64 90 92 94 96 58 60 62 64 98 100 102 104 58 60 62 64 108 110 112 114 116 118 120 H1H7968P | 122 124 126 128 130 | _132 134 136 H1H7969P | 138 140 142 144 146 | _148 150 152 H1H7970P | _154 156 158 160 162 | _164 166 168 H1H7971P_|_170 172 174 176 178 | _180 182 184 H1H7972P | 186 188 190 192 194 | 196 198 200 HTM7804N_|_202 204 206 208 210 | 212 214 216 HIM7814N_| 218 | 220 222 224 226 | 228 230 232 H1M7832N | 234 | 236 238 240 242 | 244 246 248 252 254 256 258 | 260 262 264 268 270 272 274 276 278 280 284 286 288 200 | 202 204 296 H2M7806N | 298 300 302 304 306 | 308 310 312 H2M7807N_|_ 314 316 318 320 322 | 324 326 328 H2M7809N_|_ 330 332 334 336 338 | 340 342 344 H2M7810N_|_ 346 348 350 352 354 | 356 358 360 H2M7811N_|_ 362 364 366 368 370 | 372 374 376 H2M7812N_| 378 380 382 384 386 | 388 390 392 Table 2: Nucleic Acid Sequence Identifiers of anti-STEAP2 antibodies SEQ ID NOs: Antibody H1H11878P 17 19 21 23 25 27 29 31 H1H11880P 33 35 37 39 41 43 45 47 49 51 53 55 57 59 B81 B63 65 B87 689 71 57 59 61 63 73 75 77 79 57 59 B81 B63 81 83 85 87 57 59 81 B63 89 91 93 o5 57 59 B81 B63 97 99 101 103 57 58 [| 63 115 117 119 H1H7968P 121 123 125 127 129 131 133 135 H1H7968P 137 139 141 143 145 147 149 151 H1H7970P 153 155 157 159 161 163 165 167 H1H7971P 169 171 173 175 177 179 181 183 H1H7972P 185 187 189 191 193 195 197 199 HAM7804N 201 203 205 207 209 244 213 215 H1M7814N 217 219 221 223 225 257 229 231 HI1M7832N 233 | 235 237 239 241 243 245 247 H2M11162N 249 251 253 255 257 259 261 263 H2M11163N 265 267 269 £274 273 275 277 279 H2M11164N 281 283 285 287 289 291 293 295 H2M7806N 297 299 301 303 305 307 309 311 H2M7807N 313 | 315 317 319 321 323 325 327 H2M780SN 329 331 333 335 337 339 341 343 H2M7810N 345 347 349 351 353 355 357 359 H2M7811N 361 363 365 367 369 371 373 375 Antibody >Y | HCVR | HCDR1 | HCDR2 | HCDR3 | LCVR | LCDR1 | LCDR2 | LCDR3 | Designation HiH11243N | 1 3 5 _ 9 11 13 15 H1H11878P_| 17 19 21 23 25 27 29 31 H1H11880P_| 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 57 59 61 63 73 75 77 79 57 59 61 63 81 83 85 87 57 59 61 63 89 91 93 95 57 59 61 63 97 99 101 103 57 59 61 63 107 109 111 113 _| 115 117 119 H1H7968P _ _ 121 123 125 127 129 | _131 133 135 H1H7969P _ _ 137 139 141 143 145 | 147 149 151 H1H7970P__ _ 153 155 157 159 _ 161 163 165 167 H1H7971P__ _ 169 171 173 175 177 | _179 181 183 H1H7972P__ _ 185 187 189 191 193 | 195 197 199 HIM7804N__ _ 201 203 205 207 209 | 211 213 215 HIM7814N_ 217 | _219 221 223 225 | 227 229 231 HIM7832N__ 233 | _ 235 237 230 241 243 245 247 H2M11162N_| 249 | 251 253 255 257 | 259 261 263 H2M11163N_|_265 | _ 267 269 271 273 |_275 277 279 H2M11164N_|_ 281 283 285 287 289 | 291 293 295 H2M7806N__'_207 | _ 299 301 303 305 | 307 300 311 H2M7807N_ 313 | 315 317 319 321 323 325 327 HOM7809N_ 329 | _ 331 333 335337 | 339 341 343 H2M7810N__ 345 | 347 349 351 353 | _ 355 357 359 H2M7811N__ _ 361 363 365 367 _ 369 | 371 373 375

[0355] Anti-MET Antibodies Suitable for Protein-Drug Conjugates

[0356] In some embodiments, the antibody is an anti MET antibody. According to certain embodiments, protein-drug conjugates, e.g., ADCs, according to the disclosure comprise anti- MET antibody. In some embodiment, the anti- MET antibody may include those described in US 2018 / 0134794.

[0357] In some embodiments, the antibody is an anti-MET / MET bispecific antibody, which comprises a first antigen-binding domain (D1) which specifically binds a first epitope of human MET and a second antigen-binding domain (D2) which specifically binds a second epitope of human MET. In some embodiment, the anti-MET / MET bispecific antibody may include those described in US 2018 / 0134794.

[0358] In certain embodiments, D1 and D2 domains of an anti- MET / MET bispecific antibody are non-competitive with one another. Non-competition between D1 and D2 for binding to MET means that, the respective monospecific antigen binding proteins from which D1 and D2 were derived do not compete with one another for binding to human MET. Exemplary antigen- binding protein competition assays are known in the art.

[0359] In certain embodiments, D1 and D2 bind to different (e.g., non-overlapping, or partially overlapping) epitopes on MET.

[0360] In one non-limiting embodiment, the present disclosure provides protein-drug conjugates comprising a bispecific antigen-binding molecule comprising: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human MET; and wherein D2 specifically binds a second epitope of human MET.

[0361] Anti- MET / MET bispecific antibodies may be constructed using the antigen-binding domains of two separate monospecific anti-MET antibodies. For example, a collection of monoclonal monospecific anti-MET antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise against one another for cross-competition to a MET protein. If two different anti-MET antibodies are able to bind to MET at the same time (i.e., do not compete with one another), then the antigen-binding domain from the first anti-MET antibody and the antigen-binding domain from the second, non-competitive anti-MET antibody can be engineered into a single anti-MET / MET bispecific antibody in accordance with the present disclosure.

[0362] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. As will be made evident by the present disclosure, any antigen binding construct which has the ability to simultaneously bind two separate, non-identical epitopes of the MET molecule is regarded as a bispecific antigen-binding molecule. Any of the bispecific antigen-binding molecules described herein, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology) as will be known to a person of ordinary skill in the art.

[0363] The bispecific antigen-binding molecules, which comprise a first antigen-binding domain (D1) which specifically binds a first epitope of human MET and a second antigen-binding domain (D2) which specifically binds a second epitope of human MET, may be referred to herein as “MET / MET bispecific antibodies,” “MET x MET bispecific antibodies,” “MET / MET,” “MET x MET” or other related terminology. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO:2109. In some embodiments, the second epitope of human MET comprises amino acids 305-315 and 421-455 of SEQ ID NO:2109. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO:2109; and the second epitope of human MET comprises amino acids 305-315 and 421-455 of SEQ ID NO:2109.

[0364] Exemplary antigen-binding domains (D1 and D2) that can be included in the MET x MET bispecific antigen-binding molecules provided herein include antigen-binding domains derived from any of the anti-MET antibodies disclosed herein. For example, the present disclosure includes MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0365] Also provided herein are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0366] Provided herein are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCVR and an LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 3 paired with any of the LCVR amino acid sequences listed in Table 3. According to certain embodiments, the present invention provides MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 3.

[0367] Also provided herein are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0368] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0369] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0370] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0371] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0372] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 3 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0373] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 3 paired with any of the LCDR3 amino acid sequences listed in Table 3. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 3.

[0374] Also provided are MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a set of six CDRs (i.e.,, HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-MET antibodies listed in Table 3.

[0375] In a related embodiment, the present disclosure provides MET x MET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair as defined by any of the exemplary anti-MET antibodies listed in Table 3.

[0376] The MET x MET bispecific antigen-binding molecules provided herein may comprise a D1 antigen-binding domain derived from any of the anti-MET antibodies of Table 3, and a D2 antigen-binding domain derived from any other anti-MET antibody of Table 3. Non- limiting examples of MET x MET bispecific antibodies of the present disclosure are depicted in Figure 10. Figure 10 is a matrix illustrating the components of 272 exemplary MET x MET bispecific antibodies. Each numbered cell of the matrix (numbered 1 through 272) identifies a unique bispecific antibody comprising a “D1” antigen binding domain and a “D2” antigen binding domain, wherein the D1 antigen binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDRs from the corresponding anti-MET antibody listed along the Y-axis, and wherein the D2 antigen binding domain comprises the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or CDRs from the corresponding anti- MET antibody listed along the X-axis. Thus, for example, the MET x MET bispecific antigen- binding molecule “number 10” shown in the matrix comprises a D1 antigen-binding domain comprising an HCVR / LCVR pair, or 6-CDR set, from the exemplary anti-MET antibody H4H13290P2, and a D2 antigen-binding domain comprising an HCVR / LCVR pair, or 6-CDR set, from the exemplary anti-MET antibody H4H13321P2.

[0377] As a non-limiting illustrative example, the present disclosure includes MET x MET bispecific antigen binding molecules comprising a D1 antigen-binding domain and a D2 antigen- binding domain, wherein the D1 antigen binding domain comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092, or a set of heavy and light chain CDRs (HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016-2018-2094-2096- 2098, and wherein the D2 antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092, or a set of heavy and light chain CDRs (HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2038-2040-2042-2094-2096- 2098. An exemplary MET x MET bispecific antibody having these sequence characteristics is the bispecific antibody designated H4H14639D, also referred to as bispecific antibody No. 2076, which comprises a D1 derived from H4H13306P2 and a D2 derived from H4H13312P2.

[0378] Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences for anti-MET and MET / MET antibodies

[0379] Table 3 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-MET antibodies described herein. (As noted above, all anti-MET antibodies of the present disclosure possess the same light chain variable region, and thus the same light chain CDR sequences as well). The corresponding nucleic acid sequence identifiers are set forth in Table 4. Table 3: Amino Acid Sequence Identifiers SEQ ID NOs: 2094 2096 2098 2094 2096 2098 2094 2096 2098 2094 2096 2098 2094 2096 2098 2094 2006 2098 2094 2006 2098 2094 2006 2098 2094 2096 2098 2036 | 2038 2040 2042 || 0092 | 2094 2098 2098 2094 2096 2098 2094 2096 2098 2094 2096 2098 2094 2096 2098 2094 2096 2098 2094 2096 2098 Antibody — Cena | HCVR | HCDR1 | HCDR2 | HCDR3 || LCVR | LCDR1 | LCDR2 | LCDR3 1958 1960 1962 || 2002 | 2094 2006 _ 2098 HatazotP2 | 1964 | 1986 1988 1970 || j00p | nossa 2006 2008 HartazesP2 | 1972 | 1974 1976 1978 || j00p | 200s 2006 2008 Hatazeor2 | 1980 | 1982 1984 1986 || s00p | sos4 2006 2008 Ha1a300P2 | 1988 | 1990 1992 1994 || s00p | sos4 2006 2008 HaH1a301P2 | 1996 | 1998 2000 2002 || s00p | sosa 2006 2008 Ha1a302P2 | 2004 | 2008 2008 2010 || s00p | sos4 2006 2008 HataaosP2 | 2012 | 2014 2016 2018 || s00p | sosa 2006 2008 HataaooP2 | 2020 | 2022 2024 2026 || j00p | sos4 2006 2008 HaHtaa11P2 | 2028 | 2030 2032 2034 || j00p | sos4 2006 2008 HaH13312p2 | 2036 | 2038 2040 2042 fl oop | s0s4 209s 2088 H4H13313P2 | 2044 | 2046 2048 2050 [| oop | s094 209s 2088 HaH13316P2 | 2052 | 2054 2056 2058 [| oo0p | s0s4 2098 2088 HaH13318P2 | 2080 | 2082 2084 2086 [| oop | 2034 2098 2088 HaH13319P2 | 2088 | 2070 2072 2074 [| jp | 2084 2098 2088 HaH13325P2 | 2076 | 2078 2080 2082 fl oop | gosa 209s 2098 HaH13331P2 | 2084 | 2086 2088 2000 [ hogy | cogs 2008 200s Table 4: Nucleic Acid Sequence Identifiers SEQ ID NOs: Antibody — Designation | HCVR | HCDR1 | HCDR2 | HCDR3 || LCVR | LCDR1 | LCDR2 | LCDR3 HaH13200P2 | 1955 | 1957 1959 1961 || so91 2003 2005 2087 | SEQ ID NOs: 2003 2005 2097 | 2003 2005 2097 | 1987 | 1989 1991 1993 [| 5501 2093 2005 2097 | 2003 2005 2097 | 2003 2005 2097 | 2003 2005 2097 | 2003 2005 2097 | 2003 2095 2097 | 2003 2095 2097 | 2003 2095 2097 | 2003 2095 2097 | 2003 2095 2097 | 2003 2095 2097 | 2077 2079 2081 || 5091 2093 2095 2097 | Antibody — I Designation | HCVR | HCDR1 | HCDR2 | HCDR3 [| LCVR | LCDR1 | LCDR2 | LCDR3 1965 1967 1969 | ooo 2008 2085 2007 | Har132osP2 | 1971 1973 1975 1977 |[ poor poss goes poor | Hari13200P2 | 1979 | 1981 1983 1985 |[ hoor poss goes poor | Hari13300p2 | 1987 | 1989 1991 1993 || soi 003 2095 2007 | Har13301P2 | 1995 | 1997 199 2001 || jor 003 2095 2007 | Har13302p2 | 2003 | 2005 2007 2009 || j091 003 2095 2007 | Har1aa0ep2 | 2011 2013 2015 2017 || jor 003 2095 2007 | Hari13000P2 | 2019 | 2021 2028 2025 || j091 003 2095 2007 | Haras1P2 | 2027 | 2029 2081 2033 || sog1 00s 005 2007 | Har13s12p2 | 2085 | 2087 2000 2041 || poor 00s 005 2007 | Har13313p2 | 2043 | 2045 2047 2049 || sog1 pops p05 2007 | Har13s1P2 | 2051 2053 2055 2057 || poor pops 005 2007 | Har13318p2 | 2059 | 2081 2068 2065 || poor pops 005 2007 | Har13019p2 | 2067 | 2089 2071 2078 || sog1 00s 005 2007 | Harnaszsp2 | 2075 | 2077 2079 2081 || poor pops goes 2007 | H4H13331P2 | 2083 | 2085 2087 2089 || 2091 2083 2005 2007 | H4H13331P2 | 2083 | 2085 2087 2089 || 5091 2003 _ 2005 2097 |

[0380] Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. “H4H"), followed by a numerical identifier (e.g. “13290,” “13291,” “13295,” efc.), followed by a “P2” suffix, as shown in Tables 3 and 4. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., “H4H13290P2,” “H4H13291P2,” “H4H13295P2," efc. The prefix on the antibody designations used herein indicate the particular Fc region isotype of the antibody. In particular, an “H4H” antibody has a human IgG4 Fc (all variable regions are fully human as denoted by the first 'H' in the antibody designation). As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse 1gG4 Fc can be converted to an antibody with a human 1gG1, efc.), but in any event, the variable domains (including the CDRs) —which are indicated by the numerical identifiers shown in Tables 3 and 4 — will remain the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain. Antibody conjugation

[0381] Techniques and linkers for conjugating to residues of an antibody or antigen binding fragment are known in the art. Exemplary amino acid attachments that can be used in the context of this aspect, e.g., lysine (see, e.g., US 5,208,020; US 2010 / 0129314; Hollander ef al., Bioconjugate Chem., 2008, 19:358-361; WO 2005 / 089808; US 5,714,586; US 2013 / 0101546; and US 2012 / 0585592), cysteine (see, e.g., US 2007 / 0258987; WO 2013 / 055993; WO 2013 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO 2011 / 130598; US 2013 / 0101546; and US 7,750,116), selenoysteine (see, e.g., WO 2008 / 122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non-natural amino acids (see, e.g., WO 2013 / 068874, and WO 2012 / 166559), and acidic amino acids (see, e.g., WO 2012 / 05982). Lysine conjugation can also proceed through NHS (N-hydroxy succinimide). Linkers can also be conjugated to cysteine residues, including cysteine residues of a cleaved interchain disulfide bond, by forming a carbon bridge between thiols (see, e.g., US 9,951,141, and US 9,950,076). Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008 / 0305497, WO 2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127- 130) and disulfide linkers (see, e.g., WO 2013 / 085925, WO 2010 / 010324, WO 2011 / 018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313). Site specific conjugation techniques can also be employed to direct conjugation to particular residues of the antibody or antigen binding protein (see, e.g., Schumacher et al. J Clin Immunol (2016) 36 (Suppl 1): 100). In specific embodiments discussed in more detail below, Site specific conjugation techniques, include glutamine conjugation via transglutaminase (see e.g., Schibli, Angew Chemie Inter Ed. 2010, 49 ,9995).

[0382] Payloads according to the disclosure linked through lysine and / or cysteine, e.g., via a maleimide or amide conjugation, are included within the scope of the present disclosure.

[0383] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where Step 1 is lysine-based linker conjugation, e.g., with an NHS-ester linker, and Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0384] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where Step 1 is cysteine-based linker conjugation, e.g., with a maleimide linker, and Step 2 is a payload conjugation reaction (e.g., a 1,3- cycloaddition reaction).

[0385] In some embodiments, the protein-drug conjugates of the present disclosure are produced according to a two-step process, where Step 1 is transglutaminase-mediated site specific conjugation and Step 2 is a payload conjugation reaction (e.g., a 1,3-cycloaddition reaction). Step 1: Transglutaminase Mediated Site Specific Conjugation

[0386] In some embodiments, proteins (e.g., antibodies) may be modified in accordance with known methods to provide glutaminyl modified proteins. Techniques for conjugating antibodies and primary amine compounds are known in the art. Site specific conjugation techniques are employed herein to direct conjugation to glutamine using glutamine conjugation via transglutaminase (see e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0387] Primary amine-comprising compounds (e.g., linkers L1) of the present disclosure can be conjugated to one or more glutamine residues of a binding agent (e.g., a protein, e.g., an antibody) via transglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennler et al., Protein Conjugate Chem. 2014, 25, 569-578, and WO 2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be coupled to a primary amine linker compound. Briefly, in some embodiments, a binding agent having a glutamine residue (e.g., a gIn295, i.e. Q295 residue) is treated with a primary amine-containing linker L1, described above, in the presence of the enzyme transglutaminase. In certain embodiments, the binding agent is aglycosylated. In certain embodiments, the binding agent is deglycosylated.

[0388] In certain embodiments, the binding agent (e.g., a protein, e.g., an antibody) comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the binding agent comprises two heavy chain polypeptides, each with one gin295 residue. In further embodiments, the binding agent comprises one or more glutamine residues at a site other than a heavy chain 295.

[0389] In some embodiments, a binding agent, such as an antibody, can be prepared by site-directed mutagenesis to insert a glutamine residue at a site without resulting in disabled antibody function or binding. For example, included herein are antibodies bearing Asn297GIn (N297Q) mutation(s) as described herein. In some embodiments, an antibody having a gin295 residue and / or an N297Q mutation contains one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to transglutaminase and therefore capable of conjugation to a linker or a linker-payload. An exemplary naturally occurring glutamine residue can be found, e.g., at Q55 of the light chain. In such instances, the binding agent, e.g., antibody, conjugated via transglutaminase can have a higher than expected LAR value (e.g., a LAR higher than 4). Any such antibodies can be isolated from natural or artificial sources.

[0390] In certain embodiments of the disclosure, the linker-antibody ratio or LAR is from 1,2,3,4,5,6, 7, or 8linker L1 molecules per antibody. In some embodiments, the LAR is from 1 to 8. In some embodiments, the LAR is from 1 to 6. In certain embodiments, the LAR is from 2 to 4. In some cases, the LAR is from 2 to 3. In certain cases, the LAR is from 0.5 to 3.5. In some embodiments, the LAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the LAR is 2. In some embodiments, the LAR is 4. Step 2: Payload Conjugation Reaction

[0391] In certain embodiments, linkers L1 according to the present disclosure comprise a branching unit B, which comprises at least one reactive group B’ capable of further reaction after transglutamination. In these embodiments, the glutaminyl-modified protein (e.g., antibody) is capable of further reaction with a reactive payload compound or a reactive linker-payload compound (e.g., L2-P as disclosed herein), to form a protein-payload conjugate. More specifically, the reactive linker-payload compound L2-P may comprise a reactive group B” that is capable of reacting with the reactive group B’ of the linker L1. In certain embodiments, a reactive group B' according to the present disclosure comprises a moiety that is capable of undergoing a 1,3- cycloaddition reaction. In certain embodiments, the reactive group B’ is an azide. In certain embodiments, the reactive group B” comprises an alkyne (e.g., a terminal alkyne, or an internal strained alkyne). In certain embodiments of the present disclosure the reactive group B’ is compatible with the binding agent and transglutamination reaction conditions.

[0392] In certain embodiments of the disclosure, linker L1 molecules comprise a branching unit B which comprises one reactive group B’. In certain embodiments of the disclosure, linker L1 molecules comprise a branching unit B which comprises more than one reactive group B.

[0393] In certain embodiments, the reactive linker-payload L2-P comprises one payload molecule (n = 1). In certain other embodiments, the reactive linker-payload L2-P comprises two or more payload molecules (n 2 2). In certain embodiments, the reactive linker-payload L2-P comprises from 1 to 12 payload molecules, or from 1 to 10 payload molecules, or from 1 to 8 payload molecules, or from 1 to 6 payload molecules, or from 1 to 4 payload molecules, or from 1 to 2 payload molecules.

[0394] In certain embodiments, the reactive linker-payload L2-P comprises one payload molecule. When such L2-P is reacted with a BA-L1-B, the DAR will be about equal to the LAR of the BA-L1-B. For example, if L2-P comprising one payload molecule is reacted with a BA-L1-B having a LAR of 4 (e.g., via Q295 and N297Q transglutamination), the resulting protein-drug conjugate will have a DAR of 4.

[0395] In certain embodiments, the reactive linker-payload L2-P comprises 2 payload molecules. When such L2-P is reacted with a BA-L1-B, the DAR will be about 2 times the LAR of the BA-L1-B. For example, if L2-P comprising 2 payload molecules is reacted with a BA-L1-B having a LAR of 4 (e.g., via Q295 and N297Q transglutamination), the resulting protein-drug conjugate will have a DAR of 8.

[0396] For example, if L2-P comprising 3 payload molecules is reacted with a BA-L1-B having a LAR of 8 (e.g., via Q295 and N297Q transglutamination of a branched L1-B unit comprising 2 groups B), the resulting protein-drug conjugate will have a DAR of 24.

[0397] In certain embodiments of the disclosure, the drug-antibody ratio or DAR (e.g., abbreviated as the lower case letter n) is from about 1 to about 30, or from about 1 to about 24, or from about 1 to about 20, or from about 1 to about 16, or from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or about 1, 2, 3, 4, 5, 8, 7, or 8 payload molecules per antibody. In some embodiments, the DAR is from 1 to 30. In some embodiments, the DAR is from 1 to 24. In some embodiments, the DAR is from 1 to 16. In some embodiments, the DAR is from 1 to 8. In some embodiments, the DAR is from 1 to 8. In certain embodiments, the DAR is from 2 to 4. In some cases, the DAR is from 2 to 3. In certain cases, the DAR is from 0.5 to 3.5. In certain cases, the DAR is from 10 to 14. In certain cases, the DAR is from 14 to 18. In certain cases, the DAR is from 20 to 24.5. In some embodiments, the DAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the DAR is 2. In some embodiments, the DAR is 4. In some embodiments, the DAR is 8. In some embodiments, the DAR is 12. In some embodiments, the DAR is 16. In some embodiments, the DAR is 24.

[0398] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)x)n (A), wherein BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is a first linker; Bis a branching unit comprising at least one adduct of a group B’ and a group B”; L2 is a second linker covalently attached to the branching unit B via at least one group B”; EAE M is absent or ke , where R, R’, and R” are independently at each occurrence hydrogen or a C1-Cy4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent comprising a structure according to Formula (P): Wor” Apr F TT ARE ~AX ®) k and m are independently an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NHz) with a compound L1-B, wherein the branching unit B comprises the at least one group B’, b) contacting the product of step a) with k or more equivalents of a compound L2-(-M-Dxd)m, wherein the linker L2 comprises at least one group B”, N=N HH wherein one of the groups B’ and B” is selected from -N3 and N~N and the other mY Sr aC AF TN of the groups B’ and B” is selected from gm oe ; and Neer where Q is C or N; and ¢) isolating the produced compound of Formula (1).

[0399] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)k)n (A), wherein BA is an antibody or an antigen-binding fragment thereof, Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one adduct of a group B’ and a group B” as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above; M is BAK absent or & , where R, R’, and R” are as described above; Dxd is an anti- tumor agent comprising a structure according to Formula (P): Kon? dle ge 1 AAA F N oo A (P), k and m are independently an integer from 1 to 12, and nis an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least one group B’, with k or more equivalents of a compound L2-(-M-Dxd)m, wherein the linker L2 comprises at least one group B” capable of covalently binding with the group B’, + { N=-N wherein one of the groups B’ and B” is selected from -N3 and NN ; and the other TR Nid TE i of the groups B’ and B” is selected from ~§—f oo ; and Neer where Q is Cor N: thereby producting L1-B-(-L2-(-M-Dxd)m)k; b) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gln (BA-GIn-NHz) with the L1-B-(-L2-(-M-Dxd)m)x product of step a), and ¢) isolating the produced compound of Formula (1).

[0400] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd))n wherein BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is a first linker; B is a branching unit comprising at least one adduct of a group B’ and a group B”; L2 is a second linker covalently attached to the branching unit B via at least one group B" SAK M is absent or 4 , where R, R’, and R” are independently at each occurrence hydrogen or a C1-Cy4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent comprising a structure according to Formula (P): SN PEN lt y rT SONS ARE “od 0 ®), k is an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NH.) with a compound L1-B, wherein the branching unit B comprises the at least one group B’, b) contacting the product of step a) with k or more equivalents of a compound L2- M-Dxd, wherein the linker L2 comprises the at least one group B” capable of covalently attaching to the group B’, N=N <p wherein one of the groups B’ and B” is selected from -Nj and N-N and the other of the groups B’ and B” is selected from $= A ; and SN where Q is C or N; and ¢) isolating the produced compound of Formula (1).

[0401] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)«)a (I), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one adduct of a group B’ and a group B” as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above; M is si AK absent or fe , where R, R’, and R” are as described above; Dxd is an anti- tumor agent comprising a structure according to Formula (P): np x = TT Oo 0 i N- ANF “~~ F N \ ee — oH Bb (P), kis an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least one group B’ with k or more equivalents of a compound L2-M-Dxd, wherein the linker L2 comprises at least one group B”, thereby producting L1-B-(-L2-M-Dxd)x, wherein one of N=N Sp {oo ON-N the groups B’ and B” is selected from -Nsand ~~ N-N and the other of the groups B’ and B” Ne ,where QisCorN; TN Nui, TF ! is selected from =f oA ; and Ne b) contacting, in the presence of a transglutaminase, the binding agent BA comprising at least one glutamine residue Gin (BA-GIn-NHy) with the L1-B-(-L2-M-Dxd)« product of step a), and ¢) isolating the produced compound of Formula (1).

[0402] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (l11): BA-(GIn-NH-L2’-P)), (111), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L2'-P is H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p as described above, and n is an integer from 1 to 30; Q 0 SP1 is absent or a first spacer unit selected from the group consisting of % Ae Q n i and B2 is absent or a branching unit; SP2 is absent or a second spacer unit selected from the group consisting of a C1.s alkyl, -(CH2-CH2-O)\~, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2)-, -NH-(CHz),-C(O)-, -NH-(CH2- CH2-O)v-, -NH-(CH2-CH2-O)~C(O)-, -NH-(CH2-CH2-O)-(CH2)u-, -NH-(CH2-CH2-O)v~ (CH2),~-C(O)-, -(CH2)y-NH-C(O)-, -NH-(CH2)y-NH-C(O)-, -NH-(CH2)-C(O)-NH-, or combinations thereof: wherein subscripts u and v are independently an integer from 1 to 8: AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit selected from the group consisting of, wo 7 o : £7) 4 tA A ~Y ww N o SP3 is absent or a third spacer unit selecte ne a HN of Ap Fy , wherein R. is independently at each occurrence absent A 5 fo wl, ol at I. I H O-, H O° How Ag HO. Ag LP Pa or a group selected from OH and OH SAK M is absent or & , where R, R’, and R” are independently at each occurrence hydrogen or a C4-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): pS pe. wh A ~~ ~ < P), p is an integer from 1 to 30; wherein the method comprises the steps of: b) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gln (BA-GIn-NH2) with the L2'-P, and ¢) isolating the produced compound of Formula (III).

[0403] In one aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd))a 0, wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one group B' as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above, wherein the group B’ and the group B” form 0 | a : ANA the at least one adduct as described above; M is absent or fe , where R, R’, and R” are as described above; Dxd is an anti-tumor agent comprising a structure according to Formula (P): Li Pe rT DOGO AA JILL 5 — ne (P), kis an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the binding agent BA comprising at least one glutamine residue Gin (BA-GIn-NH.) with a compound L1-B, wherein the branching _— a N unit B comprises the at least one group B’ selected from -Ns, raf “te N=N, Se 40% ( | N=N ~~ J thereby nraducing RA-Gln-NH-1 1-B- I thereby producing BA-GIn-NH-L1-B; b) contacting the product of step a) with k or more equivalents of a compound L2-M-Dxd, wherein the linker L2 comprises at least one group B” capable of covalently binding with the group B’, and ¢) isolating the produced compound of Formula (1).

[0404] In another aspect, the present disclosure provides a method of producing a compound having a structure according to Formula (I): BA-(GIn-NH-L1-B-(-L2-M-Dxd))a 0, wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one group B' as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above, wherein the group B’ and the group B” form 2 l R si KZ 3 7 ® the at least one adduct as described above; M is absent or fe , where R, R’, and R” are as described above; Dxd is an anti-tumor agent comprising a structure according to Formula (P): Hort A r TT £NF y \_ / \ AK or en oo o (P), kis an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least ce SAAT pe] one group B’ selected from -Ns, ~f=f fr N-N , with k or more equivalents of a compound L2-M-Dxd, wherein the linker L2 comprises at least one group B” capable of covalently binding with the group B’, thereby producting L1-B-(-L2-M-Dxd)x: b) contacting, in the presence of a transglutaminase, the binding agent BA comprising at least one glutamine residue Gin (BA-GIn-NHy) with the L1-B-(-L2-M-Dxd)« product of step a), and ¢) isolating the produced compound of Formula (1).

[0405] In one embodiment, the glutamine residue GIn is naturally present ina CH2 or CH3 domain of the BA. In another embodiment, the glutamine residue Gin is introduced to the BA by modifying one or more amino acids. In one embodiment, the Gin is Q295 or N297Q.

[0406] In one embodiment, the transglutaminase is microbial transglutaminase (MTG). In one embodiment, the transglutaminase is bacterial transglutaminase (BTG).

[0407] In one embodiment, M is absent. In another embodiment, M-Dxd is has a structure selected from the group consisting of AS iD TY Pah selected from the group consisting of © H Rm 0 yo Ar NH N N rN 0 CL F N 4 7 0 Ne OHO and Aallbvl amd wheres «a. ranrccante tha me = H ) - NH Ye } o oNH NX 0 or wo Ns EN a] on, Fe, | SN F LY = A NL , pe | or OHO and oH ©, wherein R is a hydrogen or a C1-C4 alkyl, and where .amn represents the point of attachment to L2.

[0408] In one embodiment, the compound L2-Dxd has a structure selected from the group . Jad, - hr ITA ad hf Kak - OS min I 1 HY 5 1 YC X Whe ‘ -~ FN SL | A S=hony NY NY NH oO oO 0 mL Audopoty SF & A ro Y BM 25 oY x A J NL OA SECA S RS HON YOY EY 4 So Ps RY 4 AN - ong AX 27 Fa, 2 W 3 vo. = So Hoody J I Ty — i A Nemo a Aen AAAI 0 YY Ss © © o grr 3 ZN . Nn . o = ep O a LA a a o 4 i B® Seoghiyrokghoady, Two NY 2 ¥y ory Ott Hor Seon oO 7 oO LA 9 G o ¥ ; u ! Na etry ; ory oA S rn? A) A A CH YT HOA yy AH OH © oye Ho ° ©. =o Sk Ao HE Ho = A AA Meh, FO SOY Sos a o ~~, Sagi On mga on i An 2 ° Ap I LITE A wo Sp, Ay A hoody, Sa YY a Ln 2 Y tal re y J Rel, Ay Ory YY Ong Y vy Hem got py HO" Te o o G ~Q Kio, > =o CY ow bl 3 CY Yo | or NN NON Ng NON NEA AN A ANS ¢ rel A | ¥ ial H { % r Noe I wore Sing b | —_— — Sv Jt } arse S AST J EG *M1 ~ No - lm Sy Vo No | “yn, N a ; J took, a OONY Xr Nok JL ALC ; rg yg © aN £,, F ws Ny Ya No Sb, : Fmptondy Sm AO ag ~AA A LL NY Loo SIE oT ] TTI Sphesmmmnn I K LT i. yy a ~ Lh —_— -- ji Hoo XL : a i oY At BREE Pp Y On & N ¥ W J Onn ~ Sy ” HIN yo Y Ny C LA, 0) x or a pharmaceutically acceptable salt thereof.

[0409] In one embodiment, the compound L2-Dxd encompasses an optional branching unit B2. In such embodiments, the compound L2-Dxd comprises 2 or more Dxd units.

[0410] In one embodiment, the branching unit B2 has a structure selected from the group consisting of: Os, 9 54 8 K ~~ AO “7 N=N ONY ox To A ca ume SN Be ox Hel A £ 9 1 \ ! o NN PNY oY os *%- FREUNY LAP Fu Ay a Co 7 0 Lo Lo rs oo y MY Ye Kee 7 o i ] w Gr F rt and oY

[0411] In one embodiment, the compound L2-Dxd has a structure selected from the group consisting of: ~ pat yA 4, of Oy ¥ orion ly rer oh © ji -~ oq? Le, - ee) LN > Chomgornominnimon tI Ato, © oe “ AN | Tie k oN 0 ro NO NH LY a 9 SSN, H ro Ou NH © 9 u ps Noe, o . oo Lop vy | Semon Na, A a 0 CW 1 Syl H o o H ] fe LOY aC er, / Ey oo. SE 2 | i C0 y POH EB Sy i I o Oy NH oT Sut? Spo Hh © 9 9 | J Ho LK N 2 i CNTY onan ligk La XID Q Q ° J CEE o Z oh JL © 9 hit Aon Seu, H pom e % : x oH D Xa OY : y CH, 2 Ay - 3 4 3 5 &) 2 Pe WH Q o H Khoo, . SY y " i 1 i or = f # : A HL, A Q } Ou mnAg ‘ Omg " Yr : A SNR § 0 9 ™ i i nn LCL ~ Hoo CY Ferg p ST CN il ~ op a HN - ~o HN Sg Ong . OF "NH t I O. g fondo t,, i o OY r Ls 1 0 5 HN, ) bomen Ay I a4 EB 1 5 4g a Q 2 o Lg Hook, nL ! SORT 6 NT, Cr hn a wl IT Pop ~ tJ a i g oN o. oO! A Op, oy 2 oN Pay 5° 2 5 3 HX y™ 0 Aaa, food, 2 =o monk LE OTT Sead ~ 0 Sy an Ppa, ate ror ht Oy = L3- I RB SSAA) IF ann Sng Ne ; A i gy Son I oon ou Re oo pe q - J oo ay $8 VS " o , com fo My, 2 de Mngt TY XE SGLEA & “OH oN NH Y Nak, ua 1 J jg ~~ nh I ez Hn ~ PEE a oY wo Oo ah on pw he OA pAymmron / v 3 H (po 1, . 3 M3 HN [NN Ay 0 \, BOINSCALE Ras Jur oY ry q : & oe HO | NA, > o y a Q ery o : o Ags Fo 0, Xn 1 Oy mO. He 2 5 ; oie FA I 5 , ~ ~ 9 2 Li 1 Cob ~ 9 Sg H a \, I NT NH, H ? GTN 0 PN No, : I TY TW w Ogy NH \ Hal” On J i : . arn OOK Syn o) NN Spo 3 HN, 1 4 i 4 oO fel hp A ~ 3 AAT [3 H ] ° : ~ oN , Pon 5 1 < NN Y -: 9 H 8 gh Li A Cry as Hak, fas AS © A FEY Co : ~ ~ ad he 1 va he aon ~ a Sy, y oN Pon ~ Sy = . WL 8 ’ oo gph = : gy fA a. * rs yr SR Sod H Nr T So | J: bY & } NT UNHy 4 Po = Linn lod Lh 2 : Hedy {, X or ’ : a Aer pd T 0H 3 : pe RS % N NH 1 Ay a I) Ro 3 room Ao dy 3 rnp A wy SG YIVTCY X AE tg A LL, A So rctmgmin i t / No ea Ne [ 4. "ommnded LY Sn TALC oh BN LA, ¥ J goed LN oA © i YOY Ce Sdn wo 9 Aor Sh ¢ Ho oo y Vomongled LA | Saag ed re Te h an, or Et Sond 0. A, N < : y J PAYY & : rtrd, ~ "ar FI ytd a i H i ¥ TTT 5 omni 207 x a, ‘ i In ’ YE ry “3 & pr. oe Sows aa ) 6 Lo LA Jb A Se Xx o oon NH ~ Sutalyh Nr Le y el x rrp lr ® RA A S ny : \ ¥ a Ea Sw oy . . Jel, | NY y pte dh LL ~y x Nn DY C3 Dd Pom ~ Q Sr p— Sow on A A ’ ELT 8 LT, X Q é Pon Sr edd } } J Hoo = Le Ln LLY Front HL ? Pon Se, Sy ~ 2 ww Aor She ee Yr 7 Be 0 A 1 Y T. y ° rey ™i C1 YP SA Pro uy 1, ) XL { + | 5 Eo T or oe 9 o i Oe WIT 0 ene 4 2 Q gS Cr pe o YA $f oh ox Nw ~ rp A adage? on ~ og Sw H, Pon 5 a DR Sf eh ror 4 3 0 o é Pom “on 3 POA Oy 5 ~~ 1 i f NOY 8 er TLL AL Y 7 Aon Shoes Sy ° Q Qt LY 8 Ory J oyiel lt 4 = 8 | ~ ~ 3 T Pa Svs A i pm ™~ LA, AT A CY Ay G8 ’ wet dt oA a L ° Hon Se - RUN St ° ° ° oS Lh 9 nb ATH : ST Xx 3, I ! Lo o (7, : SI Wo y XJ ! oe uP AEA Ahn A 1 i hep dy 2 / i v oh mpfr dol 8.1 Oy $i 08 rw YN s LL 7 : EYE 6 Pd EN wy Wh, or ; i wy 4, $ Wg Ft Ses NETS CEE acs age Jed “YY yy rye HG, 5 , and ~ Jad, 3 png deh LI Ay (Jf SOY rh Z woe San Egat oA wf Qa tigger Fogg A LY a Ou { Ye o YY 1 crm A Y 5s vac oh re Lik 2 L, AY pe OTE rr A 1 a ne LA, XJ or a pharmaceutically acceptable salt thereof. Therapeutic Formulation and Administration

[0412] The present disclosure provides pharmaceutical compositions comprising the protein-drug conjugates of the present disclosure.

[0413] In one aspect, the present disclosure provides compositions comprising a population of protein-drug conjugates according to the present disclosure having a drug-antibody ratio (DAR) of about 0.5 to about 30.0.

[0414] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0415] In one embodiment, the composition has a DAR of about 2.

[0416] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0417] In one embodiment, the composition has a DAR of about 4.

[0418] In one embodiment, the composition has a DAR of about 6.5 to about 8.5.

[0419] In one embodiment, the composition has a DAR of about 8.

[0420] In one embodiment, the composition has a DAR of about 10 to about 14.

[0421] In one embodiment, the composition has a DAR of about 12.

[0422] In one embodiment, the composition has a DAR of about 14 to about 18.

[0423] In one embodiment, the composition has a DAR of about 16.

[0424] In one embodiment, the composition has a DAR of about 20 to about 24.5.

[0425] In one embodiment, the composition has a DAR of about 24.

[0426] The compositions of the disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0427] The dose of a protein-drug conjugate administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like. The suitable dose is typically calculated according to body weight or body surface area. When a protein-drug conjugate of the present disclosure is used for therapeutic purposes in an adult patient, it may be advantageous to intravenously administer the protein-drug conjugate of the present disclosure normally at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a protein-drug conjugate may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0428] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0429] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0430] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ |, Il and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Qaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.

[0431] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527- 1533.

[0432] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0433] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. Therapeutic uses of the protein-drug conjugates, linker-payloads and payloads

[0434] In another aspect, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful, inter alia, for the treatment, prevention and / or amelioration of a disease, disorder or condition in need of such treatment.

[0435] In one embodiment, the present invention provides a method of treating a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound (e.g., an antibody-drug conjugate, a linker-payload and / or a payload) according to the disclosure, or the composition comprising any compound according to the present disclosure.

[0436] In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating HER2+ breast cancer. In one embodiment, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful for treating prostate cancer.

[0437] In one aspect, the present disclosure provides a method of selectively delivering a compound into a cell. In one embodiment, the method of selectively delivering a compound into a cell comprises linking the compound to a targeted antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0438] In certain embodiments, the present disclosure provides a method of selectively delivering into a cell a compound having the structure P-I: und having the structure P-I: oO Ry Ha A Ko TTY Ry Rp NH Np Ne 0 FTL ° Ne Ho (PD), R. are indenandantlv a hvdraocen ar an wherein Rs, Rz, and Rs, and R4 are independently a hydrogen or an alkyl, e.g., a C4-C12 alkyl, or a C4-Cs alkyl, or a C4-Cs alkyl, or a C4-C4 alkyl, or wherein R; and Rs together form a 5-membered or a 6-membered ring, or a pharmaceutically acceptable salt thereof.

[0439] In one aspect, the present disclosure provides a method of selectively targeting an antigen on a surface of a cell with a compound. In one embodiment, the method of selectively targeting an antigen on a surface of a cell with a compound comprises linking the compound to a targeted antibody. In one embodiment, the compound is a payload as described above. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

[0440] In certain embodiments, the present disclosure provides a method of selectively targeting an antigen on a surface of a cell with a compound having the structure P-I: face of a cell with a compound having tt g ony HN A mengs® Re os “\ 0 Sol 5% ea, RR. are indenandantlv a hvdraaan ar an wherein R1, Rz, and Rs, and R4 are independently a hydrogen or an alkyl, e.g., a C4-C12 alkyl, or a C4-Ce alkyl, or a C1-Cs alkyl, or a C+-Cs alkyl, or wherein Rz and Rs together form a 5-membered or a 6-membered ring, or a pharmaceutically acceptable salt thereof. Anti-HER2 Antibody-Drug Conjugates

[0441] In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by HER2 expression or activity, or treatable by binding HER2 without competing against modified LDL, or and / or promoting HER2 receptor internalization and / or decreasing cell surface receptor number.

[0442] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation, activation and / or targeting of an immune response would be beneficial. In particular, the anti-HER2Z protein-drug conjugates, including both monospecific anti-HER2 antibodies and bispecific anti-HER2 / MERZ antibodies of the present disclosure can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved include killing of the cells expressing HERZ in the presence of effector cells, for example, by COC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing HER2 which can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[0443] In one embodiment, the protein-drug conjugates of the present disclosure (and therapeutic compositions and dosage forms comprising same) comprise a bispecific antigen- binding molecule comprising: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human HER2; and wherein D2 specifically binds a second epitope of human HER2.

[0444] In one embodiment of the above, D1 and D2 do not compete with one another for binding to human HER2.

[0445] The protein-drug conjugates of the present disclosure can be used to treat, e.g., primary and / or metastatic tumors arising in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. According to certain embodiments of the present disclosure, the anti-HER2 antibodies or anti-HER2 / HER2 bispecific antibodies are useful for treating a patient afflicted with a breast cancer cell that is IHC2+ or more. According to other related embodiments of the present disclosure, methods are provided comprising administering an anti-HER2 antibody or an anti-HER2 / HER2 antibody as disclosed herein to a patient who is afflicted with a breast cancer cell that is IHC2+ or more. Analytic / diagnostic methods known in the art, such as tumor scanning, etc., can be used to ascertain whether a patient harbors a tumor that is castrate- resistant.

[0446] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject. The term "residual cancer" means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.

[0447] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation, activation and / or targeting of an immune response would be beneficial. In particular, protein-drug conjugates comprising the anti-HER2 antibodies or anti HER2 / HER2 antibodies of the present disclosure can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by HER2 expression or activity or the proliferation of HER2+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved include killing of the cells expressing HER2 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing HER2 which can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, breast tumor cells.

[0448] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with HER2 expression (e.g., breast cancer) comprising administering one or more of the anti-HER2 protein-drug conjugates or anti-HER2 / HER2 bispecific protein-drug conjugates described elsewhere herein to a subject after the subject has been determined to have breast cancer (e.g., and IHC2+ breast cancer). For example, the present disclosure includes methods for treating breast cancer comprising administering protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding molecule or an anti-HER2 / HER2 bispecific antibody or antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0449] In certain embodiments, the present disclosure also includes the use of an anti- HER?2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by HER2-expressing cells. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-HER2 antibody or antigen-binding fragment or an anti-HER2 / HER2 bispecific antibody or antigen-binding fragment, as disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine. Anti-STEAP2 Antibody-Drug Conjugates

[0450] In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by STEAP2 expression or activity, or treatable by binding STEAP2 without competing against modified LDL, or and / or promoting STEAP2 receptor internalization and / or decreasing cell surface receptor number.

[0451] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation, activation and / or targeting of an immune response would be beneficial. In particular, the anti-STEAP2 protein-drug conjugates of the present disclosure can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by STEAP2 expression or activity or the proliferation of STEAP2+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved include killing of the cells expressing STEAP2 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing STEAP2 which can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, prostate tumor cells.

[0452] The protein-drug conjugates of the present disclosure can be used to treat, e.g., primary and / or metastatic tumors arising in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytic / diagnostic methods known in the art, such as tumor scanning, etc., can be used to ascertain whether a patient harbors a tumor that is castrate- resistant.

[0453] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject. The term "residual cancer" means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.

[0454] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with STEAP2 expression (e.g., prostate cancer) comprising administering one or more of the anti-STEAP2 protein-drug conjugates described elsewhere herein to a subject after the subject has been determined to have prostate cancer. For example, the present disclosure includes methods for treating prostate cancer comprising administering protein-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0455] In certain embodiments, the present disclosure also includes the use of an anti- STEAP2 antibody of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by STEAP2-expressing cells. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-STEAP2 antibody or antigen-binding fragment, as disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine. Anti-MET Antibody-Drug Conjugates

[0456] In certain embodiments, the protein-drug conjugates, e.g., ADCs, disclosed herein are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by MET expression or activity, or treatable by binding MET without competing against modified LDL, or and / or promoting MET receptor internalization and / or decreasing cell surface receptor number.

[0457] The protein-drug conjugates of the present disclosure (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation, activation and / or targeting of an immune response would be beneficial. In particular, the anti-MET or anti MET / MET bispecific protein-drug conjugates of the present disclosure can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by MET expression or activity or the proliferation of MET+ cells. The mechanism of action by which the therapeutic methods of the present disclosure are achieved include killing of the cells expressing MET in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing MET which can be inhibited or killed using the protein-drug conjugates of the present disclosure include, for example, lung tumor cells.

[0458] The protein-drug conjugates of the present disclosure can be used to treat, e.g., primary and / or metastatic tumors arising in the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and / or ovary. In certain embodiments, the protein-drug conjugates of the present disclosure are used to treat one or more of the following cancers: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer. Analytic / diagnostic methods known in the art, such as tumor scanning, etc., can be used to ascertain whether a patient harbors a tumor that is castrate- resistant.

[0459] In certain embodiments, the present disclosure also includes methods for treating residual cancer in a subject. The term "residual cancer" means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.

[0460] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with MET expression (e.g., lung cancer) comprising administering one or more of the anti-MET or anti MET / MET bispecific protein-drug conjugates described elsewhere herein to a subject after the subject has been determined to have lung cancer. For example, the present disclosure includes methods for treating lung cancer comprising administering protein-drug conjugate comprising an ant-MET or anti MET / MET bispecific antibody or antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., anti-androgen therapy).

[0461] For example, anti-MET antibody-drug conjugates and MET x MET bispecific antibody-drug conjugates of the present disclosure are useful for the treatment of tumors that express (or overexpress) MET. For example, the anti-MET antibody-drug conjugates and MET x MET bispecific antibody-drug conjugates may be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the anti-MET antibody-drug conjugates and MET x MET bispecific antibody-drug conjugates are used to treat one or more of the following cancers: acute myelogenous leukemia, adult T-cell leukemia, astrocytomas, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer (e.g. gastric cancer with MET amplification), glioblastomata, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), Kaposi's sarcoma, kidney cancer, leiomyosarcomas, liver cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphomas, malignant gliomas, malignant mesothelioma, melanoma, mesothelioma, MFH / ibrosarcoma, multiple myeloma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic carcinoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0462] In certain embodiments, the present disclosure also includes the use of an anti- MET antibody-drug conjugate or a MET x MET bispecific antibody-drug conjugate of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by MET-expressing cells. In one aspect, the present disclosure relates to a protein-drug conjugate comprising an anti-MET antibody-drug conjugate or a MET x MET bispecific antibody-drug conjugate, as disclosed herein, for use in medicine. In one aspect, the present disclosure relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine. Combination Therapies and Formulations

[0463] The present disclosure provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined with or administered in combination with protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads of the present disclosure include, e.g.,, a HER2 antagonist (e.g., an anti-HER2 antibody [e.g., trastuzumab] or a small molecule inhibitor of HER2 or an anti-HER2 antibody-drug conjugate, or an anti-HER2 / HER2 bispecific antibody or an anti- HER2 / HER2 bispecific antibody-drug conjugate), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as HER2 / ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRVIII (e.g., an antibody that specifically binds EGFRuvlI), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, gDC-0879, PLX-4720), a PDGFR-a inhibitor (e.g., an anti-PDGFR-a antibody), a PDGFR-B inhibitor (e.g., an anti-PDGFR- antibody), a VEGF antagonist (e.g., a VEGF-Trap, see, e.g., US 7,087,411 (also referred to herein as a "VEGF-inhibiting fusion protein"), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009 / 0142354), an Ang2 antagonist (e.g., an anti- Ang2 antibody disclosed in US 2011 / 0027286 such as H1H685P), a FOLH1 (PSMA) antagonist, a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti- STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin antibody), etc.

[0464] Other agents that may be beneficially administered in combination with the protein- drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads of the disclosure include cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, [L-13, IL-17, IL-18, or to their respective receptors. The pharmaceutical compositions of the present disclosure (e.g., pharmaceutical compositions comprising an anti-HER2, an anti-HER2 / HER2 bispecific, an anti- MET, an anti-MET / MET bispecific, or an anti-STEAP2 protein-drug conjugate (e.g., antibody-drug conjugate as disclosed herein) may also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[0465] The present disclosure also includes therapeutic combinations comprising any of the protein-drug conjugates (e.g., antibody-drug conjugates), linker-payloads and payloads mentioned herein and an inhibitor of one or more of HER2, VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRVIII, cMet, IGF1R, B-raf, PDGFR-a, PDGFR-B, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CAS, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). The antigen-binding molecules of the disclosure may also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids and / or NSAIDs. The antigen-binding molecules of the disclosure may also be administered as part of a treatment regimen that also includes radiation treatment and / or conventional chemotherapy.

[0466] The additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of an antigen-binding molecule of the present disclosure; (for purposes of the present disclosure, such administration regimens are considered the administration of an antigen-binding molecule "in combination with" an additional therapeutically active component).

[0467] The present disclosure includes pharmaceutical compositions in which protein- drug conjugates (e.g., antibody-drug conjugates), linker-payloads and / or payloads of the present disclosure are co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein. Administration Regimens

[0468] According to certain embodiments of the present disclosure, multiple doses of a protein-drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti- MET / MET bispecific, or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or a payload may be administered to a subject over a defined time course. The methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of a protein-drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti- MET / MET bispecific, or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or a payload of the disclosure. As used herein, "sequentially administering" means that each dose of a protein-drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or a payload is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of a protein- drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an ant-MET, an anti- MET / MET bispecific, or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or a payload, followed by one or more secondary doses of the protein-drug conjugate (e.g., an anti- HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti- STEAP2 antibody-drug conjugate), linker-payload and / or payload, and optionally followed by one or more tertiary doses of the a protein-drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or payload.

[0469] The terms "initial dose," "secondary doses," and "tertiary doses,” refer to the temporal sequence of administration of the protein-drug conjugate (e.g., an anti-HER2, an anti- HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti-STEAP2 antibody- drug conjugate), linker-payload and / or payload of the disclosure. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses” are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the protein-drug conjugate (e.g., an anti-HER2, or an anti-HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or payload, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of the protein-drug conjugate (e.g., an anti-HER2, an anti- HER2 / HER?2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti-STEAP2 antibody- drug conjugate), linker-payload and / or payload contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[0470] In one exemplary embodiment of the present disclosure, each secondary and / or tertiary dose is administered 1 t0 26 (e.g., 1, 1%, 2, 2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21%%, 22, 22%, 23, 23%, 24, 24%, 25, 25Y2, 26, 26%, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of a protein-drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific, or an anti- STEAP2 antibody-drug conjugate), linker-payload and / or payload which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0471] The methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and / or tertiary doses of a protein-drug conjugate (e.g., an anti-HER2, an anti-HER2 / HER2 bispecific, an anti-MET, an anti-MET / MET bispecific,or an anti-STEAP2 antibody-drug conjugate), linker-payload and / or payload. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g, 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0472] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination. EXAMPLES

[0473] The following examples illustrate specific aspects of the instant description. The examples should not be construed as limiting, as the examples merely provide specific understanding and practice of the embodiments and their various aspects. Abbreviations ADC Antibody-drug conjugate Aglycosylated antibody Antibody does not have any glycan aq. Aqueous Boc N-tert-butoxycarbonyl! Thermo Scientific Prod# 28372, containing 100 mM sodium BupH | phosphate and 150 mM sodium chloride, potassium free, pH was adjusted from 7.2 to 7.6-7.8 MQ, unless otherwise noted. COT Cyclooctynol Da Dalton DAR Drug to antibody ratio DCM Dichloromethane ~ DIBAC ~ Dibenz[b flazocine, 11,12-didehydro-5,6-dihydro- DIBAC-Suc ~~ Dibenz[b,flazocine-5(6H)-butanoic acid, 11,12-didehydro "DIBACT ~~ 3H-Benzolc}-1,2,3-triazolo[4,5-¢][1lbenzazocine, 8,9-dihydro- “DIPEA ~ TDiisopropylethylamine DMF N,N-dimethylformamide DMSO Dimethylsulfoxide ELSD Evaporating light scattering detector Equiv. Equivalent ESI Electrospray ionization aq Gram GUL azide (((8)-5-(4-azidobutanamido)-1-carboxypentyl)carbamoyl)-L- glutamic acid 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium HATU hexafluorophosphate HC Heavy chain of immunoglobulin HEK Human embryonic kidney (cells) HPLC High performance liquid chromatography hr or hrs Hours Lc Light chain of immunoglobulin LC Liquid chromatography mg milligrams min minutes mL milliliters uL microliters mM millimolar uM micromolar MS Mass spectrometry MSD Mass-selective detector MW Molecular weight NHS N-hydroxy succinimide nM nanomolar NMR Nuclear magnetic resonance PAB Para-aminobezyloxy(carbonyl) PBS 10 mM sodium phosphate buffer and 150 mM sodium chloride PBSg 10 mM phosphate, 150 mM sodium chloride, 5% glycerol PEG Polyethyleneglycol ppm Parts per million (chemical shift) PPTS pyridinium p-toluenesulfonate RP Reversed phase RT room temperature Sat. Saturated SEC Size exclusion chromatography Suc Succinic acid MTBE Tert-Butyl methyl ether TEA Triethylamine TFA Trifluoroacetic acid TG Transglutaminase THF Tetrahydrofuran

[0474] Most starting materials were commercially available from Sigma-Aldrich®, J&K®, ChemExpress®, etc. The following compounds were synthesized according to corresponding references. Structure CAS Ref. oy | LP1-1 | | Rae. | W02018089373 | CoN a Nee soy ibe on | Lat | | erie | wozotaosess | OUR Oy we y™ | LP2-2 | =? 1, 19 ey -{ 3 oA | BRA | wozotsosesrs | Noo Syn NH ro hy AeA | LP2-2 | { ~Y SONY ON | ny ar | wozorasssars | "tn? [A N NH © 7 H 9 be Hy x AKT 4 | pas | Ly ~tongn A db LS | 22241228: | wozor — | H 1 £1, on” General Methods

[0475] EXAMPLE 1: Synthesis of camptothecin derivatives (payloads) Table 5: Exemplary camptothecin analog payloads P1-P4 according to the disclosure |#| Name | Structures |eLogP] MF | MW Fo miz | salt | 194) i Purity (%) NH | 438.1 > pf MH, P1|Exatecan| ro L145 |CashezF NOs CHi0S]531.56] 7 | oar MsOH| \ et [2M+Na] OHO Pp Dxd LOD | 055 | CasHzFNaOs lte3.4g] 100 | me | NA pea.ag] 100 | 494.2 [M+H] | ww Ho © Ss o c CCD | No 5% = 580.2 [M+H], TFA | 1159.2 [2M+H] odo Ee | 060 [CasHeoF N&O. C2HF:04693.56) 97 FINE hw | 5% 620.3 [M+H] OH O | arp emg? ° NH WF | 002 | CazHuFNsOr fp10.64 88 F N' \ 2 | 3G OHO

[0476] Payload P1, Exatecan mesylate, was commercially obtained from MCE. Payloads P2 and P3 were synthesized as described in WO02015155998, incorporated herein by reference, and camptothecin derivative payload P4 was synthesized as described in Scheme 1A and according to the synthetic steps outlined in Examples 1A-1E: Scheme 1: Synthesis of Camptothecin derivatives (payloads) P3 and P4 ° odor Ss Bor pitcl rt. 4h ra frReR =R" =H. A | MeOH. THE oR g Ra i 8 Ra FmocHN. AL Aor proaciy Dir Foc IL EI senayt aioe Fmsarin ALK a y ¥ r.4h Eo frRsR =R" =H A 8 benzyl glycolate, n.2h for R= H; RR" = {CH} 3 {Rw AC for R= H; RLR" = -{CH,}y-: P43, R= H; R, R= {CHyly- Pa-1, Rw H; R, R" % -(CHa)y- P42, Rw; RR wo{CHg)y- [07 R= Hi RR" = (CH 13 TMSCIL DCM, 1. 3 h 2) Benzyl giycolate, rt, 1 h 9 Rr FrmochN. I KR CH Ay fe] HATU. DIPEA. DMF LA ant 3 P34, ReR «R =H #25 P35, RR 2 R" 8 H NA mally Pape P44, R= H; R, R = {CH}; P45, R=H:R,R = CH)» OHO PLR=R =R"=H P4, R= HR, R ® J(CHy)y 2) Benzyl giycolate, rt, 1 h 3 ke ke FiniosHN. A N° HN A 0° hi h Rr NH Rr NH “SSR o i o _ ~ 0 en OCI v » NP, Som iE FANE , DME \_ / $ DhaF \_ / \ P35, RuR aR" w H No Hath Papa ACT P45 R=H; R,R = CH» OHO PI R=R=R'=H OHO P4, R= H; R, R ® o(CHy)ye

[0477] Example 1A: Synthesis of 9H-Fluoren-9-yimethyl N-[2-(2-hydroxypyrrolidin-1-yl)- 2-oxoethyl]carbamate (P4-2) Fmock™ Q oO OH (P4-2)

[0478] To a mixture of Fmoc-Gly-Pro-OH P4-1 (0.10 g, 0.26 mmol) in dry DMF (1 mL) was added lead tetraacetate (0.14 g, 0.31 mmol). The resulting mixture was stirred at RT for 30 minutes; reaction progress was monitored by LCMS. The resulting mixture was filtered through Celite, and the filtrate was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-10% ethyl acetate in petroleum ether) to give compound P4-2 (50 mg, 53% yield) as a white solid, and no acetate intermediate was obtained. ESI m / z: 389 (M + 23)*. H NMR (400 MHz, DMSO) 57.90 (d, J = 7.4 Hz, 2H), 7.73 (d, J = 7.5 Hz, 2H), 7.47-7.37 (m, 3H), 7.33 (t, J = 7.3 Hz, 2H), 5.86 (brs, 1H), 5.48 (d, J = 4.0 Hz, 0.25H), 5.39 (d, J = 4.0 Hz, 0.75H), 4.33-4.18 (m, 3H), 3.96 (d, J = 6.0 Hz, 1.5H), 3.75 (d, J = 6.0 Hz, 0.5H), 3.59-3.33 (m, 1H), 3.22- 3.11 (m, 1H), 2.00-1.59 (m, 4H) ppm.

[0479] Example 1B: Synthesis of Benzyl 2-{[1-(2-{[(9H-fluoren-9-ylmethoxy)carbonyl] amino}acetyl) pyrrolidin-2-ylJoxy}acetate (P4-3) o (P4-3) Oy [— ~~ Sedum S a

[0480] To a solution of compound P4-2 (0.30 g, 0.82 mmol) in DCM (25 mL) was added chlorotrimethylsilane (TMSCI) (0.27 g, 2.5 mmol). The reaction mixture was stirred at RT for 3 hours; reaction progress was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was diluted with DCM (25 mL). To the solution were added benzyl glycolate (0.27 g, 1.6 mmol) and DIPEA (0.21 g, 1.6 mmol), and the reaction mixture was stirred at RT for an hour; the completion of the reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by reversed phase flash chromatography (0- 100% acetonitrile in ag. ammonium bicarbonate (0.05%)) to give compound P4-3 (0.11 g, 25% yield, with purity >99% and 50 mg, with purity 75%) as a white solid. ESI m / z: 537.3 (M + Na)*. H NMR (400 MHz, DMSOqs) & 7.91-7.89 (m, 2H), 7.74-7.69 (m, 2H), 7.63-7.48 (m, 1H), 7.42- 7.25 (m, 9H), 5.51-5.00 (m, 2H), 4.35-4.21 (m, 5H), 4.00-3.77 (m, 2H), 3.52-3.38 (m, 2H), 3.30- 3.18 (m, 1H), 2.19-1.64 (m, 4H) ppm.

[0481] Example 1C: Synthesis of 2-{[1-(2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino} acetyl)pyrrolidin-2-ylJoxy}acetic acid (P4-4) F moc” Sp gr ~e & o (P4-4)

[0482] To a solution of compound P4-3 (82 mg, 0.17 mmol) in methanol (3 mL) and THF (7 mL) was added wet palladium on carbon (10% Pd, 20 mg) under nitrogen protection. The mixture was degassed and stirred under hydrogen balloon pressure at RT for 2 hours, and the completion of the reaction was monitored by LCMS. The reaction mixture was filtered through Celite and the filtrate was concentrated jn vacuo. The residue was purified by reversed phase flash chromatography (0-100% acetonitrile in ag. ammonium bicarbonate (0.05%)) to give compound P4-4 (36 mg, 49% yield) as a white solid. ESI m / z: 447.1 (M + Na)*.

[0483] Example 1D: 9H-Fluoren-8-yimethyl N-{2-[2-({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4, 15-diazahexacyclo[14.7.1.02,14.0¢,12.08,11.02°, 4]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-ylJcarbamoyl}methoxy)pyrrolidin-1-yl]-2- oxoethyl}carbamate (P4-5) FriocHN” Aone 185 i FON RE ~n (5 5 (P4-5)

[0484] To a mixture of compound P4-4 (63 mg, 0.15 mmol) and Exatecan mesylate (66 mg, 0.12 mmol) in DMF (2 mL) were added HATU (61 mg, 0.16 mmol) and DIPEA (46 mg, 0.36 mmol), and the mixture was stirred at RT for 2 hours; reaction completion was monitored by LCMS. The reaction mixture was directly purified by reversed phase flash chromatography (0- 100% acetonitrile in ag. ammonium bicarbonate (10 mM)) to give compound P4-5 (45 mg, 44% yield) as a yellow solid. ESI m / z: 842.3 (M + H)*.

[0485] Example 1E: 2-{[1-(2-Aminoacetyl)pyrrolidin-2-ylJoxy}-N-[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.08,11.020,24] tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yllacetamide (P4) 3,20(24)-heptaen-23-yllacetamide (P4) wns 0 cr HE] Sr Ry FP AXA, el pr 5S (P4) “ft reves iran DA B AR mam BA imal

[0486] To a solution of compound P4-5 (45 mg, 54 pmol) in DCM (4 mL) was added diethylamine (20 mg, 0.27 mmol), and the mixture was stirred at RT overnight. Reaction completion was monitored by LCMS. The reaction mixture was concentrated in vacuo and the residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound P4 (9.5 mg, 28% yield) as colorless oil. ESI m / z: 620.3 (M + H)*. Table 6, below, provides cytotoxicity and ADME (absorption, distribution, metabolism, and excretion) data for payloads P1-P3 according to the disclosure. Table 6. Cytotoxicity and ADME results of Payloads. R | NH : i an 0 LN Oo Nu me Ref# Structures HCT-15 [#] Refit ICso (NM) | traaae | ICso (NM) Ratio to MMAE R [ "NY MMAE -y RE | +y +v Ratio to MMAE =v MMAE | 14.810 1x 0.946 1x | P1 | Exatecan| H 1202 | oos7x | 1734 1.833x | Pe] Dxd COCH,OH 6.995 | 0472x | 2654 2.805% P3 | wimg | 44857 | 3011x | 22161 23.426x

[0487] Here “v" stands for without verapamil and “+v’ stands for with verapamil. Verapamil is a known inhibitor of P-glycoprotein and may function to block P-glycoprotein- modulated efflux.

[0488] Example 2: Linker2-Payloads

[0489] Example 2A: Linear Linker2-Payloads (LL2P)

[0490] Table 7, below, provides structures for exemplary linear linker2-payloads (LL2P) according to the disclosure. Table 7. Structures of Linear Linker2-Payloads (LL2P) # | (P) Structures Ee | pt | Pa So ; Cry te ANA Ao A ° wo] NY LIN i | Lp2 | Pa foo, , WS edRVe ass ans Fels i hn dpmonnann Sy oe ; prs © 2 H eben ALK Lak, oh Amidon dy, Y | pa | Pa | pa | P3| o Lo Lo o p Peryhprg yet 7 } pf ° i ° wo. X, tryed NY o Cr OH ANTS Z NS Shop BH TN Y o . o ode nA iad, { { Ho] : HN Y © pei pA i 7 CNH E 9 Q o ery =o | oY JJ F ee Hoy o H Q H 0 o AY x 7 Q © Pe So | oT ~ Tq hataastecaapTes T oO OH NN W o 0 W 0 Nop ey ° pe ° A oO PN 0 OH i ra : > H 2 9 H ¢ Neo . AP wg ° fA foo A o r 7 Qu xX Ho A Ng PS BH | LP12] Pa | NH ] A, 0... fo TY Q RL rv - Nm A, =Y ~ Not [P13] Pa | —_ = 3 0 og loo lA rss on 2OY & TY vl TT ~ 0 Sho, 5 | LP14] P3 | Y, ~ oo Sh | Lp1s| pa | Yo Ng Sy To To y v A Hook HA Op TaN AXA C TY ot ore] wendy oy 2G Ya | - : rm" [p17] Ps | Keds RE Or BEE 5 i, & “i £ 2 g Fd peg ON Lo TN PV. foil o . Co domi Tei 3 TTY yd | Pia | Ps | oY Arioons ery ! oF + yw YT [p19] pa | LL ~ 0 Sel, i = Lopes 3 | TE I TL TY = O ee Orgs fe g I 84 A La A [D / L joven] SYS YT fi co | Lp21 | P3 | YY ~ o CA if NH; 5 ppg Tf | 22] pa | On Fommmongied i LIC TY SB : 22 ELLE An a he XJ EL

[0491] Table 8, below, provides the chemical properties of exemplary linear linker2- payloads (LL2P) according to the disclosure. Table 8. Chemical properties of Linker2-Payloads (LL2P) Linker Purity Payloads Jd oF | miz # | re Payloads | cLogP | MF MW % name ( ) cor. = P3 |p] = ‘ (Gly- CogHgoF N11019.C2H 698.8 PEG4 NHoHo- | 072 | FiO: 51054 | 98 | wiz - AMY" boi iB eh athod liao 18] WILLD PEG4 NHcHo- | 072) Fs: | 151054 | 96 | ize | Dxd) biBAC. P38 _ | LP2| Jt SAN (Gly- . | 760.5 PEGE oho. | 148 | CnHuFNuOs | 151965 | 95 | zen 1.46 * ; | CoHotFNi2Ore | 1519.65 | 95 | oaize | Dxd) P3 COT- (Gly- 915.3 [es] SOF | Lov. | 1.84 | CuHaFNiOn | ota95 | so hn Dxd) P3 DIBAC- ~~ (Gly- 1128.4 jpg] DE | | 112 | CotFNOr [112816 | so (208 Dxd) COT- P3 1156.3 (Gly- EDA- (G- 573 | CuHeFN-On | 1156.14 | 97 | 1156.14 | NHCH,0- | “Vie | wseliszl INTA18 | 110. al >i (M+H) | Les | i | n B Dxd) COT- P3 (Gly- 066 | CoHuFNOw | 114216 | >95 11433 | 1142.16 | i PY Sunt EDA \HCH,O- | 088 | CesHoFN:Owo | 1142.16 | >95 (M+H) | ps EDA- (GLC)PAB | Dxd) Lp7| cot P4 pr| GaG | (@-Preo- | CasHssFNsOrz 955.3 Dxd) | (M+H) GGG | Dx) | rill 1001 INGVTZ (M+H) xd) DiBAC. | P4 Co 592.1 FY. 1 ides, |. | (Gly-Pro0- | Ce2HgsFN7O14 | (fragme 4p Miles, ips] Bias: | (Gly-Pro0- | CezHssFN:O14 | (rag Dxd) nt) P3 ly- (Gly ¥ CsaHs7FNsO12 1005.0 NHCH:0- | AS21571 INgVA12Z (M+H) | pe | cor-aer | NHGHO- | Dxd) cor. ps3 ~ (Gly- [P10] PABIGLC | NHGHRO- | = (Gly- 1156.3 BGLC | NHCHO- | CseHa2FN7O19 (M+H) ) way Dxd) P3 CoT- | (Gly CeeHaeFN-O1e 1142.3 NHCH:0- | ogliBal INAS (M+H) COT- (Gly- jun PAB(GLC) | NHCHZO- | Nv) Dxd) cor. Ps (Gly ¥ CssHasFNsO2o | 607.4 NE (Gly- 607.4 |LPiz| PAB(GLC | not CesHasFNsOz0 Wi2+H) P3 NH2- PEG2- ~(Gly- i CssH7oFN11O15 | 573.0 LX (Gly- PEG2- oo [Leal voPAR | NHCH: Aly WE PEG2- NHCH:O- | CssH7oFN11O15 | (M / 2+H) vePAB Dxd) P3 NH2- PEG2- (Gly 1 CeaoH77FN12018 | 637.5 NHCH,0- NRZ- © | PEG2 | NHCH.O- [cP OEvePAB | a PEG2- Rr CeoH77FN12018 V / o+H OEVCPAB | Nip | aie P3 NH2- PEG2- | ~(Gly- 1 CaoH77FN1201s | 637.4 NHCH-0O- NF2- (Gly- | 5| PEC2 NHGH,O- just | ‘EvcPAB | on Mi - Ci H77FN120 PEG2 NHCH:O- | CeoH77FN1201e | (M / 2+H) EVGPAB | 3k P3 Nz 6169 PEG4- (Gly | CsgH7sFN11017 J ont as [XL io PEG4- OLD. |Letel vepag | NHCH: | CseH7sFN11047 MIL | (M / 2+H) vcPAB WHE Sew Dxd) Mc(3)- an JA PEG4- P3 FAS (Gly- 692.4 |tP17| veraga- | So. CoshlssFN+2020 | wiz+th NHCHz- | NSH eh Dxd P3 |Lp1g| a NHCH:O- | Dxd) NH2- —— 6815 Nba 681.5 [Pt PEG4- P3 CesHasFN12020 | (M / 2+H) PEvVCPAB (Gly- NHCH-0- Dxd) COT. P3 763.5 Stir! |= (Gly: PEG4- |, ,., ‘J. |, -028 | C74Hg7FN 12022 1525.7 NHCH,0- COIT- |LP20 PEG4- OEVcPAB | Dxd) =” AND ol leh dol PE34 NHCH:O- | 0.28 | Cr4He7FN12022 1625.7 | | wzsh | EvcPAB Dxd) NH2- P3 - PR INP l&= (Gl - PEG4- GW 451 | CesHesFNuOs ~~ 1361.4 | | 681.4 NHCH-0- NHz- |LP21 PEG4- LEvcPAB | Dxd) = AMY" - Man: PEG4 NHCH:O- | 451 | CeaHasFN12020 1361.4 | | 2+ | EvcPAB Dx) COT- P3 ) _ = (Gl - PEG4- | GY gos | C74Ha7FN 12022 1525.7 LOT- Gh ] (Gly- } 763.5 |Lp22 FECL Noho. | 028 | CruHerFNuOz 15257 | 24H) EvcPAB D xd

[0492] *Mixture of lactone and ring-opening products. Values of MW and m / z are data of the corresponding lactone.

[0493] Example 2B: Branched Linker2-Payloads (BL2P)

[0494] Table 9, below, provides structures for exemplary branching units B1-B5 according to the disclosure. Table 9. Structures of Branching Units B1-B5 B1 B2 B3 B4 B5 5 wi | San ; . hh SN |, au By | ; | Structure UY | Ay dpa Ee C “5 4 ol B be i a # RS a,

[0495] Table 10, below, provides structures for exemplary branched linker2-payloads (BL2P) according to the disclosure. Table 10. Structures of Branched Linker2-Payloads (BL2P) name LP ¥ name | eLogP | Structures ENB BN Bug et odd Ade de afar tynpSetad pl ps 8 ht Ay 4 Ye ny LP = P23 es amr eo XT SC PEG4- GGFG- NHCH2- PEG2- vePAB-G- GGFG- NHCHz- Dx — Dxd ) 5 > = vp ordepedy fe roti Y gr Wd coT-B2- Wo Lh K ’ NH: oN A PEG2- i VCPAB-G- = 5, od CS y HCH2- . 0 enpredynpivoky 2m Diz Uk ey hye (I Q NAAT bt al toy FO | CH2- rv . o aeindooti, 5 pe fond & I OY 5 N i PEG2- "I 1 50 OP 2 Hy pfn dy, he | ge rd : eet 5 Dio at COL Ig y an iH ’ PEG2- tg 7 yw A DEVCPAB- OS NHCH2- Dxd]2 yz & y oo By LA i: — ert jy Th, x a A, O xX Looted 5% i WAT rhea Ye | argh EA S ory KS Ho Gh Xr ee ee : a. oo 3 ARE Pipi poe INH- PEG4- Po She ~ TL — mt mmm— TT. TIE Lo dy pram Ip IIE IS DIBAC- Pp FRY, cor suc-B2- - TE Xy ow ; INH- - 7 : PEG4- | ¢ Fon Ss 0 Snir 33 y & x 1 grriealy S b Dxd]2 bors db A ICTTY ye of PAB-G- DRY Cow Tote d. (5m Vora SOY [NISUcaasaasdsttol Dxd]2 bpm Ad Os ox Ll, “ vePAB-G- i, NN “ - Te EEE iat eo aN oe orp Ae $- mn I SF vy A A | B3-NH- hid WL or PEG2- i” re yo a Tee rent NHCH2- oi 2 of 3.03 L Qo rare $e Dxd]2 do A Xr RCT wy An vo 0 befall , a NHCH2- Cal iota d 533 C gr moi dy, a0 ¥ Dxdj2 I ALT ry Poles fel, x She, ~ | LP28 rp EF rey be 2 | core 5 SGA 3 he 3 yp ar She - PEG2- pdm) DEVCPAB- IN i of aE Hind oo | wcHz- ap & oh 4 5. Dxd]2 : | aLP29 INH- gt Wik oo PEG2- 2 fat as LA, xy DEVGPAB- pdm hi i G- s yr ¢ «7% | NHCH2- * Wr ad Lyra SE ra On mag iE Sa, 5 x oa’ Mg Dxcl2 ergy oy ho She pe NF2- Heer PEG3- Cee pepe TCOT-B3- Fe Lr IS Sat | INH- | stems os do LE xr o | 0, Degen nd] es y PEvCPAS: v ia ae - termi, I LOT NHCH2- GL ar | Dxd2 A x epg gripe le, gro ld 8 ALS pT | | agri 2 eT a LE ao ry " prestige 3 AI i ¥ TOT ony oy a So ye J CR dab 53. | dio X > eo | DIBAC- snp fl Hey 2S suc-B3- ey . ¢ 5 i AL xr INH- ZL Torr oo be y PEG2- OA Aimminn Ca LP30 | og cpas- | I Ls od, { . | | < orn OY Ph NHCH2- TT o Pati oo AN Y | LP31 toed, 3 erg I | Lr ab Te LA x LEVCPAB- | HATO ) . ) > i rs 3 Pyroedy fd | Dep Lorna 4 Oy SE xX ied, SS TIvTCr, LE An pe A Y | Lpaz2 o i % 3 EY. § of egypt SN COE opi A Ley x sal | INH- ; ! 3 & “i (LE PEG2- , Out Poy A J eo a Cy Arsnaond oor s ’ of (Fy 8 ppp d 3. 2 3 . w hh eo hy Cmoranghal 5 AAS TX OE [3 5 © yo pad - Ea Ni ¥ NH2- PEGS- TCOT-B3- [NH- 3 PEG2- LEVcPAB- G- NHCH2- Dxd]2 NH2- PEG3- 2 4 TCOT-B3- eh gC pe INH- | resem Foegeroes Ct opm + | aLpas | pecz. HEE eS non] all * LEVCPAB- TTT i : yor, a predped 3g So wan SE A LITTER Sly r cr PIETY ? Seats | >on 4 AF | -3.41 Frees Heys | 8 7 x Wo Ht ; ™ tI, Agony pi rT on FEE Ady | ; TE XE Aa omg tn 2 | -4.94 COT-B3- [NH- PEG2- GGFG- NHCH2- Dxd]2 mt ATH Be 3 5 Tr Yr A Bee i rn b Ba pHi I‘ 0 XE | ~ / Lo ~ ram HP id : 4 A yf Fore oS | “eo . _& 3,8 Rotnd, pe | nu ’ , ” 2 to Hongo rAd A 8” ; ; Y Ye % fo . re Nh INH- oo py oe ice: [359 (7 iY ; yr; ore Lh | NrcHz- Tomah . & : oH Dxd]2 I ¥ B a 5 & N To Set . 4 GA Jo J Cg a : ori ’ ~~ i 5 ~ 3 ok 3- © thn 2 OY LER TY ce oor He Nh x Co fy a1 32 | il, tok, > {> “ | “fmoniny red, Xo LT 5 + =r Yy 2) 7 LTT, EG2- a » hi | -3.59 Ty ARS | tod " $i | CH gli tf, Ct. & NY CAH Dxdj2 T Le Mo Nga! din de, cor- ont I~ ATTY fy | PEG2:B4- | yh ry re ia INH- 3 § Ao X PEG2- ABE moms IL wr I LEVCPAB- | : LW ~ oto dl, 3 | a 2 $Y 4 8 Cry pl HE, Formed Ls. 2 AJ Y eats | LP39 0 erate 3 me _ tgp LOY x 35 | PEG2:B4- | . ob He i. & [NH- ao gr Lf ’ Y CEVCPAB- | 2.63 Ot antes et . 7 - A - Ye ICTY 257 Dxdj2 Telly Cr * ory wl cort- Yoyo ott PEG2:B4- | , ch Pe NA ! NH oS AS o yw Hore | 2.63 Oye > , ) Ys | S ore Sony on oye Ay Se J Ag Ao Se : = AHA 2 | TCOT- | Y ttt J lk 4, 5 PEG2-B4- HEN SLC i “I 0 |e | 2 OH, 4 30 Re Arion. a] S TdT | NHCH2- 3 UA, oF A ® 5 A co Hoo Ay, S30 SAA Gr | ory 5, 14 Foe Gh AJ HP g rt Ie Ny Shitting 1 Smt, A, (Se - Ave 47 bey SA | Fo hw ke ATC e rp EY se 5 SOL, y . os Y ) Son vr fied, PEG2-B5- | . ¢ os 0 4 ry he NH- J mmm fon Hamp? Hey ¥ Uk Pecz. 884 (Ty heey TY ¥ LA, x LEVCPAB- | “© [ 3 w: . Y G } 3 NHCH2- % sp preg, 3% Dxd]3 Y. wm o ’ &- | oo ~~ S A wn Se, wy? Dxdll3 3 : a « ? kW Layo preyed oe mph TY x 538 pe Lh % NH2- oat Xp ord A PEG3- SERRE TT Se TE9% ES Td or PEG2-B5- i : oe wngty L , fo te Sp 2 | aLP42 | peg, | -11.22 | i ordre £30 857 Lh Tas - TT Sry oo EY el Ire NT NH2- oe SANE SA 3 oy 3, PEG3- 7 oe pi x TCOT- tym : ] Poss. grip | n LA vpn, yp ppdgngonody fe | - . of cage pen Tn ET 3 Xa, PEe2. | 1122 | She a Sy an LEVCPAB- Jha s wu 3 ) wn reer Bondy BT NHCH2- soni bn ETT Dxd]3 TY GA soo nih deed wnt tp tr pe 2g wha Yon LY

[0496] Table 11, below, provides the chemical properties of exemplary branched linker2- payloads (BL2P) according to the disclosure. Table 11. Chemical properties of Branched Linker2-Payloads (BL2P) # LP name | eLoaP | MF MW # LP name | cLogP MF MW Mass | Lp23 | COPE COT 2.17 | CrazHiusFaNzzOus | 2658.9 | 867.6 (M / 3+H) COT-B2-[NH-PEG2-vcPAB- | Lp24 { SE 1.80 | CraaHissFaNmOu | 2549.7 | 850.8 (M / 3+H) LP1 = GGFG-NHCH2-Dxd LP2 = NH2-PEG4-GGFG- NHCH2-Dxd COT-B2-[NH-PEG2-vcPAB- 1.80 G-NHCH2-Dxd]2 ! | COT-B2-[NH-PEG2- 3.80 PEvcPAB-G-NHCH2-Dxd]2 : | Lp27 COT-B2-[NH-PEG2- [pas | RE 3.80 | CrouHoF:N2sOo | 2808.0 | 936.7 (W3+H) 717.3 (WI4+H) DIBAC-suc-B2-[NH-PEG4- | _ (ring-open form) [Pos | erect | 1.25 | CuushraFaNaiOss | 2849.1 0 latone form B3-[NH-PEG2-vcPAB-G- [p27 | { -C2VCPABC, 422 | CraaHisoF2N2sOss | 2560.8 1280.9 (Wi2+H) | COT-B3-[NH-PEG2- [Lpos | SE ap FEE “4.15 | CuuatisaF2NutOss | 2983.2 | 995.2 (W / 3+H) | B3-[NH-PEG2-vcPAB-G- 4.22 NHCH2-Dxd]2 | | COT-B3-[NH-PEG2- 4.15 PEVcPAB-G-NHCH2-Dxd]2 : NH2-PEG3-TCOT-B3-[NH- | PEG2-PEVcPAB-G-NHCH2- | -8.21 Dxd]2 | pas NH2-PEG3-TCOT-B3-[NH- | 9LP29 | PEG2PEVCPAB-G-NHCH?: | 8.21 | CastaosFaNOuo | 3201.4 | S012 THAZHY: Dxd]2 S{3uH) 786.4 (W / 4+H) (both ring-open); 781.8 (V / 4+H) DIBAC-suc-B3-[NH-PEG2- [Pao | PESELBINIEE | ve | CussHurFaNerOuo | 3108.3 fn 1036.2 (M / 3+H) | lactone B3-[NF-PEG2-EVePAB-G- | pas { 2IEVCPABC, 8.57 | CreaHiraFaNsOu | 2619.0 940.5 (V / 3+H) COT-B3-[NH-PEG2- Paz | ee | 418 | CraaHussFsNasOsc | 2083.2 | 995.5 ( / 3+H) POET ™ fe Sd l= Sgt gl: C2 Read ™[ PREP | ape | PEG2--EvcPAB-G-NHCH2- | 8.21 | CrsoMz0¢F2N3oOus 3201.4 801.0 (M / 4+H) Yrie- B3-[NH-PEG2--EvcPAB-G- 857 NHCH2-Dxd]2 : | COT-B3-[NH-PEG2- 4.15 LEvcPAB-G-NHCH2-Dxd]2 : NH2-PEG3-TCOT-B3-[NH- | PEG2-'EvcPAB-G-NHCH2- | -8.21 Dxd]2 [Pas | DIBAC-suc-B3-[NH-PEG2- 3.41 LEvcPAB-G-NHCH2-Dxd]2 : | Lpas 786.5 (M / 4+H) (both ring-open), 1042.3 (M / 3+H) | Cis1H187F2N27043 | 3106.3 | (mono ring- open), 1036.1 (M / 3+H) | lactone | CraoessFaNasOso | 2436.6 | 812.7 (M / 3+H) COT-B4-[NH-PEG2- | Lp3s SE ob | 2% | CrasHiroFsNasOsz | 2010.1 | 970.8 (M / 3+H) COT-B4-[NH-PEG2- [Lpar | EE rey -3.59 | CrasHirsFaNzsOuz | 2010.1 | 570.6 (Wi3+H) us - HaooF 2NasO. | 3157.4 936.7 (M / 3+H) | LPas | LEVCPAB-G-NHCH2-Dxd]2 | “+55 | Cras 2 Near COTPESIBANA 14 HiasF2N4O: | 27508 917.3 (M / 3+H) [Lp3o | LEVCPAB.GNHeio ot | 2:63 | CraaHisaFaN24Osa | COT-B3-[NH-PEG2-GGFG- 4.94 NHCH2-Dxd]2 : COT-B4-[NH-PEG2- 3.59 PEVCPAB-G-NHCH2-Dxd]2 : | COT-B4-[NH-PEG2- 3.59 LEVCPAB-G-NHCH2-Dxd]2 : | COT-PEG2-B4-[NH-PEG2- 455 'EVCPAB-G-NHCH2-Dxd]2 : | COT-PEG2-B4-[NH- 263 LEVCPAB-G-NHCH2-Dxd]2 : NH2-PEG3-TCOT-PEG2- | apo | B4INH-EVCPAB-G- | 6.69 | CrioHresFaNasOiz | 2069.2 | 991.2 (M / 3+H) NHCH2-Dxd]2 COT-PEG2-B5-[NH-PEG2- | Lpat LEVCPAB.G-NHCH2-Dxal3 | 6:84 | CarohersFaNosOss | 4426.7 | 1107.6 (M / 4+H) NH2-PEG3-TCOT-PEG2- | aLp4z | B5-[NH-PEG2--EVCPAB-G- | -11.22 | Carel FaNiaOss | 4644.9 | ts NHCH2-Dxd]3 :

[0497] EXAMPLE 3: Synthesis of vcPAB-carbamate linker-payloads

[0498] Linker-payloads LP1 and LP2 were synthesized as described in Scheme 2 and in Examples 3A-3C (for LP1) and 2D (for LP2), below. Starting materials L1-1 (CAS 2226472-26-8) and L2-3 (CAS 2226472-28-0) were synthesized according to WO2018089373A2, incorporated by reference herein in its entirety. Scheme 2. Synthesis of vcPAB-carbamate linker-payloads NL 0 eon i A - NT y LJ WL ho TY Ne oo o oy H a H Sr on Ll, Apo d l LIAS Toa a cPAB WH T H Shoes : SE in. Bf SH a Tm Cy Aro TD Ah ° Ca TB DFEADME Rh oY DMAP. DIFEA, DMF Li L12 Ld tt. overnight L214 12.2 boo o it gg? H Ys Angra? Lp J J, Nod Ps OH LIPEA. DMF NO: ) Ys 8 orl ribo yr § NF ~ og 1 In it, overnight L432 ~ H L23 2 Q mod 9 WH 2 al egy wien te (CF 2 0 Cre oH, 5, H J 2 Ay Abd (Xx wpa, = 3 vv W Wo LP *- o © Sy? “NH, H LP2

[0499] Example 3A: N-[(15)-1-{{(1S)-4-(Carbamoylamino)-1-{[4-(hydroxymethyl)phenyl] carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amide (L1-2) o Lu a Sy ok ANOS Aone — sony ry Not By TH ~ 0 SP y pe (L1-2)

[0500] To a solution of compound L1-1 (0.17 g, 0.33 mmol) in DMF (10 mL) were added DIPEA (0.13 g, 1.0 mmol) and vcPAB (0.13 g, 0.34 mmol) successively, and the reaction mixture was stirred at RT for an hour. Reaction completion was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-80% acetonitrile in water) to give compound L1-2 (0.18 g, 70% yield) as a colorless oil. ESI m / z: 791.3 (M + H)*. '"H NMR (400 MHz, DMSQus) 5 9.91 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.61 (t, J= 56 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 5.98 (t, J = 5.6 Hz, 1H), 5.42 (s, 2H), 5.10 (brs, TH), ), 4.43 (s, 2H), 4.39-4.37 (m, 1H), 4.30-4.21 (m, 2H), 3.87 (d, J = 14.8 Hz, 1H), 3.75(d, J = 14.8 Hz, 1H), 3.62-3.58 (m, 2H), 3.50-3.46 (m, 12H), 3.43 (t, J = 6.0 Hz, 2H), 3.27- 3.22 (m, 2H), 3.06-2.92 (m, 2H), 2.41-2.32 (m, 2H), 2.26-2.05 (m, 3H), 1.99-1.66 (m, 6H), 1.62- 1.55 (m, 3H), 1.44-1.35 (m, 3H), 0.89 (d, J = 6.8 Hz, 3H), 0.83 (d, J= 6.8 Hz, 3H) ppm.

[0501] Example 3B: {4-[(2S)-5-(Carbamoylamino)-2-[(28)-2-{1-[2-(cyclooct-2-yn-1-yloxy) acetamido]-3,6,9, 12-tetraoxapentadecan- 15-amido}-3-methylbutanamido]pentanamido] phenyl} methyl 4-nitrophenyl carbonate (L1-3) SoA 0 Ne JL 2 DED BORA FEO. Ow. 0 oe ; No, A AY Ap gn ~Ay y Ay 2 Ses ~ 2 Sy SNM, - Ho (L1-3)

[0502] A suspension of compound L1-2 (80 mg, 0.10 mmol), DMAP (12 mg, 0.10 mmol) and DIPEA (26 mg, 0.20 mmol) in dry DMF (5 mL) was stirred at RT for 10 minutes before the addition of bis(4-nitrophenyl) carbonate (61 mg, 0.20 mmol). The reaction mixture was stirred at RT for 2 hours. Reaction completion was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-80% acetonitrile in water) to give compound L1-3 (53 mg, 55% yield) as a white solid. ESI m / z: 956.3 (M + H)*.

[0503] Example 3C: {4-[(2S)-5-(Carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1- yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido] phenylimethyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0%,14.04,13.08,11.020 #]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- ylJcarbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1)

[0504] To a yellow solution of compound L1-3 (16 mg, 17 pmol) and P3 (12 mg, 17 umol) in dry DMF (2 mL) was added DIPEA (6.5 mg, 51 umol), and the clear reaction solution was stirred at RT for 2 hours. Reaction completion was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-60% acetonitrile in ag. TFA (0.01%)) to give linker-payload LP1 (15 mg, 63% yield as TFA salt) as a white solid. ESI m / z: 698.8 (M / 2 + H)*. TH NMR (400 MHz, DMSOqe) 5 9.99 (s, 1H), 8.80 (t, J=6.8 Hz, 1H), 8.50 (d, J =9.2Hz, 1H), 8.12 (d, J= 7.2 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.62- 7.58 (m, 3H), 7.42 (t, J = 6.0 Hz, 1H), 7.31 (s, 1H), 7.28 (d, J = 8.4 Hz, 2H), 6.53 (brs, 1H), 5.98 (t, J= 5.2 Hz, 1H), 5.63-5.57 (m, 1H), 5.46-5.37 (m, 3H), 5.21 (s, 2H), 4.93 (s, 2H), 4.63 (d, J = 6.4Hz, 2H), 4.41-4.35 (m, 1H), 4.29-4.21 (m, 2H), 4.02 (s, 2H), 3.87 (d, J = 14.4Hz, 1H), 3.75 (d, J = 14.8 Hz, 1H), 3.63-3.58 (m, 4H), 3.50-3.48 (Mm, 12H), 3.46-3.41 (m, 2H), 3.27-3.24 (m, 2H), 3.23-3.12 (m, 2H), 3.07-2.91 (m, 2H), 2.47-2.45 (m, 0.5H), 2.41-2.33 (m, 4.5H), 2.25-2.04 (m, 5H), 1.99-1.69 (m, 9H), 1.63-1.54 (m, 3H), 1.44-1.33 (m, 3H), 0.88-0.82 (m, 9H) ppm. (The proton of TFA was not observed). "°F NMR (376 MHz, DMSQOus) 5-74 (TFA), -111 (Ar-F) ppm.

[0505] Example 3D: {4-[(2S)-2-[(28)-2-[1-(4-{2-Azatricyclo[10.4.0.0% {]nexadeca- 1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9, 12-tetraoxapentadecan-15- amido]-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,235)- 10-ethyl-18-fluoro- 10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo [14.7.1.0%,4.04,12.08,"".0%,*|tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl carbamoyl} methoxy)methyllcarbamoyl}methyl)carbamate (LP2) * A XL OT Hyiedy =r Are A! A) he, ~ LA N” NH; N 2

[0506] Following the procedure to make LP1 except substituting L2-3 for L1-3, linker- payload LP2 (12 mg, 46% yield) was obtained as a mixture of the lactone product (LP2, pictured above) and the ring-opening product (LP2-RO, pictured below) as a white solid after purification by reversed phase flash chromatography (0-100% methanol in ag. ammonium bicarbonate (10 mM)). , y o ( a : = ) 6 a dM N. . i’ ie = mmo 1 f or "( = 5 AA ALL = 9 BY 4 9 o£ oO a MC - Da of g o Se {LP2-RO)

[0507] Lactone LP2: HPLC purity: 67%, retention time: 7.41 min, ESI m / z: 507.3 (M / 3 + H)*, 760.5 (M / 2 + H)*; Ring-opening product LP2-RO: HPLC purity: 33%, retention time: 6.61 min, ESI m / z: 513.3 (M / 3 + H)*, 769.5 (M / 2 + H)*.

[0508] Lactone product and ring-opening product mixture 'H NMR (400 MHz, DMSQOqe) & 9.99 (s, 1H), 8.80 (t, J = 6.4Hz, 1H), 8.50 (d, J = 8.8Hz, 1H), 8.12 (d, J = 7.2Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.80-7.75 (m. 2H), 7.69-7.67 (m, 1H), 7.63-7.58 (m, 3H), 7.51-7.46 (m, 3H), 7.45 7.33 (m, 3H), 7.32-7.26 (m, 4H), 6.53 (s, 1H), 5.98 (t, J = 6.0 Hz, 1H), 5.63-5.57 (m, 1H), 5.42 (s, 4H), 5.21 (s, 2H), 5.03 (d, J = 14.0 Hz, 1H), 4.93 (s, 2H), 4.63 (d, J = 6.8 Hz, 2H), 4.41-4.35 (m, 1H), 4.25-4.21 (m, 1H), 4.02 (s, 2H), 3.62-3.57 (m, 5H), 3.48-3.45 (m, 12H), 3.31-3.28 (m, 2H), 3.23-3.14 (m, 2H), 3.11-3.07 (m, 2H), 3.05-2.98 (m, 1H), 2.96-2.91 (m, 1H), 2.60-2.55 (m, 1H), 2.46-2.44 (m, 1H), 2.39 (s, 3H), 2.35-2.33 (m, 1H), 2.26-2.15 (m, 3H), 2.03-1.94 (m, 2H), 1.88- 1.67 (m, 4H), 1.63-1.57 (m, 1H), 1.46-1.33 (m, 2H), 0.88-0.81 (m, OH) ppm. "SF NMR (376 MHz, DMSOgs) 6-111 ppm.

[0509] Linker-payloads LP16 and LP17 were synthesized as described in Scheme 3 and in Examples 3E-3F (for LP16) and 3G (for LP17), below. Scheme 3. Synthesis of LP16 and LP17. A LL wo TRAE oy Al Tan Lo 2 EigNH. DME, 11. 2 h, Nyy LGR 0 0 Ye > PENN WL wo w 3 TRY Y Loam © Ne ’ boi, S ~ SN No Sm, 853971-51.3 AAI eS DIFCA, CMF a th Soe owe og EW . oo % repay 2X o° Apert el LAAS : oY 0 CECE A Sha, - r Sit eg” LPIBE Y « Fmoc tI Neg, Hp wpa ys piety

[0510] Example 3E: {4-[(29)-2-[(2 S)-2-Amino-3-methylbutanamido]-5- (carbamoylamino)pentanamido]phenyl}methyl N-({[({{[(108,23S5)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-0xa-4,15-diazahexacyclo[14.7.1.02,14,04,13,08,11.02 24]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methylJcarbamoyl}methyl) carbamate (LP16-1) ° Heh HAN Sg ” Noy " wo ZI Q Cr a A WK ANE NNR Li F =

[0511] To a solution of payload P3 (0.11 g, 0.15 mmol) in DMF (2 rns added DIPEA (39 mg, 0.30 mmol) and Fmoc-vePAB-PNP (CAS: 863971-53-3, 77 mg, 0.10 mmol), and the reaction mixture was stirred at room temperature for an hour until the mixture turned clear and P was totally consumed according to LCMS. The resulting solution was separated by reversed phase flash chromatography (0-70% acetonitrile in ag. TFA (0.01%)) to give Fmoc-LP16-1 (98 mg, ESI m / z: 494 (Moxo + H)*, 714.2 (M — Moxo + H)* as a light yellow solid, which was dissolved in dry DMF (4.5 mL). To the solution was added diethylamine (0.5 mL) slowly, and the reaction mixture was stirred at room temperature for 2 hours until Fmoc was totally removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-40% acetonitrile in ag. TFA (0.01%)) to give LP16-1 (TFA salt, 45 mg, 41% yield from P3). ESI m / z: 493.1 (M / 2 + H)*. TH NMR (400 MHz, DMSO) & 10.20 (s, 1H), 8.80 (t, J=6.8 Hz, 1H), 8.68 (d, J = 7.6 Hz, 1H), 8.50 (d, J = 8.8 Hz, 1H), 8.09-8.01 (m, 3H), 7.79 (d, J = 10.8 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 6.0Hz, 1H), 7.31 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 6.53 (s, 1H), 6.05 (t, J = 5.6 Hz, 1H), 5.63-5.57 (m, 1H), 5.54-5.45 (m, 2H), 5.42-5.41 (m, 2H), 5.21 (s, 2H), 4.93 (s, 2H), 4.63 (d, J =6.4 Hz, 2H), 4.55-4.50 (m, 1H), 4.02 (s, 2H), 3.69-3.61 (m, 3H), 3.24-3.12 (m, 1H), 3.08-2.94 (m, 2H), 2.40 (s, 3H), 2.23-2.18 (m, 2H), 2.14-2.03 (m, 1H), 1.90-1.81 (m, 2H), 1.78-1.68 (m, 1H), 1.84-1.53 (m, 1H), 1.47-1.36 (m, 2H), 0.96 (d, J = 3.2 Hz, 3H), 0.94 (d, J = 3.2 Hz,, 3H), 0.87 (t, J = 7.6 Hz, 3H) ppm. (Proton of TFA was not revealed.) "°F NMR (376 MHz, DMSQqe) 6 at -111, -73 ppm.

[0512] Example 3F: {4-[(25)-2-[(2S)-2-(1-Amino-3,8,9,12-tetraoxapentadecan-15- amido)-3-methylbutanamido]-5-(carbamoylamino) pentanamido]phenyl}methyl N-({[({{(10S,235)- 10-ethyl-18-fluoro-10-hydroxy-198-methyl-5,2-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,12.08,11.02° #]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- ylJcarbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP16) rep IX : x ike I~ . { i or - ‘ . a N, (Jo he xX 2 > ea A I y <Q ; th a C : A 4 Fa Ho F

[0513] To a solution of LP16-1 (16 mg, 15 umol) in anhydrous DMF (1 mL) — DIPEA (4 mg, 29 pmol) until pH value between 8.0 and 9.0, and then a solution of compound If (CAS 1314378-14-7, 9 mg, 15 pmol) in anhydrous DMF (1 mL) was added into the reaction solution. The mixture was stirred at room temperature for an hour until starting materials were totally consumed according to LCMS. The resulting solution was separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP16f (18 mg, ESI m / z: 728.3 (M / 2 + H)*) as a white solid, which was dissolved in anhydrous DMF (1.9 mL). To the solution was added diethylamine (0.1 mL), and the yellow reaction solution was stirred at room temperature for half an hour until Fmoc was totally removed according to LCMS. The resulting solution was concentrated in vacuo and the the residue was purified by prep-HPLC (10-95% acetonitrile in ag. formic acid (0.01%)) to give linker-payload LP16 (6 mg, 34% yield) as a light yellow solid. ESI m / z: 616.9 (M / 2 + H)*. "H NMR (400 MHz, DMSOye) & 10.03 (s, 1H), 8.80 (d, J =6.8 Hz, 1H), 8.51 (d, J= 9.2 Hz, 1H), 8.41 (s, 1H), 8.15 (d, J = 6.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 6.8 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 6.4 Hz, 1H), 7.32 (s, 1H), 7.27 (d, J = 8.0 Hz, 2H), 6.56-6.50 (m, 1H), 6.05-6.01 (m, 1H), 5.63-5.58 (m, 1H), 5.43 (s, 4H), 5.21 (s, 2H), 4.93 (s, 2H), 4.63 (d, J = 6.8 Hz, 2H), 4.42-4.35 (m, 1H), 4.23 (t, J = 7.6 Hz, 1H), 4.02 (s, 2H), 3.63-3.58 (m, 4H), 3.53-3.49 (m, 12H), 3.46-3.43 (m, 2H), 3.18-3.15 (m, 2H), 3.06-2.93 (m, 2H), 2.79-2.76 (m, 2H), 2.42-2.36 (m, 5H), 2.24-2.12 (m, 2H), 2.03-1.93 (m, 1H), 1.89-1.81 (m, 2H), 1.74-1.65 (m, 1H), 1.65-1.54 (m, 1H), 1.48-1.31 (m, 3H), 0.89-0.82 (m, 9H) ppm. "°F NMR (400 MHz, DMSO4s) 5-111 ppm.

[0514] Example 3G: {4-[(2S)-5-(Carbamoylamino)-2-[(2 S)-2-{1-[3-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)propanamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3- methylbutanamido]pentanamido]phenylimethyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.08,11.02°, #]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methylJcarbamoyl}methyl) carbamate (LP17) a o H o 5 ° i Cry ) AO gO A Ay F 2 Ny NA H " Hog H L 0 ¥ = a 7 B nN Voy PR = oO Sete, H (LP17)

[0515] To a yellow solution of LP16-1 (30 mg, 30 pmol) in anhydrous DMF (1.5 mL) were added DIPEA (8 mg, 62 umol) and la (CAS: 756525-89-2, commercial, 16 mg, 30 umol), and the reaction mixture was stirred at room temperature for an hour until starting materials were totally consumed according to LCMS. The resulting mixture was directly purified by prep-HPLC (0-100% acetonitrile in ag. TFA (0.05%)) to give LP17 (TFA salt, 15 mg, 38% yield) as a light yellow solid. ESI m / z: 692.4 (M / 2 + H). 'H NMR (400 MHz, DMSQus) 5 9.99 (s, 1H), 8.79 (t, J = 6.8 Hz, 1H), 8.50 (d, J = 8.4 Hz, 1H), 8.11 (d, J=8.0 Hz, 1H), 8.02 (t, J = 6.0 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 6.0 Hz, 1H), 7.32 (s, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.00 (s, 2H), 6.52 (br s, 1H), 5.98 (t, J = 6.0 Hz, 1H), 5.63-5.57 (m, 1H), 5.43- 5.41 (m, 4H), 5.21 (s, 2H), 4.93 (s, 2H), 4.63 (d, J = 6.8 Hz, 2H), 4.43-4.34 (m, 1H), 4.25-4.21 (m, 1H), 4.02 (s, 2H), 3.61-3.57 (m, 6H), 3.49-3.48 (m, 12H), 3.21-3.12 (m, 4H), 3.05-2.91 (m, 4H), 2.49-2.39 (m, 5H), 2.33 (t, J = 7.2 Hz, 2H), 2.22-2.14 (m, 2H), 2.02-1.94 (m, 1H), 1.89-1.80 (m, 2H), 1.75-1.65 (m, 1H), 1.65-1.54 (m, 1H), 1.49-1.32 (m, 2H), 0.87-0.82 (m, 9H) ppm. (Proton of TFA was not revealed.) °F NMR (376 MHz, DMSQOus) 6-111, -73 ppm.

[0516] Linker-payloads LP1, LP2, LP13, LP14, LP15, LP19, LP20, LP21, and LP22 were synthesized as described in Scheme 4 and in Examples 3H-3AR, below.

[0517] Scheme 4. Synthesis of linear vcPAB-Linker-P3 LP1, LP2, LP13-LP15, and LP19- LP22 Hi 2 vd fa ea LPI. m=4,Y=2COT [ ] AN. i w 3 TO yA I on io . ] LPI 22, Eros c N HE i Ay Z PEA mony erm Ya, 0. Re { 2 Jol ; 3 H Lorch ede cor N y AN A A iC . No _teimesvegme | H + i N ~ i HATU, DIPEA, DMF . SN 0 oO Sv nme Wah A or Ng NT NH, ~ 0 BELLA LIE, o 4, 4 © No AL ATU, DIPEA, DHF AL 7 he: man Soe NE, 17 BOGGS UL iieOF, 2 Dall pes, pyr LP15-2, n= 1 * 8 m=2 Y= Fmoe HATU. DIPEA. DMF CEvePAB n= 1, Re 3h LP12, 020 m=4.Y=COT 4. 3h TN og Aig MN NE LE LP13.2,n=0,m=2Y =Fmoc ~ vePAB, n= 0 1) BoC“ Gli OMe}-CH. LPt4-2, n=“ R- m=2Y =Fmoc ATU. DIPEA DMF | OFyopAB. n= 1, R- 3h LP12,n=0m=4,Y=COT t.3h 2) TFA, DCM. 1. 3 h. LP2-Z, n=0 m=4,Y = DIBAC 2) TFA, DCM, 21, 2 h, TET) LP19-2, n=,“ R m=4,Y = Fmoc 4.3h. N 2) TFA, DCM. 1. 3h, LP22,n=0,m=4,Y = DIBAC 2 TPA, DCM, 1, 2h % LP19:2,n=1,% R m=4,Y = Fmoc ) LEvePAB, n=1," § EE yt CTE LP20.2. n=1,"R-m=4,Y=COT P22. n=1, "8. m=4, Y= Fmoc LP22-2 n=1, "= S.m=2& Y=COT “2 1 ia i An OTE Ey wr, 2 SAS ENT te a Ne, TR Bi, DBE ThE Nv CT OR DIPEA. HOBY, DMF 9 s LB 1.2h a PN nA ie wy: He Xu LO oe 7 H + TELA, So ~o pa ~ © Sy Led far Y = Foe, = 0: Et,NH, DMF. rt. 1 h tory = Fmoc n= 1 JorY = COT ar DIBAC n= 1 Eh Sz N £9. LIOR THE 1, 2h, w No Sg LP#-4 Bh Tor'Y = Frc, v= 0: Evi. DME. 2. 1h, for ¥ = Foe, n= 3 . Q 1 piperidine, DAF. © h 1 K od, 3 Up 780 LOH THE 1 Q SN Sar f . N o 3 Y 4 A ory ¥ iY us PO +4 for = COT ar DIBAC. n = 0 skip his step VS Neg L. HO NYY Ey mg Uc 4 LE N LOB THE 1.21 Soo Sy on ¥ Q a H ol 0 NS Se a YO He 8, AS Hoo A N + N “CN - N Tl Hol H tT Gg S HO" No Sy Rou, = ~ 0 Sy P12, n=0m=2.Y=H LP14,n=1" R.m=2 Y=H LP18. n=1* 8. m=2 Y=H Pi. n=0m=4 Y=00T LP2.n=0,m=4 Y= DIBAC NN | Y=oOT= {J 2 P19, n=1,* R-m=4 Yuh LP. n=1*R- m=4 Y=COT P21. n=1,"8-m=4Y=H LP22.n=1.%8--m=4Y=00T ’ oo Se 4 3 : 7 a

[0518] Example 3H: Methyl (4R)-4-amino-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl) phenyl]carbamoyl}butyljcarbamoyl}-2-methylpropyllcarbamoyl}butanoate (PEvcPAB) SRE FY ~oAy Yop PE > 0 Sa H (°EvcPAB)

[0519] To a solution of vePAB (0.25 g, 0.85 mmol) in DMF (3 mL) were added DIPEA (0.37 g, 2.9 mmol) and HATU (0.25 g, 0.95 mmol), and the mixture was stirred at room temperature for 10 minutes before the addition of Boc-DGIlu(OMe)-OH (0.40 g, 1.1 mmol). The reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was purified by reversed phase flash chromatography (0-70% acetonitrile in water) to give Boc-DEvePAB (0.35 g, ESI m / z: 623.4 (M + H)*) as a white solid, which was dissolved in DCM (5 mL). To the solution was added TFA (1.5 mL), and the reaction mixture was stirred at room temperature for 3 hours until Boc was totally removed, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (10-40% acetonitrile in water) to give PEvcPAB (0.27 g, 48% yield) as a white solid. ESI m / z: 523.4 (M + H)*.

[0520] Example 3: Methyl (45)-4-amino-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl) phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoate (EvcPAB) ~oRp LL WA Sy NH; H (“EvcPAB)

[0521] Following the similar procedure as PEvcPAB except using Boc-LGlu(OMe)-OH instead of Boc-DGIu(OMe)-OH, intermediate “EvcPAB (0.18 g, 49% yield) was obtained as a light yellow solid. ESI m / z: 523.3 (M + H)+, 545.3 (M + Na)*.

[0522] Example 3J: General procedure for LP#-2 and synthesis of LP13-2, LP16-2, LP1- 2 and LP2-2

[0523] To a solution of LP13-1, LP16-1, LP1-1 or LP2-1 (1.0 equiv.) in DMF (0.25 mM) were added DIPEA (3.3 equiv.) and HATU (1.0 equiv.), and the mixture was stirred at room temperature or 10 minutes before the addition of vePAB or EvePAB (1.1-1.5 equiv.). The reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in water) to give linker LP#-2 (LP13-2, LP16-2, LP1-2, or LP2-2) as a white solid.

[0524] Example 3K: (9H-Fluoren-9-yl)methyl N-{2-[2-(2-{[(1S)-1-{[(19)-4- (carbamoylamino)- 1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2- methylpropyljcarbamoyl}ethoxy)ethoxy] ethyl}carbamate (LP13-2) Oo on Frm 0cHNT Ou, f IPN Xo Lott LC : ~, nN x ” ~ 0 Syn (LP13-2)

[0525] Following the general procedure using vePAB (0.85 g, 2.2 mmol) and LP13-1 (0.60 g, 1.5 mmol), compound LP13-2 (1.0 g, 87% yield) was obtained as a white solid. ESI m / z: 761.3 (M + H)*, 783.3 (M + Na)*.

[0526] Example BL Methyl (4R)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl] carbamoyl}butyllcarbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2- ({[(9H-fluoren-9-yl)methoxy] carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP14-2) i SE Lye Berges is N 7 Ay Cc ~ a Sy, H (LP14-2)

[0527] Following the general procedure starting from LP14-1 (0.19 g, 0.37 mmol) and DEvCcPAB (0.14 g, 0.34 mmol), linker LP14-2 (0.20 g, 66% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-70% acetonitrile in water). ESI m / z: 904.4 (M + H)™. (LP15-2)

[0528] Example 3M: Methyl (48)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl] carbamoyl}butyllcarbamoyl}-2-methylpropyllcarbamoyl}-4-(3-{2-[2- ({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (LP15-2) 9 wo 0 TY oH rosy I A A o C ol, ~grhn Lo 0 NTH: (LP15-2)

[0529] Following the general procedure starting from LP13-1 (0.45 g, 1.1 mmol) and LEvcPAB (0.65 g, 1.2 mmol), linker LP15-2 (0.70 g, 69% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-100% acetonitrile in water). ESI m / z: 904.5 (M + H)*. 'H NMR (400 MHz, DMSQus) 6 9.94 (s, 1H), 8.15-8.07 (m, 2H), 7.91-7.87 (m, 2H), 7.78 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 7.2 Hz, 2H), 7.55-7.52 (m, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.35-7.30 (m, 3H), 7.23 (d, J = 8.4 Hz, 2H), 6.00-5.93(m, 1H), 5.42 (bs, 1H), 4.42 (s, 2H), 4.39- 4.34 (m, 2H), 4.31-4.27 (m, 2H), 4.24-4.16 (m, 2H), 3.60-3.56 (m, 5H), 3.47 (s, 3H), 3.36-3.34 (m, 2H), 3.14-3.10 (m, 2H), 3.04-3.00 (m, 1H), 2.96-2.91 (m, 1H), 2.44-2.38 (m, 2H), 2.35-2.29 (m, 4H), 2.00-1.95 (m, 1H), 1.92-1.86 (m, 1H), 1.77-1.66 (m, 2H), 1.61-1.55 (m, 1H), 1.46-1.34 (m, 2H), 1.25-1.18 (m, 1H), 0.87-0.80 (m, 6H) ppm.

[0530] Example 3N: N-[(18)-1-{[(1S)-4-(Carbamoylamino)-1-{[4-(hydroxymethyl) phenyl] carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amide (LP1-2) 1 aN 9 or EEO. PANN 0. e [A NO y Anny On ~ Ay Y v a 7 Ni ~ o Sy n (LP1-2)

[0531] Following the general procedure starting from LP1-1 and vePAB (3.3 g, 8.0 mmol), linker LP1-2 (4.3 g, 68% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-100% acetonitrile in water). ESI m / z: 791.5 (M + H)*.

[0532] Example 30: Methyl (4R)-4-{[(18)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl] carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H- fluoren-9-yl)methoxy] carbonyl}amino)-3,8,9, 12-tetraoxapentadecan- 15-amido]butanoate (LP19- 2) (L19-2) a , ; 5 5 0H H 2, AO Frost mo Og On A ry 0 cL [SS wh NN (

[0533] Following the general procedure starting from DEvcPAB (0.17 g, 0.34 mmol) and LP16-1 (0.17 g, 0.34 mmol), linker LP19-2 (0.18 g, 53% yield) was obtained as a light yellow solid. ESI m / z: 993.5 (M + H)*.

[0534] Example 3P: Methyl (4R)-4-{[(18)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl] carbamoyl}butyl]carbamoyl}-2-methylpropyljcarbamoyl}-4-{1-[2- (cyclooct-2-yn-1-yloxy) acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoate (LP20-2) N Ha? mit By WE Be o Sym NN © iE yon onpfomommngmonp ih i AT o o & "uP io Sy Crs MH ] (LP20-2)

[0535] Following the general procedure starting from PEvcPAB (0.28 g, 0.54 mmol) and LP1-1 (0.25 g, 0.48 mmol), compound LP20-2 (0.20 g, 44% yield) was obtained as a light yellow solid. ESI m / z: 934.5 (M + H)".

[0536] Example 3Q: Methyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl] carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H- fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9, 12-tetraoxapentadecan-15-amido]butanoate (L...

Claims

WHAT IS CLAIMED IS:

1. A compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)i)n (A), wherein: BA is an antibody or an antigen-binding fragment thereof: Gln is a glutamine residue; L1 is absent or a first linker: Bis a branching unit comprising at least one adduct of group B’ and group B”, wherein AN ; wd. one of the groups B’ and B” is selected from -Na and N-N : and the other of the Ne 00e ACo = Nyt ' b groups B’ and B” is selected from fg oe and No , Where Q is CorN; L2 is a second linker covalently attached to the branching unit B via the at least one group B”; M is absent or a moiety having the structure Is , where R, R’, and R” are independently at each occurrence hydrogen or a C1-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Fg? SA k is an integer from 1 to 12; m is an integer from 1 to 30, and nis an integer from 1 to 30.

2. A compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)i)n (I), wherein: BA is an antibody or an antigen-binding fragment thereof; Gln is a glutamine residue; L1 is absent or a first linker; B is a branching unit comprising at least one adduct of group B’, where the group B’ is ye N=N {Th | A . wn, ; = ha = - 2 and Ns selected from -Nj, —$~=3—, hor N-N ;and \— / ,whereQisC orN; L2 is a second linker covalently attached to the branching unit B via at least one group B”, wherein the group B’ and the group B” form the at least one adduct; M is absent or a moiety having the structure + ,whereR, R’, and R” are independently at each occurrence hydrogen or a C4-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Rap ~N NH 7 1 Ih I F N PR, (P); k is an integer from 1 to 12, and nis an integer from 1 to 30. 3 The compound of claims 1 or 2, wherein the BA is an antibody or an antigen-binding fragment thereof.

4. The compound of any of claims 1-3, wherein the BA is an anti-HER2 antibody, an anti- STEAP2 antibody, an anti-MET antibody, an anti-EGFRVIII antibody, an anti-MUC16 antibody, an anti-PRLR antibody, an anti-PSMA antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2 / HER2 bispecific antibody, an anti-MET / MET bispecific antibody, or an antigen-binding fragment thereof.

5. The compound of any of claims 1-3, wherein the BA is an anti-HER2 / HER2 bispecific antibody.

6. The compound of claim 5, wherein the anti-HER2 / HER2 bispecific antibody comprises: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human HER2; and wherein D2 specifically binds a second epitope of human HER2. Zi The compound of claim 8, wherein D1 and D2 do not compete with one another for binding to human HER2.

8. The compound of any of claims 1-4, wherein the BA is an anti-STEAP2 antibody comprising an HCVR / LCVR amino acid sequence pair as set forth in Table 1.

9. The compound of claim 7, wherein the anti-STEAP2 comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:2 / 10; 18 / 26; 34 / 42: 50 / 58: 66 / 58; 74 / 58: 82 / 58: 90 / 58; 98 / 58; 106 / 114; 122 / 130; 138 / 146; 154 / 162; 170 / 178; 186 / 194; 202 / 210; 218 / 228; 234 / 242; 250 / 258; 266 / 274; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

10. The compound of any of claims 1-4, wherein the BA is an anti-MET / MET bispecific antibody.

11. The compound of claim 10, wherein the anti- MET / MET bispecific antibody comprises: a first antigen-binding domain (D1); and a second antigen-binding domain (D2); wherein D1 specifically binds a first epitope of human MET; and wherein D2 specifically binds a second epitope of human MET.

12. The compound of claim 11, wherein D1 and D2 do not compete with one another for binding to human MET.

13. The compound of any of claims 1-4 and 10-12, wherein the BA is an anti-MET / MET bispecific antibody comprising an HCVR / LCVR amino acid sequence pair as set forth in Table 3.

14. The compound of any of claims 1-4 and 10-13, wherein the BA is an anti-MET / MET bispecific antibody comprising a D1 antigen-binding domain and a D2 antigen-binding domain, wherein the D1 antigen binding domain comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2012 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2014-2016- 2018-2094-2096-2098, and wherein the D2 antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2036 / 2092, or a set of heavy and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 2038-2040-2042-2094-2096-2098.

15. The compound of any of claims 1-4 and 10-13, wherein the BA is an anti-MET / MET bispecific antibody H4H14639D, which comprises a D1 derived from H4H13306P2 and a D2 derived from H4H13312P2.

16. The compound of any of claims 1-9, wherein the glutamine residue Gin is naturally present in a CH2 or CH3 domain of the BA.

17. The compound of any of claims 1-9, wherein the glutamine residue Gin is introduced to the BA by modifying one or more amino acids.

18. The compound of claim any of claims 1-9, wherein the Gln is Q295 or N297Q.

19. The compound of any of claims 1-18, wherein the BA targets a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

20. The compound of any of claims 1-19, wherein L1 comprises C1.s alkyl, phenyl, -NH-, - C(0)-, -(CH2)y-NH-C(Q)-, -(CH2)u-C(O)-NH-, -(CH2-CH2-O)\~, -(CH2)u-(O-CH2-CH2)\~ C(0)-NH-, a peptide unit comprising from 2 to 4 amino acids, or combinations thereof, each of which may be optionally substituted with one or more of -S-, -S(02)-, -C(0)-, - C(02)-; and CO2H, wherein subscripts u and v are independently an integer from 1 to 8.

21. The compound of claim 1, wherein L1 is selected from the group consisting of: 0 SCI Soro 7 i 4 NG o ~1 fe ~F SON Frog “eh “5 f+ le H seo Sogn OI, os ~NAE Ghar Erber SLL falar oY a X e i LAT 5, I . wo hd NTR », v fe i ~ , rp 5 and pr H a _ i S & , wherein Ra is a group rans-cyclooctene, a maleimide, an amine comprising an alkyne, an azide, a tetrazine, a trans-cyclooctene, a maleimide, an amine, a ketone, an aldehyde, a carboxylic acid, an ester, a thiol, a sulfonic acid, a tosylate, a halide, a silane, a cyano group, a carbohydrate group, a biotin group, a lipid residue and wherein subscripts x, n, p and q are independently an integer from 0 to 12, and combinations thereof.

22. The compound of any of claims 1-21, wherein B comprises one adduct of group B’.

23. The compound of claim 22, wherein -NH-L1-B’ is selected from the group consisting of: Hy EE Ay Nog nO gr BY) H AO - NN nN Ser On ye 7 arg 8) Fon Ag Ong On ga Oe AB) HOO ol od J Ar eos LN o 2 & a a" ’ H Lm y SP My rE) H Ayr Apaturgro " Ho” © Ne) 8 or { LX a AK Bog y TY IN NH no N where the Fi is the amino point of attachment to the glutamine residue of the BA. 24, The compound of claim 23, wherein the group B’ is an azide (-Na), and the adduct of the group B’ comprises a triazole.

25. The compound of any of claims 1-21, wherein B comprises two adducts of group B'.

26. The compound of any of claims 1-21, wherein B comprises three adducts of group B’.

27. The compound of any of claims 1-21, wherein B comprises at least four adducts of group B.

28. The compound of any of claims 1-21, wherein B comprises a group selected from the group consisting of: A er er busts Raul ls 0 HR Jy 2 ») H Bh -( i ~~ ” o PE) o Fr ©). lp I Pg Lot ra £ on “yi 9 A he, = . - on a th) abe d R 4 Fox 5 £ Fu, F 2027 0, 4B) 3 Ng - er TE [a ad wo ho, CY Sg Ee) _ a wii fE oo G oe i Selig) \ i No - (8) ozs {8 [NPN § OFF o gw oe y A fc i eo and “®) | where (B’) comprises points of attachment of the adduct of the group B’ and the group B”.

29. con of claim 28, wherein B is selected from the group consisting ¢ 9 Ayr oN * Ae N 1, Ayrton wt Br Saga a 4 er 5) Woe, Our, F) 0 A we ~ ye \ RN ~y pO gr (8) 9 Onn q ~ ~ hE: i @ > a Fomor 6 oxlmgnm 1 & # 0. Mo, P::) Arto, Ny TNE ery ®) 4 NH Sy © Pre Z Zant, ® x 2 TR a Pe ®), 3 8) > oo oN Co Fa Py ~ Ea {t:}] ig ll 0” AONB) A) OB) 30. compound of claim 1, wherein -NH-L1-B is selected from the group cons 9 . Apne Km Omg, oy ant N 4 Ayrton, Sgr On, rgd ” hp On gO YOON H Woe Ae Omg On Os, of Wi Ou gy Te o J Froggy. { H N Hg H ry Vosagr Ongena AB) a o A Sar On gy Anema Igd N | H Wg, H oR) a LE | 6 Angra Sgr mone N NC T 3 yr N 18) BY ENN. a “ Pm oP ’ 2 oal, ad B = J I (8) ~(B) AnOmgrnlo mgm Bam AO 2 N H 1 fn RE 2 hy 8, NN re i 5 4d ot gd gh LG A Nord 7 oy oR 5 Nt hs J® ONAN NABY brady Lae Kg Og BY : — AH where the HS is the amino point of attachment to the glutamine residue of the BA.

31. The compound of claim 30, wherein the group B' is an azide (-Ns), and the adduct of the group B’ and the group B” comprises a triazole.

32. The compound of any of claims 1-31, wherein the adduct of the group B’ and the group B” has a structure selected from the group consisting of: Tm Nd i On CN Al \ JO DA ae 0 NS Dx , and wherein Qis C or N.

33. The compound of any of claims 1-32, wherein M is absent. 8 | H ? so AE : 1 # 34. The compound of any of claims 1-32, wherein M is & , where R, R’, o i wi A and R” are hydrogens at each occurrence, i.e. M is Es rR # } ! 2 AE A 1 a 35. The compound of any of claims 1-32, wherein M is de , where R is AX hydrogen and R’ and R” together form a 5-membered ring, i.e. M is leit 36. The compound of any of claims 1-35, wherein n is 2.

37. The compound of any of claims 1-35, wherein n is 4.

38. The compound of any of claims 1-35, wherein n is 8.

39. The compound of any of claims 1-35, wherein n is 12.

40. The compound of any of claims 1-35, wherein n is 16.

41. The compound of any of claims 1-35, wherein n is 24.

42. The compound of any of claims 1-41, wherein L2 has a structure according to Formula (L2): B’-8P1-B2-(-SP2-AA-SP3), (L2), wherein: B” is a group capable of covalently attaching to the group B’; SP1 is absent or a first spacer unit; B2 is absent or a branching unit; SP2 is absent or a second spacer unit; AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit covalently attached to the Dxd, and p is an integer from 1 to 12.

43. The compound of claim 42, wherein the at least one group B” is selected from the group N=N +h QO TN 7 i. a, N— x consisting of -N3, ~~ N-N | f= H, gi for , and combinations thereof. 44, The compound of any of claims 42 or 43, wherein SP1 is absent or selected from A Oo the group consisting of ok the group consisting of & ~~" © © , C16 alkyl, -(CHz2-CHz-O)y-, -NH-, - C(O)-, -NH-C(O)-, -NH-(CHz)u-, -NH-(CH2),-C(O)-, -NH-(CH2-CH2-O)v-, -NH-(CH2-CHa- O)-C(0)-, -NH-(CHz-CH2-0)\-(CHz)u-, -NH-(CH2-CH3-0)y-(CH2)u-C(Q)-, -(CH2),-NH- C(0)-, -NH-(CHz),-NH-C(O)-, -NH-(CH2),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8. 45, The compound of any of claims 42-44, wherein B2 is absent or selected from the group ey NY i 3 Ok S55 5% A Fram Bay od B 5 © consisting of os Ar oA 46. The compound of any of claims 42-44, wherein SP2 is absent or selected from the group consisting of a Ci. alkyl, -(CHz-CH2-O)y~, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2).-, -NH- (CH2)y-C(0)-, -NH-(CHz-CH3-O)y-, -NH-(CH2-CH;-0),-C(0)-, -NH-(CH2-CH3-O)\-(CHz),-, - NH-(CH2-CH2-0)y-(CH2)u-C(O)-, -(CH2)u-NH-C(O)-, -NH-(CH2),-NH-C(O)-, -NH-(CHa).- C(0O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8.

47. The compound of any of claims 42-45, wherein AA is a peptide unit comprising from 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, phenylalanine, and citrulline, and combinations thereof.

48. The compound of claim 47, wherein AA is valine-citrulline, valine-alanine, or phenylalanine-lysine. 49, The compound of claim 47, wherein AA is selected from the group consisting of glycine- glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)), L-glutamic acid-valine-citrulline (\EVC), and D-glutamic acid-valine-citrulline (PEVC).

50. The compound of any of claims 42-49, wherein SP3 is absent or selected from the group consisting of No An und of any of claims 42-49, wherein SP3 is a te 2s HL 7 ; & A on 7 a AN ; of I I , and combinations thereof, wherein Rc is independently ba A % tL Ye Ho HOY, ses at each occurrence absent or a group selected from OH and HO. 0 ¥ HO, a o NP. OH 51. The compound of any of claims 1-50, wherein the M-Dxd has a structure selected from the group consisting of © Ho nA g ui 2 NT Ay ey? o NH & N- Nard ~ 5s , and 56 , wherein R is a hydrogen or a C4-Cs alkyl, and where an represents the point of attachment to L2 or L2'.

52. The compound of claim 1 or 2 having a structure: LT OE a or hrp ody Fe BA, [Ng . 3 ey > | oo A Lo J a 8 OY a8 Fad SRS, O IT 330 - Ne rv oe ll od | She CT = / , 53. The compound of claim 1 or 2 having a structure: “J hy LA Sy Ny . . a a 3 Yo ogre ey 9 Hop Rou 3% phim ann EEO p Jest LS TA 5 BA, Og N kt y Ty : ” > | werd 9 i =o A ao SY wg Lied ; nd enn Kp LOT SSS H * “x a F fa 54. The compound of claim 1 or 2 having a structure: ~N Ny . WL xr. A sh AAO T~Aoo J ° aatane FE WNUNIN HIE Pease pas Jessy ; ~ Yh, ; 55. A compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)i)n (1), wherein: BA is an antibody or an antigen-binding fragment thereof: Gln is a glutamine residue; L1 is absent or a first linker: Bis a branching unit comprising SR Pr TE 0 N 3 \ ] DO + | Dr , or 3 pa Os or N : @ | Ay i s A AE y ¥ > M is absent or a moiety having the structure & , where R, R’, and R” are independently at each occurrence hydrogen or a C4-Cq alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent having a structure according to Formula (P): Ro? ; Pl Or o A 5 A k is an integer from 1 to 12, and nis an integer from 1 to 30.

56. A compound according to Formula (L2-P), (L2’-P), or (L2"-P): B"-SP1-B2-(-SP2-AA-SP3-M-Dxd)p (L2-P), H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd), (L2'-P), maleimide-N-SP1-B2-(-SP2-AA-SP3-M-Dxd), (L2"-P), wherein: N=N 4 pe CC NeN AY ’ B” is selected from the group consisting of -Ns, NN . gm mg or and ee and U Q . ) i - neo Iss SP1 is absent or a first spacer unit selected from the group consisting of ~ ~ a we and © SP2 is absent or a second spacer unit selected from the group consisting of a C1.¢ alkyl, «(CH2-CH2-O)s-, -NH-, -C(O)-, -NH-C(O)-, -NH-(CHz)u~, -NH-(CH2),-C(O)-, -NH-(CHz- CHz-0)y-, -NH-(CHz-CH2-0),-C(Q)-, -NH-(CHz-CHz-0)-(CHz),-, -NH-(CHz-CH-0),- (CH2)s-C(0)-, -(CH2)u-NH-C(O)-, -NH-(CH2)u,-NH-C(O)-, -NH-(CH2)u-C(Q)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8: AA is absent or a peptide unit comprising from 2 to 4 amino acids; fe He PS A ¥% LOPES LL 3 PY P * * 7 NS T H © a a rR NINN X , wherein Rc is independently at each occ H O, H O° Hon A HO, re o NCE or a group selected from OH and OH EAE M is absent or kb , where R, R’, and R” are independently at each occurrence hydrogen or a C4-Cy4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): pe a 1 © RE — 0 (P), and p is an integer from 1 to 12.

57. The compound of claim 56, wherein AA is a peptide unit comprising from 2 to 4 amino acids selected from glycine, valine, phenylalanine, proline, glutamic acid, lysine, alanine and citrulline, and combinations thereof.

58. The compound of claim 57, wherein AA is valine-citrulline, valine-alanine, or phenylalanine-lysine.

59. The compound of claim 57, wherein AA is selected from the group consisting of glycine- glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO: 2113)), glycine-glycine-phenylalanine (GGF),glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO: 2114)) and glutamic acid-valine-citrulline (EVC).

60. The compound of claim 56, wherein the compound has a structure according to Formula (L2-P) selected from the group consisting of: oy Sgn A 3 Orr Hol H ° : 1 Ue © a Kio o ! hdr AL ALOT iY X 0 a] vy ~ Py TT o ° o of PRT 5 J HO. 50 9 5 9 HO, Ag YY / 7 iY § a © ory My # TN 4 Neorg ba Au ZN “Ho. Bl ou 9 Ha, A, No OH. “op oN NO LOY 2 HON 2 [oy Re # — | Naor 0 mod AL ASE 0K, CXYTEY NY 7 grrr Cpt > ~ Sr 3 ] Oo OH F ZN NA i i x i oA i No, ~~! = oy ey oY - o z GOH HO. on x Oo OH F 0 3 Q_ i N ; on 8 oH Lg HJ co 0 o Q H H Ne, JL Noo, = YT o TN ~ HO ~~ YOM OH nr Ho on rt g nd af i 2 ENP hy, Agony, J 3 a S$ SoA o PY Qq N Koro A, J =a o Arfmmmngigh Li AIIHY YS | [ LIV YCy & soo Sy ia To. oo {a ~ FE. J 8 TN Tu | (mgm ly Li 407 VY AF LA 1 PFvVYE x bo Sw, y oo H o 2 | or a pharmaceutically acceptable salt thereof. The compound of claim 56, wherein the compound has a structure according to Formula (L2-P) or (L2'-P) selected from the group consisting of: io 5 + ho 0 wi = Ye erm Ip AY Jeon x ot Vo No | “yn, N —- © H iL AoA, LC Lr Ty He Xu. Ay ° T HARASS Ee 44H X A LK, : HOT TO Ny Ya No Sb, ° “To T§.a PnP SE Braas ans Jsst 1° o 9 [FI epiondy, FC pte AK SOT SE Hy THY ~ © LE, H Ye ~ 1 Sy NH; or a pharmaceutically acceptable salt thereof.

62. The compound of any of claims 56, wherein the compound has a structure according to Formula (L2-P), (L2-P) or (L2"-P) selected from the group consisting of: o o ) a ’ a AIA RE He Re yy IT CEY SSE Cr iL SO “rh - J ad HW uw 0 Noe 9 Sq A ] : Sm Sh Y td A ond, Os 8 oom In i ’ - Cry ng ray Y & A 5 , a § ERATE Sy, H H > N\ IS N=ory Hy ’ LN o 9 H BN Roy, ; 0 a ore WY | bimonAy N., A 3 So - un o Q Sys e H z Noy 9 LQ Ay ds wi epi LO ° J I oh ol = oan 5 Sh EAN OA - F SA" 0 0 Sl 0 | Og NH iJ ~~’ pt Hh 0 o o : A Hoo XL 3 9 0 YY NH {mon led Lt LO H Qo A eR 8 q ®! 1 tN - Aon Sg Sy pom e % : x oH D Xa OY : y CH, 2 Ay - 3 4 3 5 &) 2 Pe WH Q o H Khoo, . SY y " i 1 i or = f # : A HL, A Q } Ou mnAg ‘ Omg " Yr : A SNR § 0 9 ™ i i nn LCL ~ Hoo CY Ferg p ST CN il ~ op a HN - ~o HN Sg Ong . OF "NH t I O. g fondo t,, i o OY r Ls 1 0 5 HN, ) bomen Ay I a4 EB 1 5 4g a Q 2 o Lg Hook, nL ! SORT 6 NT, Cr hn a wl IT Pop ~ tJ a i g oN o. oO! A Op, oy 2 oN Pay 5° 2 5 3 HX y™ 0 Aaa, food, 2 =o monk LE OTT Sead ~ 0 Sy an Ppa, ate ror ht Oy = L3- I RB SSAA) IF ann Sng Ne ; A i gy Son I oon ou Re oo pe q - J oo ay $8 VS " o , com fo My, 2 de Mngt TY XE SGLEA & “OH oN NH Y Nak, ua 1 J jg ~~ nh I ez Hn ~ PEE a oY wo Oo ah on pw he OA pAymmron / v 3 H (po 1, . 3 M3 HN [NN Ay 0 \, BOINSCALE Ras Jur oY ry q : & oe HO | NA, > o y a Q ery o : o Ags Fo 0, Xn 1 Oy mO. He 2 5 ; oie FA I 5 , ~ ~ 9 2 Li 1 Cob ~ 9 Sg H a \, I NT NH, H ? GTN 0 PN No, : I TY TW w Ogy NH \ Hal” On J i : . arn OOK Syn o) NN Spo 3 HN, 1 4 i 4 oO fel hp A ~ 3 AAT [3 H ] ° : ~ oN , Pon 5 1 < NN Y -: 9 H 8 gh Li A Cry as Hak, fas AS © A FEY Co : ~ ~ ad he 1 va he aon ~ a Sy, y oN Pon ~ Sy = . WL 8 ’ oo gph = : gy fA a. * rs yr SR Sod H Nr T So | J: bY & } NT UNHy 4 Po = Linn lod Lh 2 : Hedy {, X or ’ : a Aer pd T 0H 3 : pe RS % N NH 1 Ay a I) Ro 3 room Ao dy 3 rnp A wy SG YIVTCY X AE tg A LL, A So rctmgmin i t / No ea Ne [ 4. "ommnded LY Sn TALC oh BN LA, ¥ J goed LN oA © i YOY Ce Sdn wo 9 Aor Sh ¢ Ho oo y Vomongled LA | Saag ed re Te h an, or Et Sond 0. A, N < : y J PAYY & : rtrd, ~ "ar FI ytd a i H i ¥ TTT 5 omni 207 x a, ‘ i In ’ YE ry “3 & pr. oe Sows aa ) 6 Lo LA Jb A Se Xx o oon NH ~ Sutalyh Nr Le y el x rrp lr ® RA A S ny : \ ¥ a Ea Sw oy . . Jel, | NY y pte dh LL ~y x Nn DY C3 Dd Pom ~ Q Sr p— Sow on A A ’ ELT 8 LT, X Q é Pon Sr edd } } J Hoo = Le Ln LLY Front HL ? Pon Se, Sy ~ 2 ww Aor She ee Yr 7 Be 0 A 1 Y T. y ° rey ™i C1 YP SA Pro uy 1, ) XL { + | 5 Eo T or oe 9 o i Oe WIT 0 ene 4 2 Q gS Cr pe o YA $f oh ox Nw ~ rp A adage? on ~ og Sw H, Pon 5 a DR Sf eh ror 4 3 0 o é Pom “on 3 POA Oy 5 ~~ 1 i f NOY 8 er TLL AL Y 7 Aon Shoes Sy ° Q Qt LY 8 Ory J oyiel lt 4 = 8 | ~ ~ 3 T Pa Svs A i pm ™~ LA, AT A CY Ay G8 ’ wet dt oA a L ° Hon Se - RUN St ° ° ° oS Lh 9 ss Oey SIA yy 2 i] ! Eo I omg oN pA LJ SAAS of C y ae efron AIR LIS ° NY oS a Fond 3 y ce ¥ ! 1 o o Qo < i i 4 5 Z Se Lt iN A 8,1 Yr SS ~My NY { of ( Y i re LL, Xr , and LS pong Jp A fT IE 54 STITT T © wry So On B $ a g 2 Rs AIEEE a A oo pyecagotid, S Pyreamm Ad onpmmanpi hy i A VY ¥ So be wh Li. 5 & 7 Hm dad, 3 1 4 4 Cer SpE TTT et ee LA, A 63. An antibody-drug conjugate according to formula (I1): Ab-(GIn-NH-L1-B-(SP1-B2-(-SP2-AA-SP3-M-Dxd)i)p)a (II), wherein Ab is an antibody; Gln is a glutamine residue; L1 is absent or a first linker; Bis a branching unit comprising at least one adduct of group B’ and group B”, where “ f Ji ke I N=N, 1 0m TY the group B’ is selected from -Na, fof for N=-N Nr LL I N=N_ 7 COSNAAY SN the group B’ is selected from -Na, f= for N-N Nr wherein B’-SP1-B2-(-SP2-AA-SP3-M-Dxd)p is the compound according to Formula (L2-P) according to any of claims 55-60, and wherein the compound of formula (L2-P) is covalently attached to the antibody via the adduct of the group B’ and the group B”; k is an integer from 1 to 12, p is an integer from 1 to 30, and n is an integer from 1 to 30.

64. An antibody-drug conjugate comprising an antibody and a linker-payload, wherein the linker-payload comprises the structure: so A o H o PY oo o I Oy eA nomenon fA x ) NF H H o % H . a ~ o o Sy o F , or o H Q Q WE 5 Wo Ory N Ny \ H 3 “ty IN oH AA N,, A ° \ nd H H H No ° Ja ANN Ns ~ 0 a where «an represents the point of attachment to the antibody, directly or through a second linker, or a pharmaceutically acceptable salt thereof.

65. A composition comprising a population of compounds according to any one of claims 1- 61, having a drug-antibody ratio (DAR) of about 0.5 to about 30.

0.

66. The composition of claim 64 having a DAR of about 1.0 to about 2.

5.

67. The composition of claim 66 having a DAR of about 2.

68. The composition of claim 64 having a DAR of about 3.0 to about 4.

5.

69. The composition of claim 68 having a DAR of about 4.

70. The composition of claim 64 having a DAR of about 6.5 to about 8.

5.

71. The composition of claim 70 having a DAR of about 8.

72. The composition of claim 64 having a DAR of about 10 to about 14.

73. The composition of claim 72 having a DAR of about 12.

74. The composition of claim 64 having a DAR of about 14 to about 18.

75. The composition of claim 74 having a DAR of about 16.

76. The composition of claim 64 having a DAR of about 22 to about 24.

5.

77. The composition of claim 76 having a DAR of about 24.

78. A compound having a structure according to Formula (P-1): oR 7 R. HN KAA 0 Pl Hy op Ry Rp NH be RS 3, oO FONT PA 4 © Ne HS (PH), AR. ara indenandantlv a hvAdranan or a wherein Ry, Rz,R3, and Rs are independently a hydrogen or a C4-C4 alkyl, or wherein R2 and Rj form a 5-membered or a 6-membered ring, or a pharmaceutically acceptable salt thereof.

79. The compound of claim 64, wherein the compound is 64, wherein the compound is A HN AS om, 20 Fa NF NF oH Ney ako, or a pharmaceutically acceptable salt thereof.

80. An antibody-drug conjugate comprising an antibody, a linker, and a payload, wherein the payload is the compound of claim 79.

81. An antibody-drug conjugate comprising an antibody, a linker, and a payload, wherein the 82. A compound having a structure according to Formula (P2): payload is IS NAN 0 "! No 7 ¥ A \ Nef OH ©, or a pharmaceutically acceptable salt thereof, where ww represents the point of attachment to the linker. A compound having a structure according to Formula (P2): Q wy NT “or pP AN 0 Wp Ng “FP FWY Se oH (P2), an ora C.-C. alkvl ar a nharmacaiitica wherein R is a hydrogen or a C+4-Cs alkyl, or a pharmaceutically acceptable salt thereof.

83. The compound of claim 80, wherein the compound is 80, wherein the compound is o Flic ANH | pL JR & es 5 ey A : or a pharmaceutically acceptable salt thereof.

84. A pharmaceutical composition comprising the compound according to any one of claims 78-83, and a diluent, a carrier, and / or an excipient.

85. A method of treating a condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the compound according to any one of claims 1-61 or 78-83, or the composition of any one of claims 64-71 or 84.

86. The method of claim 85, wherein the condition is cancer.

87. The method of claim 86, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, liver cancer, or brain cancer.

88. The method of claims 86 or 87, wherein the condition is HER2+ breast cancer.

89. A method of selectively delivering a compound into a cell, wherein the compound is according to any one of claims 78-83.

90. A method of selectively targeting an antigen on a surface of a cell with a compound, wherein the compound is according to any one of claims 78-83.

91. The method of claims 89 or 90, wherein the cell is a mammalian cell.

92. The method of any of claims 89-91, wherein the cell is a human cell.

93. The method of any one of claims 89-92, wherein the cell is a cancer cell.

94. The method of claim 93, wherein the cancer cell is selected from the group consisting of a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a lung cancer cell, a liver cancer cell, or a brain cancer cell.

95. A method of producing a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m)i)n (A), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker; B is a branching unit comprising at least one adduct of a group B’ and a group B”; L2 is a second linker covalently attached to the branching unit B via at least one group B”: AK M is absent or b , where R, R’, and R” are independently at each occurrence hydrogen or a Ci-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent comprising a structure according to Formula (P): Wy ~~ A LA r “ 5 AX k and m are independently an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NH,) with a compound L1-B, wherein the branching unit B comprises the at least one group B’, b) contacting the product of step a) with k or more equivalents of a compound L2-(-M-Dxd)m, wherein the linker L2 comprises at least one group B”, {Ms { N=N wherein one of the groups B’ and B” is selected from -N3 and N-N ; and the other Ne Ae . Aa, . Ne” of the groups B’ and B” is selected from =f or sand eo where Q is C or N; and ¢) isolating the produced compound of Formula (1).

96. A method of producing a compound having a structure according to Formula (A): BA-(GIn-NH-L1-B-(-L2-(-M-Dxd)m))n (A), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one adduct of a group B’ and a group B” as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above; M is si AK absent or fe , where R, R’, and R” are as described above; Dxd is an anti- tumor agent comprising a structure according to Formula (P): >. 0 LA CO | N— TE . & 2 = n (P), k and m are independently an integer from 1 to 12, and nis an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least one group B’, with k or more equivalents of a compound L2-(-M-Dxd)m, wherein the linker L2 comprises at least one group B” capable of covalently binding with the group B’, {Ms {f N=N wherein one of the groups B’ and B” is selected from -Nj and N=N : and the other uh, wld 2 Ned TF of the groups B’ and B” is selected from ff er ; and \o where Q is Cor N: thereby producting L1-B-(-L2-(-M-Dxd)m)k; b) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gln (BA-GIn-NH3) with the L1-B-(-L2-(-M-Dxd)m)x product of step a), and ¢) isolating the produced compound of Formula (1).

97. A method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd)i)n (0, wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker; Bis a branching unit comprising at least one adduct of a group B’ and a group B”; L2 is a second linker covalently attached to the branching unit B via at least one group B”; SAK M is absent or & , where R, R’, and R” are independently at each occurrence hydrogen or a C4-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; Dxd is an anti-tumor agent comprising a structure according to Formula (P): pS pe. wh A ~~ ~ < P), k is an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NHz) with a compound L1-B, wherein the branching unit B comprises the at least one group B’, b) contacting the product of step a) with k or more equivalents of a compound L2- M-Dxd, wherein the linker L2 comprises the at least one group B” capable of covalently attaching to the group B’, N= 0 “4 i wherein one of the groups B’ and B” is selected from -N3 and N-N ; and the other ~ OY = LIC Tx of the groups B’ and B” is selected from f= Ao ; and Nr where Q is C or N; and ¢) isolating the produced compound of Formula (1).

98. A method of producing a compound having a structure according to Formula (1): BA-(GIn-NH-L1-B-(-L2-M-Dxd))a (I), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L1 is a first linker as described above; B is a branching unit comprising at least one adduct of a group B’ and a group B” as described above; L2 is a second linker as described above covalently attached to the branching unit B via at least one group B” as described above; M is 8 | 3A AK 3 vw | absent or be , where R, R’, and R” are as described above; Dxd is an anti- tumor agent comprising a structure according to Formula (P): Ws, 0 TY rT ™ NSN 0 ] Ne Fa Pa o 4 -\ , 3 — H 6 (P), kis an integer from 1 to 12, and n is an integer from 1 to 30, wherein: the method comprises the steps of: a) contacting a compound L1-B, wherein the branching unit B comprises the at least one group B’ with k or more equivalents of a compound L2-M-Dxd, wherein the linker L2 comprises at least one group B”, thereby producting L1-B-(-L2-M-Dxd)x, wherein one of Ne=N 4 { N-N the groups B’ and B” is selected from -Nsand ~~ N-N : and the other of the groups B’ and B” oo NE = NN , where Qis CorN; = st is selected from Bote “for : and o b) contacting, in the presence of a transglutaminase, the binding agent BA comprising at least one glutamine residue Gin (BA-GIn-NHy) with the L1-B-(-L2-M-Dxd)x product of step a), and ¢) isolating the produced compound of Formula (1).

99. A method of producing a compound having a structure according to Formula (III): BA-(GIn-NH-L2*-P)), (111), wherein BA is an antibody or an antigen-binding fragment thereof; Gin is a glutamine residue; L2-P is H2N-SP1-B2-(-SP2-AA-SP3-M-Dxd)p as described above, and n is an integer from 1 to 30; Q 0. SP1 is absent or a first spacer unit selected from the group consisting of % A a a i and B2 is absent or a branching unit; SP2 is absent or a second spacer unit selected from the group consisting of a C1. alkyl, -(CH2-CH2-O)+~, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2)y-, -NH-(CH2),-C(O)-, -NH-(CH>- CH2-O)v-, -NH-(CH2-CH2-O)~-C(O)-, -NH-(CH2-CH2-O)\-(CHz2)u, -NH-(CH2-CH2-O)\~ (CH2)u-C(O)-, -(CH2),-NH-C(O)-, -NH-(CH32),~-NH-C(0)-, -NH-(CH3),-C(O)-NH-, or combinations thereof; wherein subscripts u and v are independently an integer from 1 to 8: AA is absent or a peptide unit comprising from 2 to 4 amino acids; SP3 is absent or a third spacer unit selected from the group consisting of, we 7 5 HY a A SE RE Sr H 3 SP3 is absent or a third spacer unit selecte eo Po LOPE LP # ~~ Fa # SE , wherein R¢ is independently at each occurrence absent '® AN QO TP Hi Oy Hi Op How A HO... Ao Hi AN Hi SAAN or a group selected from OH and OH eK M is absent or k , where R, R’, and R” are independently at each occurrence hydrogen or a C1-C4 alkyl, or wherein R’ and R” together form a 5-membered or a 6-membered ring; and Dxd is an anti-tumor agent having a structure according to Formula (P): Fg? AN = As ~ ~A ®). p is an integer from 1 to 30; wherein the method comprises the steps of: b) contacting, in the presence of a transglutaminase, the BA comprising at least one glutamine residue Gin (BA-GIn-NH.) with the L2’-P, and ¢) isolating the produced compound of Formula (III).

100. The method of any one of claims 95-98, wherein the glutamine residue Gln is naturally present in a CH2 or CH3 domain of the BA.

101. The method of any one of claims 95-98, wherein the glutamine residue Gin is introduced to the BA by modifying one or more amino acids.

102. The method of any one of claims 95-98, wherein the Gin is Q295 or N297Q.

103. The method of any one of claims 95-98, wherein the transglutaminase is microbial transglutaminase (MTG).

104. The method of any of claims 95-103, wherein M is absent, or wherein the M-Dxd has a A &, ty Fore o NH ore i Ne Jo NE ” oc Nae and ing re selected from the group consisting of 5 Narn, 0 yo £m NH Ji be hn O LL 2 F NNT QO Ne OH © | and A Bam arr REn Aro Mr Biel Bane wine wherein R is a hydrogen or a C1-C, alkyl, and where ~~ represents the point of attachment to L2.

105. The method of any of claims 95-104, wherein the compound L2-Dxd has a structure selected from the group consisting of: NC ho NS Aogiody FC oY ~~ orem SL hry N o | CH \ HS' | 4 ~' A Bo ELWYNN lA linn I, I all Suns pe i oA, eho A Ee ” “atl did # Be 2m “Y 9 u 9 HO, Ao AN Ap iO miei LI @ . de") 4 pe Nee gh 0 Au 7 Tro. y 9 1 9 H ¢ HOW, yng ay ANON, OT HO” NN Gl ox HNTO ial % 4 5 Ne Ag I AeA Athen, x. iP © © IE Sof fv at 5 I~ Meteo La Q © y 5 Nagra ’ NL Yo HOSS An Co OH ZN “dt 1 gw. % Step ey 1 NYY NY i 5 iP J oF AN or HO. ‘SH Om oH © HOW A oH ago _ o Ca PS IN SS thot ii ; A A [ Y ry Aa a Nos EF J A. un of / 0 ANS HG oy ig ! JE a, 0 oy on WN ~. 2 | Cepia igh 0 2 Y al © a / < hay " o . > . N orks Lh NS N . Jot XL, 3 1 Lk . sm tnd LOY NA |g fT AAT X A = Ar | - 2 No SA Hoo Se No 1 0 - H o 4 | or a pharmaceutically acceptable salt thereof.

106. The method of any of claims 95-104, wherein the compound L2-Dxd has a structure selected from the group consisting of: —_ — Yop ror wren J HY 5 “ J ow A he ~ Syn EH, ~ Syn To —o T% ~~ SY Le pion A he a LL d 3 A re J ow, pe 1 ° 9 9 ¢ yo rs { Arete RA AL 9 A= Sa TY 1 0 a 7° Nr ~ 0 Sg H TY ~ AL Ta HN or a pharmaceutically acceptable salt thereof.

107. The method of any of claims 95-104, wherein the compound L2-Dxd has a structure selected from the group consisting of:: = ok i » Emr SI OR Bees RE be) . I) A ] : Sm Sh Y td A ond, Os 8 oom In i ’ - Cry ng ray Y & A 5 , a i ERATE Sy, H H > N\ IS N=ory Hy ’ LN o 9 H BN Roy, ; 0 a ore WY | bimonAy N., A 3 So - un o Q Sys e H z Noh 9 LQ Ay Hag wi epi LO o J I oh ol = oan 5 Sh EAN ONAN - F SRA" 0 0 Sl 0 | Og NH iJ ~~’ pl Hh 0 o o : A Hoo XL 3 9 0 YY NH {moan ld A AC H o i de® 8 q ®! 1 tN - Aon Sg Sy pom e % : x oH D Xa OY : y CH, 2 Ay - 3 4 3 5 &) 2 Pe WH Q o H Khoo, . SY y " i 1 i or = f # : A HL, A Q } Ou mnAg ‘ Omg " Yr : A SNR § 0 9 ™ i i nn LCL ~ Hoo CY Ferg p ST CN il ~ op a HN - ~o HN Sg Ong . OF "NH t I O. g fondo t,, i o OY r Ls 1 0 5 HN, ) bomen Ay I a4 EB 1 5 4g a Q 2 o Lg Hook, nL ! SORT 6 NT, Cr hn a wl IT Pop ~ tJ a i g oN o. oO! A Op, oy 2 oN Pay 5° 2 5 3 HX y™ 0 Aaa, food, 2 =o monk LE OTT Sead ~ 0 Sy an Ppa, ate ror ht Oy = L3- I RB SSAA) IF ann Sng Ne ; A i gy Son I oon ou Re oo pe q - J oo ay $8 VS " o , com fo My, 2 de Mngt TY XE SGLEA & “OH oN NH Y Nak, ua 1 J jg ~~ nh I ez Hn ~ PEE a oY wo Oo ah on pw he OA pAymmron / v 3 H (po 1, . 3 M3 HN [NN Ay 0 \, BOINSCALE Ras Jur oY ry q : & oe HO | NA, > o y a Q ery o : o Ags Fo 0, Xn 1 Oy mO. He 2 5 ; oie FA I 5 , ~ ~ 9 2 Li 1 Cob ~ 9 Sg H a \, I NT NH, H ? GTN 0 PN No, : I TY TW w Ogy NH \ Hal” On J i : . arn OOK Syn o) NN Spo 3 HN, 1 4 i 4 oO fel hp A ~ 3 AAT [3 H ] ° : ~ oN , Pon 5 1 < NN Y -: 9 H 8 gh Li A Cry as Hak, fas AS © A FEY Co : ~ ~ ad he 1 va he aon ~ a Sy, y oN Pon ~ Sy = . WL 8 ’ oo gph = : gy fA a. * rs yr SR Sod H Nr T So | J: bY & } NT UNHy 4 Po = Linn lod Lh 2 : Hedy {, X or ’ : a Aer pd T 0H 3 : pe RS % N NH 1 Ay a I) Ro 3 room Ao dy 3 rnp A wy SG YIVTCY X AE tg A LL, A So rctmgmin i t / No ea Ne [ 4. "ommnded LY Sn TALC oh BN LA, ¥ J goed LN oA © i YOY Ce Sdn wo 9 Aor Sh ¢ Ho oo y Vomongled LA | Saag ed re Te h an, or Et Sond 0. A, N < : y J PAYY & : rtrd, ~ "ar FI ytd a i H i ¥ TTT 5 omni 207 x a, ‘ i In ’ YE ry “3 & pr. oe Sows aa ) 6 Lo LA Jb A Se Xx o oon NH ~ Sutalyh Nr Le y el x rrp lr ® RA A S ny : \ ¥ a Ea Sw oy . . Jel, | NY y pte dh LL ~y x Nn DY C3 Dd Pom ~ Q Sr p— Sow on A A ’ ELT 8 LT, X Q é Pon Sr edd } } J Hoo = Le Ln LLY Front HL ? Pon Se, Sy ~ 2 ww Aor She ee Yr 7 Be 0 A 1 Y T. y ° rey ™i C1 YP SA Pro uy 1, ) XL { + | 5 Eo T or oe 9 o i Oe WIT 0 ene 4 2 Q gS Cr pe o YA $f oh ox Nw ~ rp A adage? on ~ og Sw H, Pon 5 a DR Sf eh ror 4 3 0 o é Pom “on 3 POA Oy 5 ~~ 1 i f NOY 8 er TLL AL Y 7 Aon Shoes Sy ° Q Qt LY 8 Ory J oyiel lt 4 = 8 | ~ ~ 3 T Pa Svs A i pm ™~ LA, AT A CY Ay G8 ’ wet dt oA a L ° Hon Se - RUN St ° ° ° oS Lh 9 SHS 5 Ce AY Oy Sad, Ho © pA BR ood WX JA Lf, XJ rT } ; o 2 Ara . g . o 0 aoe only 0 Adm Inno i AL J ALJ ° VY 2 Bol 2 < a 7 uo o 8 AN A [a Sel Xr Ca H 4 ol ° o Qo 4 } tod 5S L 4G FEcad ans gests ~My HY t ( TY ! oe Wh Xr , and am Lor A ys ! Ha JT Ad LTT Je a &e ’ y r : we ous 17% She GF forgo A ¢ Ae min r d : ‘ : 8 mlm 0 8 mm fo Hd Se k TY A : PL o - a Lh ) & ey tid (3 WPELIET cas aap deste A i £ 5 wor Shen ” J

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