NZ793865A - Antibody drug conjugates comprising sting agonists - Google Patents

Abstract

The present disclosure provides scaffolds and antibody-drug conjugates (ADCs) comprising a stimulator of interferon genes (STING). The present disclosure also provides uses of the ADCs in treatment, e.g., treatment of cancer.

Description

The present disclosure provides scaffolds and antibody-drug ates (ADCs) comprising a stimulator of interferon genes (STING). The present disclosure also provides uses of the ADCs in treatment, e.g., treatment of cancer.

NZ 793865 AN'I‘IBQDY DRUG CGNJ‘UGA’I'ES CGR/H’RISING STING 'I‘S D APPLECATIONS [001} This application claims priority to, and the benefit of US Provisional Application No, 63/004,108 filed April 2, 2020, U.S. Provisional Application No. 63/040,755 filed June 18, 2020, and US. Provisional Application No. 63/111,820 filed November 10, 2020. The contents of each of these applications are hereby incorporated by reference in their entireties.

INCORPORA’I‘ION BY REFERENCE 0F SEQUENCE LISTING [002} The contents of the text file named “MRS N—033m001WOMSeqListtxt”, which was created on March 31, 2021 and is 49 K8 in size, are hereby incorporated by reference in their entirety.

BACKGROUND Stimulator of interferon genes (STING) is a or in the endoplasmic reticulum that ates innate immune sensing of cytosolic en derived" and selwaN'A. STING is a 378 amino acid protein, which mainly contains three structural domains: (i) N-terminal transmembrane domain (aa 1—154); (ii) central globular domain (aa 155—341); and (iii) Cuterminal tail (aa 342— 379). STING may form symmetrical dimers combined with its ligands in Vnshaped conformation, yvhile not completely covering the bound ligands A STING agonist can hind into the pocket region of STING. However, the STING activation process is easily inhibited in some severe disease ions, resulting in the inactivation of the STING pathway. Therefore, screening and designing potent STING agonists is of great ance for cancer immune therapy and other infectious diseases treatments, ing, but not limited to, obesity, liver injury, sugar—lipid lism, and virus ion, Specific targeting of immune ys presents opportunities for cancer therapy, potentially offering greater icity than cell population-based therapeutic ches [004} Antibodysdrug conjugates (ADCs) are comprised ot‘a drug like small molecule, covalently linked to an antibody. The antibody represents a targeting mechanism tuned to a specific site of action, Upon reaching the site, the ADC is designed to release a small molecule, the drug, allowing it to perform its ed function in a targeted manner, as opposed to diffusing systemically through the entire body of the subject, This targeted approach allows for treatment with drugs that would otherwise require doses so high as to be toxic when administered systemically. [005} A key feature of the innate immune system is the recognition and elimination of n substances. identification of these pathogenic invaders occurs through host recognition of ionarily conserved microbial structures known as pathogen— associated molecular patterns (PAMPs). Host recognition may occur by multiple pathways, such as activation of pattern recognition receptors {FREQ}, which ultimately lead to downstream signaling events and culminate in the mounting of an immune response. {006} The antibody—drug conjugates of this sure modulate the activity of STING, and accordingly, may provide a beneficial therapeutic impact in treatment of diseases, disorders and/or conditions wherein modulation of STING lator of Interferon Genes) is beneficial, includino, but not limited to, inflammation, allergic and autoimmune diseases, infectious diseases, cancer, prencancerous syndromes, and as vaccine adjuvants. There remains a need for new inimunotherapies for the treatment of diseases, in particular cancer.

SEWER/EARY In some aspects, the present disclosure provides a conjugate of Formula (l): Al—(iflig m coitus (l) or a. pliarinaceutically able salt or solvate thereof, n: PERM denotes a protein—based recognition—in olecule; LC, when present, is a. linker unit; A1 is a divalent linker moiety connecting the PERM to LC when LC is present, or to B when LC is absent; D is a STlNG agonist drug ; and dis is an integer ranging from about i to about 204 [008} In some aspects, the present disclosure provides a scaffold useful for ating with a PERM, wherein the scaffold is ofForrnula (II): p 1~D (H) or a pharrnaceutically acceptable salt or sol'vate thereof, wherein: PERM s a protein—based recognition—rnolecule; LC, when present, is a linker unit; All is a monoValent linker rnoiety comprising a functional group capable of forrning a nt bond with a functional group of the PERM; D is a STING agonist drug moiety; and dis is an integer ranging from about I to about 20. [009} In some aspects, the present disclosure provides a pharmaceutical composition comprising a conjugate described herein and one or more pharrnaceutically acceptable carriers or excipients. {010} In some aspects, the present disclosure provides a method of ting or enhancing an activity of a stimulator of interferon genes (STIN G} in a t, sing administering to the subject a conjugate described herein or a pharrnaceutically acceptable salt thereof.

[Olll In some aspects, the present disclosure es a method of preventing or treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate described herein or a pharmaceutically acceptable salt thereof. [012} In some aspects, the present disclosure provides a conjugate described , or a pliarniaceutically acceptable salt thereof, for activating or ing an activity of STING in a subject.

[Ol3l In some aspects, the t disclosure provides a conjugate described herein, or a pharmaceutically acceptable salt thereof, for preventing or treating a disease or disorder in a subject. [0i All In some aspects, the present sure provides a use ofa conjugate described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a ment for activating or enhancing an activity of STING in a subj ect, [015} In some aspects, the present sure provides a use of a conjugate described herein, or a pharrnaceutically acceptable salt tl’iereof, in the cture of a medicament for preventing or treating a disease or disorder in a subject. 6] Unless otherwise defined, 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. in the specification, the singular forms also include the plural unless the t clearly dictates otherwise, gh methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. Ihe references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will l. In addition, the materials, methods and examples are illustrative only and are not intended WO 02984 2021/025556 to be limiting. ln the case of conflict n the chemical structures and names of the compounds disclosed herein, the chemical structures will control {017} Other es and advantages of the disclosure will be apparent from the following detailed description and claims.

BREE}? DESCRlP'HQN 0%? THE FEGURES {018} Fit}. 1A plots the red object confluency as a function of time for Conjugate Sh—l and Conjugate 31‘ (each at 100, 10,, and 1 nM) and Conjugate Soul and Compound 1 (each at 100 and 11M) (conjugate concentrations were based on the payload).

[Olgl EEG. 13 plots the red object confluency as a function of time for Conjugate SE and Compound 1 (each at 100,, 10, and 1 nM) and Conjugate 8m (lOO nM) (conjugate concentrations were based on the payload). [020} PEG. 2 shows CDl44CD3+ cells in PBMCs, enriched nionocytes, and CDl6~depleted inonocyte populations.

[Ole EEG. 3A and FIG. SB plot the red object confluency as a function of time and show the killing of STING wild type (sgNT—2} and knock out (sg#3~2} SKBR3 NucRed cells respectively by PBNle for Conjugate 83—3? Conjugate 8j, Conjugate 8oz, and Compound I each at NHL 25‘, 5 and 1 nM (conjugate concentrations were based on the payload). [022} EEG. 4A plots the red object confluency as a function of the dose response of Conjugate 8a— 3, ate 8—j, Wild type Fe ’I‘rastuzumab and AAG Fe mutant Trastuzurn ab for the STING Wild type SEER?) cancer cell killing activity in SKBR3 cancer cell/PBMC co~cultures [023} Fifi. 4}} plots the red object confluency as a function of the dose response Conjugate 8a~39 Conjugate 81'; Wild type Fe Trastuzuinab and AAG Fe mutant Trastuzuinab for the STINQ knock out SKBRS cancer cell killing activity in SKBR3 cancer BMC tures.

[O24] EEG. SA plots the change of the red object confluency of OVCAR3eNucRed cancer cells as a function of time by PBMCS for Conjugate Sbwl and Compound 17 both at 20 and 4 nM (conjugate concentration based on payload tration). {025} Fit}. SB plots the change of the red object confluency of OVCAR3 d cancer cells as a. function of tinre by enriched nronocytes for Conjugate Shaft and Compound 1, both at 20 and 4 nM t:conjugate concentration based on payload concentration). {026} FIG. SILT, plots the change of the red object confluency of OVCAR3—NucRed cancer cells as a function of time by CD16 depleted nionocytes for Conjugate Sh-I and Compound I, both at and 4 nM (conjugate concentration based on payload concentration). [ ‘27] is a graph showing the anti—tunioi' efficacy of Trastuzuinab (3/0 ring/kg}, diABZI S'I'ING agonist (0/5 nig/ltg), Conjugate Sc—I (1/004 nig/kg or 3/012 nig/ltg), Conjugate Sa—I (1/003 mg/kg or 3/009 ing/kg) (all doses are given by antibody/payload) in a SKOVB aft model in mouse.

FIGS. 7An7I-I show cytokines levels for murine CXCLnIO (IP~10; ), IL~6 (), INFa (), IFNV (), CXCLI (KC) (), MIG (), MIPJa (), and KANI‘ES (FIG. '7I-I) as a function of time following administration of {liABZI STING agonist (0/5 ing/kg), Conjugate Scni (3/012 mg/kg) or Conjugate 8amI (3/009 mg/kg) (all doses are written as antibody/payload) in a SKOVB mouse model. Inserts in each plot show cytokine levels induced by Conjugate SanI and Conjugate 8c—I relative to vehicle.

FIGS. 8A~8C show the levels of mouse CXCLIO (), Interferon—{3 (FIG. SB), and III—6 () rnRNA in a SKOV3 xenograft model in mouse at 12 h and 72 h following administration of Conjugate 8c~I (3/012 ing/kg), ate 8a~I (3/009 ) So , Conjugate I (3/0 I 2 mg/kg) or Conjugate 8:3» I (3/009 nig/kg) (all doses are written as antibody/payload), [030} FIG. I) is the CD45 innnunohistocheinistry (II-IC) ng with rabbit anti—CD45 onal antibody at l2 and 72 hours for ate 8a«I (3/009 nig/kg), Conjugate Sew]. 3/012 rug/kg) or vehicle.

FIG. II} is a graph showing the circulating plasma concentrations of total antibody and conjugated drug following administration of Conjugate SawI (3/0} mg/kg) (dose is given as antibody/payload) to CB. l 7 SCID mice, {032} FIG. II shows the effect, ot‘IFNM (I129 or IFNX2(11,28 A) neutralizing antibodies (5 07 2, 0.4, 0.08 L) on the g activity of Conjugate Saws (1 nM or 0.1 nM, based on payload) in cancer cell and PBh/IC coecultures. [033} FIG. IE! is a graph showing the anti—tunioi' cy of diABZl STING agonist (0/5 ), Conjugate Sex-I (3/0l2 nig/kg), Conjugate SbuI (3/009 ing/kg} or Conjugate SI (3/012 ing/kg) (all doses are given by antihotly/payload) in a ()VCART: xenogi'aft in mouse. [034} F1C” 13 is a graph showing the anti-turner efficacy of Conjugate 80—2 (3/01 rng/lrg) or Conjugate 8c (3/01 rng/kg) (all doses are given as antibody/payload) in a ()VCAR3 xenograft in mouse. is a graph showing the anti—tunior cy of diABZl STING agonist (0/5 rng/kg), Conjugate 8cm} (1/004 nig/ltg) or Conjugate 81} (1/004 rng/kg} (all doses are given as antibody/payload) in a triple negative breast cancer xenograft in rnouse . [03 6} F1C” 15A is a graph showing the anti—tunior efficacy of Conjugate Sari-3 (1/004 mg/kg) or Conjugate 3g (088/004 ring/kg) (all doses are given as antibody/payload) in a colon cancer syngeneic mouse model.

EEG. 1513 shows the Kaplan Meier survival curves for Conjugate 80—3 (1/004 ) or Conjugate 8g 04 nig/kg) (all doses are given as antibody/payload) in a colon cancer syngeneic mouse model. [03 8} F186 16A is a graph showing anti~tunior efficacy of Conjugate 85.: (09/004 nig/lrg) (all doses are given as antibody/payload) for the dual mice in a colon cancer syngeneic mouse model [03 9] 13 is a graph showing the anti~tnntor efficacy of Conjugate 8g (09/00/41 nag/kg} (all doses are given as antihody/paylcad) when re~challenged with syngeneic inurine colon cancer cells. [040} EEG. MC is a graph g the anti—tumor efficacy of Conjugate 3g (09/004 nag/kg) (all doses are given as antibody/payload) when re—challenged with eic niurine lung cancer cells MG. 17 is a graph showing the anti—tumor efficacy of diABZl STlN 3 agonist (0/5 nig/kg) Conjugate 8&3 (55/018 trig/kg) or Conjugate 8i (32/018 nig/kg) (all doses are given as ly/payload) in a syngeneic inurine embryonic cancer model. [042} is a graph showing the antietunior efficacy of ate 20a (3/009 trig/kg), Conjugate 34a (3/01l trig/kg), ate 34 (3/0ll mg/kg), or Conjugate 20-1 (3/010 nig/kg) (all doses are given as antibody/payload) in a SK() «’3 xenograi‘t in mouse.

PK}. 19 is a graph showing the anti—tunior cy of Coniugate 28 (03/001 nig/kg or 1/003 nig/kg), Coniugate 29 1 rng/kg or 08/002 nig/kg), Conjugate 3:2 (03/001 rng/kg or 1/004 rng/kg), Conjugate 25 (03/001 rng/kg or 1/004 rng/lrg) or Conjugate 45 (03/001 mg/kg or 1/004 ing/kg) (all doses are given as antihotly/payload) in a SKOV3 xenograf‘t in mouse. [044} Fit-Sn, 20 graph showing the antiutumor efficacy of diABZI STE NG agonist (1.5 mg/kg q3dx3 or 0.128 mg/kg qu1), Compound 30 (1.5 mg/kg {13dx3 or 0.128 tug/kg qu1), Conjugate 320;: (342/0128 trig/kg qui}. XM,’1‘~1 519 (3.00 nag/kg qui), Conjugate 32—5 0004, 0300/0013, 42 or 300/0128 mg/kg qu1), Conjugate 329; (100/0039 or 300/0117 mg/kg qu1} :) (aii doses are given as antibody/payload) in a SKOV3 xenogi'aft in mouse. [045} FM}. 2114 is a graph showing the anti-tumor efficacy in a syngeneic mouse model of ate 8d—3 (1/004 mg/kg) 01' Conjugate 8k (09/004 nag/kg).

EEG. 213 shows the anti—tumor efficacy in a syngeneic mouse mode1 ofthe individual mice for Conjugate 8dr?) (1/004 nig/kg).

EEG. 21C shows the antintumor eficacy in a syngeneie mouse model of the individuai mice for Conjugate 81: (09/004 mg/kg).

EEG. 22 is a graph g the anti—tumor efficacy of Conjugate 802 (317/010 mg/kg), Conjugate Sari: (27/010 mg/kg or 081/003 mg/kg), Conjugate Sj 10 mg/kg or 081/003 mg/kg), or i 1V STING agonist (0/5 nig/kg) (ail doses are given as antibody/payload) in a SKOV?) xenograft in mouse. [049} is a graph showing the anti —tunioi efficacy of Conjugate 323%} (339/010 nag/kg), Conjugate 32a (093/0103 or 0 nag/kg} Conjugate 321: (212/010 nig/kg), Conjugate 32d (220/010 nag/kg) or diABZTE 1V STING agonist (0/5 ‘) (all doses are given as antihody/payioad) in a OVCAR3 xenogiatt in mouse. [050} is a graph showing the anti~turnor efficacy of a combination of Rituximah AF“ EPA ADC (075/0023 mg/kg) and Conjugate 863—2 (40/0126 mg/kg); 174535 ARHPA ADC (075/0.024 mg/kg); Conjugate 813—2 (20/0071 0140/0142 mg/kg); a combination of KEV/111535 A‘EKHPA ADC (075/0024 trig/kg) and Conjugate Sic-2 (40/0126 nag/kg); a ation of Rituxiinab ARHPA ADC 023 nag/kg) and Conjugate 8132 (40/0142 ing/k’g); a coi'nbination of Rituxiinab AF—HPA ADC (075/0023 mg/kg} and Conjugate 813—2 (20/0071 nag/kg); a combination of XIX/17174 535 AF—HPA ADC (075/0024 nag/kg) and Coi'ijugate 813—2 (40/0142 mg/kg}; a combination ofXM'1'—1535 A ADC (075/0024 mg/kg) and Conjugate 80-2 (20/0071 mg/kg); a combination of Xi‘x/1’17—1535 AF—HPA ADC (075/0024 mg/kg) and XM'I'—1535 (4.0/0 mg/kg); or XIX/1131535 (4.75/0 mg/kg) in a OVCAR3 xenograft in mouse. [051} 111C” 25 is a graph showing the anti—tumor efficacy of Conjugate 323) 03 mg/kg) 54 ; Conjugate 32—2 (090/003 mg/ii'g), Conjugate 88 (087/003 mg/kg), Conjugate 85 (287/010 nig/kg), Conjugate 92 (236/010 , Conjugate 100 (223/016 mg/lcg), Con} ugate 89 (099/0030 rug/kg), Conjugate 85a (2.59/0. l 0 rug/kg), Coniugate 939 (285/010 mg/lcg), or Conjugate 191 (279/010 mg/kg} in a SKOV3 aft model in mouse.

MG. 26 is a graph showing the anti-tumor efficacy of with vehicle, Conjugate 28 (099/00325 mg/kg}, or Conjugate 62 (Gill/0.9325 rug/kg) in a SKQVB xenograft model in mouse. {053} Flt}. 27 is a graph showing the circulating plasma concentrations for conjugated drug ing administration of Coniugate '28 (30/016) mg/kg) or Conjugate 62 (284/010 mg/kg) (dose is given as antibody/payload) to CB. l 7 SCID mice.

BETAILED DESCREPTION The present disclosure provides novel antihodyndrug conjugates, synthetic methods for making the conjugates or scaffolds, pharmaceutical compositions containing them, and various uses of the conjugates.

Befinitions [055} The chemical names provided for the intermediate compounds and/or the compounds of this disclosure described herein may refer to any one of the tautoineric representations of such compounds (in some instances, such alternate names are provided with the experimental). It is to be tood that any reference to a named compound (_an ediate compound or a compound of the disclosure) or a urally depicted compound (an intermediate compound or a compound of the disclosure) is intended to encompass all tautomeric forms including zwitterionie forms of such compounds and any mixture thereof.

It is to be understood that the terms “ln some embodiments’, “in some ments of the present disclosure”, and “In some embodiments of a compound ot‘the present disclosure” may be used interchangeably where appropriate. [057} The term “about”, “approximately”, or “approximate”, when used in tion with a cal value, means that a collection or range of values is included. In some embodiments, “about X” includes a range of values that are $2,592; $209.4), .-i-.-l 5%, £1096, $596, $2943, :i:l%, to. 5%, $0.294), or :i:O.l% of X, where X is a cal value. ln some embodiments, the term “about” refers to a range of values which are 5% more or less than the specified value. in some embodiments the term “about” refers to a range of values which are 29/6 more or less than the A.’ specified value. in some embodiments, the term “about” refers to a range of values which are 1% more or less than the specified value. {058} Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the ication as if it were individually recited herein. A range used herein, unless otherwise ied, includes the two limits of the range. in some embodiments, the expressions “X being an integer between l and 6” and “X being an integer of l to 6” both mean “X being l, 2, 3, 4, 5, or 6”, 17.6., the terms “between X and Y” and “range from X to Y, are inclusive ofX and Y and the integers there between.

The term ”antibody” as used herein, is used in the broadest sense and encompasses s antibody structures, including but not d to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific dies), and antibody nts so long as they exhibit the desired antigen—binding activity. The numbering of the antibody amino acids is according to Kabat EU lndex (See Kabat, EA, er all, Sequences of Protein of immunological interest, Fifth n, US Department of Health and Human Services, US Government Printing Office (1991)).

The term ”antibody fragment” refers to a molecule other than an intact antibody that ses a portion of an intact antibody and that binds the antigen to which the intact antibody binds, Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab—SH, 2; diabodies; linear antibodies; single-chain antibody molecules (eg. scFv); and niultispecific antibodies forined froi'n antibody fragrn en is [061} The term ody that binds to the same epitope" as a reference antibody as used herein, refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody bloclrs binding of the antibody to its antigen in a ition assay by 509%: or more. An ary competition assay is provided herein.

The term “class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: lgA, lgD, lgE, lgG, and lglvl, and several of these may be further divided into subclasses (isotypes), e.g., lgGi, lgGZ, lgG3, lth, lgAi, and lgAZ. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5., a, y, and u, respectively.

WO 02984 {063} The term “monoclonal antibody“ as used herein, refers to an antibody obtained from a population of substantially homogeneous dies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, eg, ning lly occurring mutations or arising during production of a monoclonal dy preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which lly include ent antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal dy preparation is directed t a single determinant on an antigen. Thus, the modifier ”monoclonal“ indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present ion may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage~display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein [064} The term "epitope" refers to the particular site on an antigen molecule to which an dy binds [065} The term “Proteinvbased recognition—molecule” or “PERM” refers to a molecule that recognizes and binds to a cell surface marker or receptor such as, a transinembrane protein, surface immobilized protein, or proteoglycan, ln some embodiments, the PERM comprises an engineered cysteine, Examples ot‘PBRMs e but are not limited to, antibodies, peptides, lipocalins, proteins, peptides or peptide , and the like. The protein-based recognition molecule, in addition to ing the conjugate to a specific cell, tissue or location, may also have certain therapeutic effect such as antiprolifer'ative (cytostatic and/or cytotoxic) activity against a target cell or pathway. The protein-based recognition molecule comprises or may be engineered to se at least one chernically ve group such as, ~COQH, primary amine, secoi'idary amine ~NHR, —Sl:l, or a chemically reactive amino acid moiety or side chains such as for example, tyrosine, histidine, cysteine, or lysine. in some ments, a PERM, may be a ligand (LG) or targeting moiety which specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. ing specific binding or cornpleaing of the ligand with its receptor, the cell is permissive for uptake of the ligand or -drug—conjugate, which is then internalized into the cell. As used herein, a ligand that “specifically binds or complexes with” or “targets” a cell surface rnolecule preferentially associates with a cell surface molecule via intermolecular forces. In some ments, the ligand can entially associate with the cell surface molecule with a Kd of less than about 50 nM, less than about 5 11M, or less than 500 pM. Techniques for measuring binding affinity ofa ligand to a cell surface molecule are well—known; for example, one suitable technique, is termed surface plasmon resonance {SPR}. In some embodiments, the ligand is used for targeting and has no detectable therapeutic effect as te from the drug which it delivers. in some embodiments, the ligand functions both as a targeting moiety and as a eutic or iniinunoinodulatory agent (6.g, to enhance the activity of the active drug or prodrug). The term “PEG unit” ss used herein refers to a polyethylene glycol subunit having the formula —~ “CHQCH2OM§ §—— . In some embodiments, the PEG unit comprises multiple PEG subunits.

The term "alkyd”, as used , represents a ted, straight or branched hydrocarbon group having the specified number of carbon atoms. The term Ci~C6 alkyl” or "CH; alkyl" refers to a methyl moiety or a straight or branched allryl moiety comprising from 2 to 6 carbon atoms. ary alkyls include, but are not limited to methyl, ethyl, n~propyl, isopropyl, n—butyl, isobutyl, s—butyl, f—hutyl, pentyl and hexyl.

The term alkyl)”, as used , represents a saturated, straight or branched hydrocarbon group having the specified number (n) of carbon atoms and one or more (up to 2n+l) halogen atoms. Examples of ”halot’Cm alkyl)" groups include, but are not limited to, “€173 (trifluoromethyl), ~CCl3 (trichloromethyl), l,l— roethyl, 2,2,2—trifluoroethyl, and hexafluoroisopropyl, [068} The term “alkenyl”, as used herein, refers to straight or branched hydrocarbon group having the specified number of carbon atoms and at least 1 and up to 3 carbon—carbon double bonds.

Examples include ethenyl and propenyl.

The term ”alkynyl“, as used herein, refers to straight or branched hydrocarbon group having the specified number of carbon atoms and at least I and up to 3 carbon-carbon triple bonds.

Examples include ethynyl and propynyl. [070} The term “alkoxy-“ or “(alkyl)oxy—”, as used herein, refers to an “alkyluoxy—” group, comprising an alkyl moiety, having the specified number of carbon atoms, attached through an oxygen linking atom. Exemplary ”Ci—4 alkoxy—” or ”(Ci—4 alkyl}oxy-' groups include, but are not limited to, niethoxy, ethoxy, u—propoxy, isopropoxy, 'xy, S—butoxy, and lubutoxy. {071} The term “halo(all«:oxy)—”, as used , represents a saturated, straight or branched hydrocarbon group having the specified number (n) of carbon atoms and one or more i up to Err-ll) n atoms, attached through an oxygen linking atoni. Exemplary “halo(C 1-4 alkoxy)-“ groups include, but are not limited to, ~00:le (difluoromethoxy), —0CF3 (trilluoromethoxy), —0Cl-l2Cli‘3 (trifluoroethoxy), and —0Cl—l(CF3)2 (hexafluoroisopropoxy).

The term “amino” as used herein refers to a suhstituent sing at least one nitrogen atom. Specifically, —NH;2, —NH(Ci.4 all<yl), alkylamino, or (CM alkyl)amino— or (CM )(Ci-4 alkyl)aminon or diallrylamino, amide", carbamidew, urea, and sulfaniide substituents are included in the term “amino”.

The term “carhocyclic group or moiety” as used herein, refers to a cyclic group or moiety wherein the ring members are carbon atoms, which may he saturated, partially unsaturated (non— aromatic) or fully rated tic). [0’74] The term "cycloallryl", as used herein, refers to a non—aromatic, saturated, hydrocarbon ring group comprising the specified number of carbon atoms in the ring. For example, the term "CS-6 cycloalkyl" refers to a cyclic group having from three to six ring carbon atoms, Exemplary ”Cs-.6 cycloalkyl“ groups include cgclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, [0’75] The term “aryl”, as used herein, refers to a group with aromaticity, including “conjugated” or multicyclic systems with one or more ic rings, which does not contain any heteroatom in the ring structure The term aryl includes both monovalent species and divalent species Examples of aryl groups include, but are not limited to, phenyl, hipheiiyl, naphthyl and the lilre. ln some embodiments, an aryl is phenyl [076} The term “l’ieterocyclic group or moiety”, as used herein, refers to a cyclic group or moiety having, as ring s, atoms of at least two different elements, wl'iich cyclic group or moiety may be saturated, partially rated romatic) or fully unsaturated (aromatic) The term "heteroatom", as used herein, refers to a nitrogen, sulfur, or oxygen atom, for example a nitrogen atom or an oxygen atom.

The term "heterocycloalkyl", as used herein, refers to a nonuaromatic, monocyclic or bicyclic group comprising 3ul0 ring atoms and comprising one or more (generally one or two) heteroatom ring members independently ed from oxygen, sulfur, and nitrogen. The point of attachment of a heterocycloalkyl group may be by ar y suitable carbon or nitrogen atom. [079} The term “lietei'oar'yl”, as used herein, refers to an ic yclic or bicyclic group comprising 5 to 10 ring atoms, including l to 4 heteroatorns independently selected from nitrogen, oxygen and sulfur, wherein at least a portion of the group is aromatic. for example, this term asses hicyclic cyclic—aryl groups comprising either a phenyl ring fused to a heterocyclic moiety or a heteroaryl ring moiety fused to a yclic moiety. The point of attachment of a heteroaryl group may be by any le carbon or nitrogen atom. [080} The terms “halogen” and , as used herein, refers to a halogen radical, for example, a fluoro, chloro, bromo, or iodo substituent. [081} The term ”one", as used herein, refers to a double—bonded oxygen moiety; for example, if attached directly to a carbon atom forms a carbonyl moiety (C=O). [082} The term “hydroxy” or ”liydi'oxyl", as used herein, is intended to mean the radical “OH, The term ”cyano", as used herein, refers to a nitrile group, “GEN. [084} The term ”optionally substitutet ”, as used herein, indicates that a group (such as an alkyl, cycloalkyl, alkoxy, heteroeycloalkyl, aryl, or heteroaryl group) or ring or moiety may he unsubstituted, or the group, ring or moiety may be tuted with one or more substituent(s) In the case where groups may be selected from a number of alternative groups, the selected groups may be the same or different, Suitable substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, allmxycarbonyloxy, aryloxycarbonyloxy, ylate, alkylcarbonyl, arylcarbonyl, alkoxycarhonyl, aminocarbonyl, alkylaminocarhonyl, dialkylaminocarhonyl, alkylthiocarhonyl, l, phosphate, phosphonato, phosphina‘to, amino ding allgylamino, dialkylamino, arylamino, diarylamino and alkydarylamino), acylamino (including alkylcarhonylaniino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfnydryl, alkylthio, arylthio, thiocarboxylate, sulfates, allrylsulfinyl, ato, sullarnoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, allrylaryl, or an aromatic or heteroaromatic y. {085} The term “independently“, as used herein, means that where more than one substituent is selected from a number of possible suhstituents, tl'iose suhstituents may he the same or different. {086} The term ”pharrnaceutically acceptable”, as used herein, refers to those compounds, conjugates, materials, compositions, and dosage forms which are, within the scope of sound niedical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0871 As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a ceutically able salt, polynioi'ph or solvate thereof, to ate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include ent of a cell in i-‘z'rro or an aninral model.

As used , the term “preventing,” “prevent,” or cting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, ion or disorder. [089} The term “subj ect” refers to an animal, preferably a mammal, most preferably a human, who has been the obj ect of treatment, observation or experiment. [090} The term “therapeutically effective amount” refers to an amount of an active compound or pharmaceutical agent, including a conjugate of the disclosure, which elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a cher, narian, medical doctor or other clinician, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. [091} A therapeutically “effective amount” is intended to mean that amount ofa conjugate that, when administered to a. patient in need of such treatment, is ient to effective treat or prevent, as defined herein. The amount of a given conjugate that will correspond to such an amount will vary depending upon factors such as the particular conjugate (eg, the potency , efficacy ( 1:71:50), and the biological l'ialf—life of the particular ate), disease condition and its severity, the identity (eg age, size and weight) of the t in need of treatment, but can heless be routinely determined by one skilled in the art. Likewise, the duration of treatment and the time period of administration (time period between dosages and the timing of the dosages, e.g., before/with/after meals) of the conjugate will vary according to the identity ofthe niammal in need of treatment (eg, weight), the particular conjugate and its properties leg, pharmacokinetic properties), disease or disorder and its severity and the specific composition and method being used, but can heless be determined by one of skill in the art. [092} The term “composition” refers to a product that includes the specified ingredients in eutically effective amounts, as well as any product that results, directly, or indirectly, from combinations of the specified ingredients in the specified amounts.

As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pliarmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient. [094} The term “STING agonist”, as used herein, refers to a compound or moiety which is capable of interacting with STING, eg, by binding to STING and/or inducing downstream signal transduction teg. characterized by tion of the molecules associated with STING function).

This includes direct phosphorylation of STING, IRES and/or NT~kB and could also include I‘o. In some embodiments, STING pathway activation results in increased production of type l interferons (mainly IFN~a and IIi'wa) and/or expression of interferon~stiniulated genes. [095} The term “STING agonist drug ”, as used herein, refers to a moiety derived from a STING t and capable of interacting with STING. In some embodiments, the STING agoni st drug moiety is a moiety derived from a STING agonist to allow the moiety being linked to the rest of a eonj ugate of the present disclosure. {0%} The ates of the disclosure are useful in methods for treating or ameliorating a viral infection, disease, a syndrome, a condition or a disorder that is affected by the agonism of STING.

Such methods comprise, consist of and/or consist essentially of stering to a subject, including an animal, a mammal, and a human in need of such ent, amelioration and/or prevention, a eutically efl'ective amount of a ate of the disclosure, or an ei'iantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof.

In some ments, conjugates of the disclosure, or an enai'itiomer, diastereomer, solvate or pharmaceutically acceptable salt form thereof are useful for treating or ameliorating diseases, syndromes, conditions, or ers such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B. [098} The terms "conj ugatets) of the disclosure" or ”conitigate(s) of the present disclosure”, as used , mean a conjugate as defined , in any form, i.e., any tautonieric form, any ic form, any salt or non—salt form (e.g as a free acid or base form, or as a salt, particularly a pharrnaceutically acceptable salt thereof) and any physical form thereof (eg including non" solid forms (e.g liquid or semi—solid forms), and solid forins (e.g amorphous or crystalline fornis, specific polymorphic forms, e forms, including hydrate forins (e.g niono—, di— and hemiu hydrates)}, and mixtures of various forms. {099} Accordingly, ed within the present disclosure are the conjugates as disclosure herein, in any salt or non-salt form and any al form f, and mixtures of various forms. While such are included within the present disclosure, it will be understood that the conjugates of the present disclosure, in any salt or nonnsalt form, and in any physical form thereof, may have varying levels of activity, different hioavailabilities and different handling properties for formulation 100 As used herein, the ex tressions “one or more of A 59 -;c one or more A » E‘ 7 B, or C 7 7 B, or C ”7 “one or more of A, B, and C,” "one or more A, B, and C,a: as selected from the group consisting of A, B, and C” ted from A, B, and C”, and the like are used interchangeably and all refer to a ion from a group consisting of A, B, and/or C i.e., one or more As, one or more Bs, one or more Cs or any combination f, unless indicated otherwise. [lOll It is understood that, throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that cornpositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited sing steps, r, it should be understood that the order of steps or order for ming certain actions is immaterial so long as the invention remains operable, Moreover, two or more steps or actions can be ted simultaneously [1 02] All tages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different es. The provided examples illustrate ent components and n'iethodology useful in practicing the t disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure. [103} All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor tloes it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

Conjugates and Scaffolds of the lh‘esent fiisclosnre In some aspects, the present sure es a conjugate of Formula (I): PERM—[Al {the m- i'DLn 5 (r) or a pharmaceutically acceptable salt or solvate thereof, wherein: PBRl‘vl s a protein—based recognition~n1oleciile; LC, when present is a linlter unit; Aj is a divalent linker inoiety connecting the PERM to LC when LC is t, or to D when LC is ahsent; D is a STING agonist drug moiety; and dis is an integer ranging from about l to about 20. [l 05} In some embodiments, the conjugate is of Formula (LA): PBRNLlAl—Dldis (LA) or a pharmaceutical ly able salt or solvate thereof. [106} In some embodiments, the conjugate is of Formula, (LB): l-LQD]d15 (LB) or a pharnraceutically acceptable salt or solvate thereof In some embodiments, the conjugate is ofForinula (l—B’): / l1\ AUKMA—j—Lm-D d . 15 (l-B’) or a pharniaceutically acceptable salt or solvate thereof. {108} In some aspects, the present disclosure provides a scaffold useful for conjugating with a PERM, wherein the scaffold is ofForrnula (ll): AV—(Lca m- oo (ll) or a pharmaceutically able salt or e thereof, wherein: PERM denotes a protein—based recognition—molecule; LC, when present, is a linlter unit, All is a lent linker moiety comprising a functional group capable of g a covalent bond with a functional group of the PERM, and D is a S'l’lNG agonist drug moiety. ln some embodiments, the scaffold is ofForniula (ll—A}: Alli) (iris) or a pharmaceutically acceptable salt or solvate thereof. [110} In some embodiments, the scaffold is of Formula (ILB): Alch—o (ll—B) or a pharma ceutically acceptable salt or solvate thereof. [l l 1] In some embodiments, the scaffold is of a ): / ll \ A llisliAw—f- 1.9—1) I (ll—B’) or a pharrnaceutically acceptable salt or solvate thereof. [l l2] It is understood that, for a conjugate of any one of Formulae (l), (la/5‘s), (fl—B), (l-B’), (ll), (ll—A), (ll-B), or (1143’), or a pharmaceutically acceptable salt or solvate thereof, variables PERM, LC, Al, Tl, MA, LD, D, and do can each be, where able, selected from the groups described herein, and any group described herein for any of variables PBRNI, LC, A Tl, M“, L”, D, and dis can be combined, where able, with any group described herein for one or more of the remainder of variables PBRlVl, LC, Al, Ti, MA, L”, D, and dis.

Variable r315 [l l 3] In some ments, dis is an integer ranging from about '2. to about l4, from about 2 to about 12, from about 2 to about 10, from about 2 to about 8, from about 2 to about 6, from about 2 to about 4, from about 4 to about l0, from about 4 to about 8, from about 4 to about 6, from about 6 to about 14., from about 6 to about 12, from about 6 to about 10, from about 6 to about 8., from about 8 to about 14, from about 8 to about 12, or from about 8 to about if). [114} In some embodiments, di 5 is an integer ranging from about 2 to about 8. [115} In some embodiments, dis is 2, 4, a, or 8. in some embodiments, dis is 6 or 8. {116} In some embodiments, tbs is 8.111 some ments, tbs is 6. ini‘iiitbies A I and/1 " In some embedimen’ts, each A1 ndently is a divaient 1inkei' moiety connecting the PERM to LC when LC is present, or to D when LC is absent. /97 I}? N W 38/43 2,. t i N’E o \< [118} In some ments, each A1 xfentiy is: O ; 0 ; \/<,Max/xi‘vflf/\C:W/xxxr wherein: R7 is 43—, "NR8, "(Cl-Cit) alkyi}, "(Ci—CmaikenyI)", "(Ct-{:10 a1kynyi)—, {Cs—Cs aikyiy , ,——0"(Cl—Cs 511131) —O-(Ci-Cm alkenyl}, ~0—(Ci—Cm alkynyt)", {Ci—Cit; alk31)-(C3-Cs ikyi): “(Ci—CmalkyDnaryig “(Ca—CmalkenyD—(Cans cycloaikyhg ~(Cszioaikenyiyaryln, n (Canm alk3'nyl)~(C3~Cg ikyl}, «(C2~Cio alkynyD—amb, ~(C3~Cg cycloaikyD—(C1C 10 311(3): - ai‘yHCi-Cm 2111(3)", —(C3~Cg eycloaik371)w(Cszm yi}, waryint'Canio aikenyi}, "(anCs c3'cioalk3’1)n(C2nCio alkynyi): ~ar3’1ntC2nCio alkynyi): -(3- to 8~n1emberedheteroeyeloaikyi): -(5- to S—niembei‘ed heteroaryi)—, “(CiowaikyDnB— to Snmembered heterocycinalkyhn, "(CinCioaikyD— (5- to 8—membered hetereaiyl}, —(_C2"C10 a1ken3’1)n(3n to Snmembei‘edhetei‘neycieakyl}, ~(C2'C10 a1ken3'1)—(S— to 8—membei‘ed hetemawi}, ~(C2—Cio a1k3'n31)~(3~ to 8~membered heterocycioaikyi}, {Cg—Cm a1k3n3i1)~(5~ to 8~niembered hetemary1}, {3— to 8—menibered heteroeyctoa}k31)~(Ci~Cm a1k31)~ ~(5 to 8membered beteroaryt)(C-~Cmalky})— ~(3 to 8membetedheterom01031133—1)(C2— Cm aikenylfi ~(5~ to 8—menibered heteroawl)—(C2~Cio aikenyt )~., {5— to 8~membered hetemaryhv {Ca-Cm 211101371)“, “(5 to 8~membered hetei'oaiyl)e(Cz~Cioaiknyi)-, ~13“C(03-(CHzCH2O)1~—(CHz)2~, ~(C112CHzO);--, or ~(CH2CHzO)r(CHz)2—, wherein the alkyl, iyl, alkynyi, cycloaikyh my}, heterocydoalkyi, 0r hetemaifl is nptionahy substituted; R8 is H, hydmxy, or (:14 alkyi; r is an integer ranging from about 1 to about 12; and * denotes attachment t0 PERM and 23* denotes attachment to LC when LC is present, er to D when LC is . [119} In some embodiments, R7 is —0—, —NR8, m alkyd}, -(C3-Cs a:ycleaiky1)—, —ary1—, ~0— {Ci—Cs aIkyE)—, alityE)—aryt—, —ai'yi—(Ci,-Cioatkyi}, -(Cj-Ct0 alkyi)-(C3-Cs cyeloalkyE)—, {Ca- Cs eyetoalkyt)-(C1~Cio alkyd}, _(3_ to Sumemheted hetei'ocyclnatkyl)—, —(5- to 8-menibered heteroamt}, -(Cj-Ct0 alkyt}—(3- to B—ntemhered heterocyetoalkyi}, —(Ct—Cio a1kyi)—(5u to 8- memberedhetei‘eai'yi}, —(3- to S—n’remhered heteteeycloalkyi)—(C14310 alkyt}, ~(5— t0 8-menibered hetei‘oaQ/‘D~(C1~Cio ethyl}, nO~C(O)n(CH2CH20)i-n(CI-12):: —(CH2CH20)r—, or ~(CH2CH20)r In some embodiments, R7 is —(Ci—Cioalkyi)~, —0—(Ci—Cs , —(CH2CH20}r—, ~0~C(O)— (CH2CH20)i-~(CH2);— or ~(CH2CH20)r—(CH2)2—. [121} In some embodiments, R7 is ~O—, “NH, —N(CH3), —CH2~, —(CH2)2—, —(_CH2)5~, «(D-{KO}— (CHzCH;zO)e—(CH2)2—, H2:O) ~(CH2)2—, '(CHZCHZO)2"(CHZ)23 —(CH2CH2:O)4U(CH2)23 or — (CH2CH20)6~(CH2)2~. a *4 i‘ \M/" xx R” N”? /' \T ‘“ . <\ [122} .

In some embodiments, each A1 independently is:. , ‘o ; O ‘ ./Q 35 If ,0 \Nr/m /E\I I, CV 3;,“ NW w i N W:\ .. \n 2, \z O O < O ‘0 ; or o , wherein R3 is H, i'iydroxy, at (:1—4 alky}; r is an integer ranging from about 4 to abeut 6; and denetes ment to PBRIVI and M s attachment to LC when LC is present, or to D when LC is absent. [123} It is understood that each A}, print to being eonnected t0 PERM, independently corresponds to a nlonovalent moiety A In some embodiments, each A1 independently is: c- o ; or o -v a wherein R’, R3, and r are as described herein; and W denOtes attachment to If“ when L‘ is present,. i . r» i . . or to B when I} is absent, In some embodiments each A1 independentiv is: (Eire/We; 0r wherein: r is an ii'iteger ranging from about 4 to about 6; and *"4 denOtes attachment to If when I} is present, or to B when I} is absent.

Variable LC [126} In some embodiments, each LC, when t, independentiy is: #-§——i\-t\iiA;i—LD—§w #3: D or "I wherein: #9 denotes attachment to A1 and ## denotes attachment to D; MA, when t, is a peptide moiety comprising at least two amino acids, T], when present, is a hiiic group; and LD is a divalent iinker moiety connecting D to MA when MA is present, or to A! when MA is ahsent. {127} In some embodiments, each LD comprises at least one cieavabie hond such that when the bond is broken, D is released in an active form for its intended therapeutic effect.

LD—E— ## [128} In some embodiments, each LC, when present, independentiy is if? (T1i l ‘ ‘ ~ # g Mn IJD #1; In some embodiments, each LC, when present, independently is t 7 _§- [129} . ialile U) [l 30} In some embodiments, eech LD independently is a divalent linker moiety connecting D to MA when M" is present, or to A1 when MA is absent, [l 31} In some embodiments, each LD ses at least one cleavable bond such that when the bond is cleaved, D is released in an active form for its ed therapeutic effect. [l 32] In some embodiments, LB comprises one ble bond, In some embodiments, LD comprises multiple cleavage sites or bonds. [l 33] It is understood that each LD, prior to being connected to D, ndently corresponds to a inoriovalent moiety IrDyi [l 34] In some embodiments, LD comprises a functional group capable of forming the cleuvable bond. Functional groups capable of forming the cleavable bond can include, for example, sull'hydryl groups to form disulfide bonds, aldehyde, ltetorie, or hydrazine groups to form hydrazoiie bonds, hydroxylamine groups to form OleB bonds, carboxylic or amino groups to form peptide bonds, carboxylic or hydroxy groups to form ester bonds, and sugars to form glycosidic bonds. [135} In some embodiments, each LU comprises a disult‘ide bond that is cleavable through disulfide exchange, an acid-labile bond that is ble at acidic pH, and/or bonds that are cleavable by hydrolases. In some ments, LD comprises a, carbemate bond tie, 4343(0)“ NR“, n R is hydrogen or alkyl or the like).

In some embodiments, the structure and sequence of the cleavable bond in L3 can be such that the bond is cleaved by the action of enzymes present at the target site. In some embodiments, the cleavable bond can be ble by other mechanisms. [137} In some embodiments, the structure and sequence of the cleave ble bonds in LB can be such that the bonds are cleaved by the action of enzymes present at the target site. In some embodiments, the cleevable bonds can be cleave ble by other mechanisms. [138} In some embodiments, the cleavable ) can be enzymatically cleaved by one or more enzymes, including a tumor—associated protease, to liberate the Drug unit or I), wherein the ooniugate of the present disclosure, or intermediate, or seaffoid thereof, is protonated in viva upon release to provide a Drug unit or D. méleE ngm\ AM a In some embodiments, eaeli LD independent is wherein: {140} LE when present, is ‘NI‘L‘E:((jEIZ(:}'{2{))p"i:(i}{2)0-2:Eq*£j(0)“, —NI—Iu(Ci—Cs aikyi)—()—C(O)—, or -NI-I—{(CI-IQCI:12())p—(CI.-Iz)o—2]q—Ct{))—NH—(Ci—C6 —()-C(O)—, wherein p is an integer ranging from about I to about 20, and q is an integer ranging from about I to about 10; each W independently is a naturai or unnatural amino acid unit; w is an integer ranging from about 0 to about 12; *** denotes attachment to MA when M" is present, or to Al when M" is absent; and **** denotes attaehment to D.

Haifa—E_______gusset-s- [141} In some embodiments, each LD ndent is . In some ***m **‘** ____ “NW-1%— embodiments, eaoh LD independent is E , In some embodiments, each LD aka-9: E LEWW 11‘*** w 3 independent is _ {1-42} In some embodiments, LE comprises at least one PEG unit. [143} In some embodiments, the PEG unit comprises at least I subunit, at least 2 subunits, at Ieast '3 subunits, at least 4 subunits, at Ieast 5 subunits, or at Ieast 6 subunits In some embodiments, the PEG unit comprises at least 4 subunits, at least 3 subunits, at Ieast 2 subunits, or at least 1 t. In some ments, the PEG unit comprises at least I subunit In some embodiments, the PEG unit comprises at least 2 subunits.

[I 44] In some ments, p is an integer g from about I to about I 5, from about I to about IO, from about I to about 9, from about l to about 8, from about I to about 7, from about l to about 6, or from about I to about 5, [I45} In some embodiments, p is an integer ranging from about I to about 6. In some embodiments, p is an integer ranging from about I to about 4, In some ments, p is an integer ranging from about I to about 2.

In some embodiments, p is 2. [147} In some embodiments, q is an integer ranging from about I to about 15, from about I to about 10, from about I to about 9, from about I to about 8, from about I to about '7, from about I to about 6, or from about I to about 5.

In some embodiments, q is l, 2, 3, 4, or 5. In some ments, q is 2 [149} In some embodiments, LE, when present, is —NI—I—(CH7.CI—I20)i~4—(CII2)2-C(O)—. In some embodiments, LE, when present, is —NH—(CI—I2CH7.O)2—(CH7.)2-C(0)—. In some embodiments, LE, when present, is —NI—I—(CH7.CI—I20)3{CI-Izha-CiOI. In some embodiments, LE, when present, is _ 2CHZG)3"(CHZ>I~C(O)". In some embodiments, LE, when present, is ~NH~(CH2CHZO)3' (CH2)2—C(0)—. In some embodiments, I1 when present, is ~NHnCH2CH20w(CII2)o-zr-C(O)n. In some embodiments, LE, when present, is "NHvCI-IzCH;zOnC(O)~. In some embodiments, LE, when present, is "NI-Iwmi—Cs —0—C(0)—. In some embodiments, LE, when present, is —NI-IICH2n CH(CH3)wOnC(O)w. In some ments, LE, when present, is ~NH"[(CH2CHZO)l~4"(CH’l)2' C(O)'NH~(CI'CS aIkyl)—Ow£l(0)n. In some embodiments, LE, when present, is "NH—CHzCHmOn (CH2)2—C(O)—NII~(CH2)2nO~C(O)n.

In some embodiments, w is an integer ranging from about I to about 12 (eg, I to 6, or I to 4, or I to 3, or I, 2, 3, 4, 5, 6, 7, 8, 9, I0, I I, 01‘ 12), [1 SI} In some embodiments, w is O, I, 2, '3, 4, 01‘ 5, In some embodiments, w is I, 2, 3, 4, or 5.

[ISZI In some embodiments, w is 1. In some ments, w is 2. In some embodiments, w is [153} In some embodiments eaeh W ndently is a natural or unnatural amino acid and/or a D or I, isomer [154} In some embodiments, each W independently is an alpha, beta, or gamma amino acid that is natural or nonsnatural. [155} In some embodiments, at least one W is a natural amino acid. In some embodiments, at least one W is a non-natural amino acid, [156} In some embodiments, WW does not comprise natural amino acids In some embodiments, ‘Ww does not comprise non-natural amino acids. {157} In some ments, WW comprises a natural amino acid linked to a non-natural amino acid. In some embodiments, WW comprises a natural amino acid linked to a Ill—isomer of a natural amino acid. [158} In some ments, WW is a dipeptide, eg, —Val—Cit-, -Phe-Lys-, -\/"aI-Ala— or Gin—Ala. {159} In scme ments, WW is a menopeptide, a dipeptide, a ptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an ectapeptide, a nenapeptide, a decapeptide, an undecapeptide, or a dedecapeptide unit. [160} In some ments, WW is a peptide (eg, a peptide of l to 12 amino acids), which is ated directly t0 D. In senie embodiments, the peptide is a single amine acid. In some ments, the peptide is a dipeptide. In some embodiments, the peptide is a ti'ipeptide. {161} In some embodiments, each amine acid in Vv’w is independently selected from alanine, B— alanine, ai‘ginine, aspai‘tic acid, asparagine, histidine, glycine glutamic acid, glutamine, phenylalanine, lysine, lencine, serine, tyrosine, thieenine, iseleucine, proline, tryptephan, , cysteine, methionine, selenecysteine, crnithine, penicillamine, amincalkaneic acid, lkyncic acid, aminealkanedieic acid, aminebenzcic acid, antinc—lietei‘ecycle—alkanoic acid, hetei‘ecycle—carbcxylic acid, citrulline, statine, diaminoalkanoic acid, and derivatives thereof.

In some ments, each amino acid in Ww is independently selected from alanine, B- alanine, arginine, aspartic acid, gine, histidine, glycine, glutamic acid, glutaniine, phenylalanine, lysine, lencine, serine, tyrosine, tlireenine, iseleucine, proline, tryptophan, valine, citrnlline, and derivatives thereof.

H63} In some embodiments, each amino acid in WW is independently selected from the proteinogenic and the non~preteinegenic amino acids, [164} In seine embodiments, each amino acid in WW is independently selected from I, or D isomers of the i‘cllcwing amino acids: alanine, [i—alanine, arginine, aspartic acid, gine, cysteine, histidine, glycine, glutamic acid, glutaniine, phenylalanine, lysine, leiicine, inethicnine, serine, i'ie, three-nine, tiyptcphan, proline, crnithine, penicillamine, amincalkynoic acid, amincalkanedicic acid, heteiecyclc- cai'hexylic acid, citrulline, statine, diamincalkaneic acid, valine, citi'ulline, and derivatives thereof. [1655} In some enibodii'nents, each amino acid in WW is independently cysteine, heniccysteine, penicillamine, cinithine, lysine, sei'ine, thi'eonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, seleneeysteine, preline, glycine, iseleucine, leucine, niethicnine, valine, lline, 0i e. {166} In some embodiments, each amino acid in We is independently selected item L—iscmei's of the fellowing amino acids: alanine, fi-alanine, ai'ginine, aspartic acid, gine, histidine, glycine, glutainic acid, glutamine, phenylalanine, lysine, leucine, sei'ine, tyrosine, tbreonine, isoleucine, tryptophan, citrulline, and valine. {167} In some embodiments, each amino acid in WW is independently selected from D—isomers of the following amino acids: e, fi-alanine, ne, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, , e, sei'ine, ne, tbreonine, isoleucine, tryptophan, citrulline, and valine. [168} In some embodiments, each amino acid in Ww is alanine, B—alanine, glycine, glutamic acid, isoglutamic acid, isoaspartic acid, valine citrulline, or aspartic acid. [169} In some embodiments, WW comprises Bnalamne. In some embodiments, W comprises ([3— ne)—(alanine). In some embodiments, V’Vw comprises (Ii—alanine) and optionally ic acid, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)w(alanine), (alanine)n(alanine), or e)w(citruline). [170} In some embodiments, V’Vw comprises (gliitamic acid)~(alanme).

In some embodiments, V’Vw comprises glutamic acid and optionally alanine, glycine, isoglutamic acid, aspartic acid, isoaspartic acid, valme, (ya.lme)~(alanine), (alanine)—(alanine), or e)~(citruline). [l 72} In some embodiments, ‘Ww comprises 2,3«diaminopropanoic acid, In some embodiments, WW comprises (R)—2,3~diaminopropanoic acid. In some embodiments, WW comprises glutamic acid, In some embodiments, WW comprises (glutamic acid)~(alanine), In some embodiments, WW comprises (glutamic acid)“(glyeineHalanine) In some embodiments, WW ses L—glutamic acid, D—glutaniic acid, (L—glutamic acid)- (L—alanine), tarnic aeid)-(Dwalanine), (Dgltnamic acid)~(balanine), arnic acid)-{D— alanine), , (L—glutamic acid)w(glycine’)“(L-alanine), D—glutaniic acid)—(glycine)-(D*a,lanine), (Ia- gltitamic acidl—(glycitie)—(D~alanine), or (D—gltitamic acidl—(glycine)—(_I_4-alanine).

In some ments, WW comprises a carbamate bond in addition to one or more amino acids, [175} In some embodiments, LD (eg, Vv’w) is selective for enzymatic cleavage (eg, by a particular enzyme). In some embodiments, the particular enzyme is a tumor—associated protease. [176} In some embodiments, LD (eg, WW) comprises a bond whose cleavage is catalyzed by cathepsin B, catliepsin C, cathepsin D, or a plasinin protease. [177} In some embodiments, LD comprises a sugar cleavage site. [178} In some emhndiments, LD comprises a sugar moiety (Su) linked Via an oxygen glyeosidic bond to a self-linniolati've group. [179} In some ments, a “self—immolatiye group” can be a til—functional chemical rnoiety that is capable of covalently linking together tliree spaced al inoieties (i.e., the sugar moiety (Via a glycosidic bond), a drug unit (directly or indirectly), and MA (directly or indirectly) when M" is present or Al when MA is absent. {180} In some embodiments, the glycosidic bond can be cleaved at the target site tn initiate a self— ative reaction sequence that leads to a release of the drug. [181} In some embodiments, each LB, when present independently is: WO 02984 >2: X *,/N\FAOW (35 ‘49 wherein: *** denotes attachment to MA, when MA is present, or to Al when MA is absent; and **** denotes attachment to D.

In some embodiments each LB, when present, independently is: wherein: *** s attachment to MA when M" is present, or to A1 when M“ is absent; an denotes attachment to D, In some embodiments, each Lg, when present, independently is: H H wherein: *** denotes attachment to M" and **** denotes attachment to D.

Variable M" 184 In some embodiments, MA ises a peatide inoiet1 31‘ of at least two amino acids. . [185} In some embodiments, amino acid is ed to herein as “AA,” and amino acids as “AA’s’? [186} In some embodiments, MA is a nioiety that is capable of forming a covalent bond with a — LD—D unit and allows for the attachment of multiple drug. [187} In some embodiments, MA comprises a single AA unit or has two or more AA units (egg, from 2 to l 0, from 2 to 6, or 2, 3, 4, 5 or 6} wherein the AA units are each independently a natural or non-natural amino acid, an amino alcohol, an amino de, a diamine, a polyamine, or combinations thereof. {188} In some embodiments, in order to have the requisite number of attachments, at least one of the AA units will have a functionalized side chain to provide for attachment of the ~LDnI) unit. In some embodiments, exemplary functionalized AA units (eg, amino acids, aniino alcohols, or amino aldehydes) include, for example, azido or alkyne functionalized AA units (eg, amino acid, amino alcohol, or amino aldehyde modified to have an azide group or alkyne group). [l89] In some embodiments, MA ses 2 to l2 AA units. In some embodiments, MA comprises 2 to l0 AA units. In some embodiments, MA comprises 2 to 6 AA units. In some embodiments, MA comprises 2, 3, 4, 5 or 6 AA units. [l 90} In some embodiments, MA has 2 AA units. In some embodiments, the e moiety has 3 AA units, In some embodiments, the peptide moiety has 4 AA units, In some embodiments, the peptide moiety has 5 AA units. In some embodiments, the e moiety has 6 AA units. [lgll In some embodiments, attachment within MA or with the other components of the conjugate, intermediate thereof; or scaffold, can be, for example, via ainino, carboxy, or other functional iti es In some embodiments, each amino acid in MA can be independently I) or L isomer of a thiol containing amino acid. In some embodiments, each amino acid in MA can be independently a D isomer of a thiol containing amino acid, In some embodiments, each amino acid in MA can be independently an I, isomer of a thiol containing amino acid. In some embodiments, the tliiol containing amino acid can be, for example, cysteine, homocysteine, or penicillamine. [193} In some ments, each amino acid in MA can be ndently the I, or D isomer of the following amino acids: alanine (including B—alanine}, arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutaniine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, phan, proline, ornithine, penicillamine, aniinoalkynoie acid, aminoalltanedioic acid, heterocyelo—carboxylic acid, citrulline, e, dianiinoalltanoic acid, stereoisoniers thereof, or derivatives thereof. [194} In some embodiments, each amino acid in MA is independently cysteine, liomocysteine, penicillamine, ornitliine, lysine, sei'ine, thi'eonine, glycine, glutaniine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, e, isoleticine, Eeucine, methionine, valine, alanine, or a stei'eoisomei's thereof. {195} In some ments, MA comprises a monopeptide, a dipeptide, tripeptide, teti'apeptide, oi' eptide. In some embodiments, MA comprises a pentapeptide. [196} In some embodiments, MA comprises at least about five amino acids (eg, 5, 6, '7, 8, 9, or l0 amino acids). In some embodiments, MA comprises at most about ten amino acids. [197} In some ments, each amino acid in MA independently is glycine, sei‘ine, glutamic acid, lysine, aspartic acid, and cysteine. [198} In some embodiments, MA comprises at least four glycines and at least one glutamic acid eg neh and glutamic acid, wherein the glutaniic acid is at any position along the peptide chain, such as, for example, (glutamic acid)n(glycine)4; (glycineHglutaniic acid)w(glycine)3; (glycinehfglutamic (glycine)z; (g1ycine)3~(g1utamic acid)n(glycine); 01’ (g1ycine)4~(g1utamic acid). [,l99} In some embodiments, MA comprises (glyt:ine)4~(gluta,niic acid). In some embodiments, the peptide moiety comprises (glutamic acid)—(glycine)4. [200} In some embodiments, MA comprises at least four es and at least one serine, eg, (glycine); and serine wherein the serine is at any position along the peptide chain, such as, for example, (serine)-{glycine)4; ne)—(serine)~(glycine)3; (glycine)2—(serine)~(glycine)2; (glycine)3~{serine)w(glyeine), or ne)4—(seriiie). [201} In some embodiments, MA ses (glycinefl—(serine). In some embodiments, the peptide moiety comprises (serine)w(glycineh. [202} In some embodiments, MA comprises (Ii-alanine)~(glycine).i*(serine) wherein the serine is at any position along the peptide chain, such as, for e, tli—alanine)~(serine)—(glycine)4; (6- alanine)-(glycine)-(sei'ine)e(g1ycine)3, {Ii-alanine)~(glycine)2~(serine)—(glycine)2; (fialaniney (gly cine)3~{sei'ine)— (glycinekor (B—alanine)-(gly cine)4—{serine). [203} In some embodiments, MA comprises (glycineh—(serine)—(g1utamic acid) wherein the serine is at any position along the peptide chain, such as, for example, (: se1‘ine}—(glycine)4—(glntamic acid}, {glycine}-(serine)—(glycine)3—(gluianiic acid); nep—(serine)-(glycine)2~(g1utamic acid); (glycine)3~{sei'ine)—(glycine)—(gltitainic acid); or (gly‘cine)4—(sei'ine)-(g1utamic acid). In some embodiments, the peptide mciety ccmpi'ises (B—alariine)—(glycine)4—(serine)~(glutamic acid) wherein the serine is at any position along the peptide chain, such as, for example, (ti—alanine} e)—(glycine)4-(gl'utairiic acid); (B—alaniiie)-(glycine}-(serine)—(glycine);—(glntamic acid); ([3— alanine)—(glycine)2—(serine)—(glycine)2—(glutaniic acid), (flaianinet(gtycineihntserineyiglycine)- mic acid); 01' (’8—alanine)-(glycine)4~(seiine)—(gltttamic acid).

In some embodiments, MA comprises (glycinefl-(serine). In some ments, the peptide moiety comprises (serine)-{gly cine)4.

In some embodiments, MA comprises (B~alanine)n(glycine)4—(se1‘ine) wherein the serine is at any position along the peptide chain.

In some embodiments, iv A ses (gly’cine)4~(serine)n(glutamic acid) wherein the serine is at any position along the peptide chain.

In some embodiments, MA ses (B—alanine)n(glycinefintsei‘iiie)~(g1ntamic acid) wherein the serine is at any position along the peptide chain.

In some embodiments, MA comprises (glutainic acid)—(glycine)i—4, wherein: the MA is attached to A1 via one of the giutamic acid; MA is attached to T1 via the glycine; and MA is attached to LD via the giutamic acid.

H we WtH o 1—4 C was [209} In some embodiments, MA comprises [210} In some embodiments, MA comprises (giutamie acid)~(glycine)4, wherein: the MA is attached to Al via the glutamic acid; MA is attached to T1 via one of the glycine; and MA is attached to LD via the giutamic acid.

H 'Jzfi‘ WtH O 4 O anus [21 1} In some embodiments, MA comprises In some embodiments, MA comprises (glutamic a.cid)~(giycine), wherein: the MA is ed to A1 via the glutamic acid, MA is attached to T1 via the glycine; and MA is ed to LB via the glutamic acid.

Q *1”: ~ - . .

In some embodiments, the e nioiety comprises, , [214} In some embodiments, MA comprises (giycine)1.4—(giutaihic acid), wherein MA is attached to A1 via one of the qjveine; MA is attached to T1 via the alutaniic acid; and MA is attached to LB L J \J via the ghitamie acid *\‘iN N h 1-4 0 mm [215}_ , A .

In some embodiments, NI comprises In some ments, MA comprises ne/34*(glutamic acid), wherein: MA is attached to A1 via the glutamic acid; MA is attached to T1 via the glycine; and MA is attached to LB via the glutaniic acid.

* E INHm ‘* 0 *VHr {217} In some embodiments, MA comprises In some embodiments, MA ccmprises (glycine}—(g1utamic acid), wherein: MA is attached to Aj Via the glycine; MA is attached to Tj Via the glutamic acid; and the MA is attached te LD via the ghitainic acid.

KEWN‘i“,_ D O *S’H’r In some embodiments, M7“ comprises [220} In some embodiments, MA ses (ginil’le)L4~(S€I‘il’1€), wherein: MA is attached te Al via one of the glycine; M“ is attached to TI via the serine; and 134‘“ is attached to L” via the .

*‘\ ’N \h 4P4 0 [WW ' and 4/; E In some embodiments, MA comprises 222 In some embodiments MA cein rises {,0} cine - serinet wherein: MA is attached to A1 via a , , . \e y the giycine; MA is ed to Tj via the serine; and MA is attached to IfD via the serine.

H i A \N//VN\(IK*X H (:3 o {223} In some embodiments, MA comprises [M.41; I In some embodiments, MA cemprises (giyciiie)4—(serine), wherein: MA is attached to Al via one of the dvcine‘ MAI: is attached ta 'I'1 via the serine; and MA is attached to LB via the serine. ., 7 * ”N /lL\** 4 O In some embodiments MA comprises . [226} In some embodiments MA comprises eytgiycineya, wherein: MA is attached t0 A1 via the serine; MA is attached t0 TI via one of the glycine; and MA is attached to LB Via the serine.

A’IEEWe.i ‘ /** 0 \tt0 1-4 [227} In some embodiments, MA comprises In some embodiments, MA comprises (seiihe)—(giycine)4, wherein: MA is attached to A1 via the ; MA is attached to T1 via one of the glycine; and NIA is attached to LB via the serine. a.» N E’W 'kiri [229} In some embodiments, MA comprises [23 G] In some einbndin'ients, M" comprises e)-(glycine), wherein: M“ is attached tn A] via the serine‘ MA is attached to T1 Via one of the “Iivcine; and MA is attached to LD via the serine. 7 23 s , g *at */ EY In some ei'nhodiments, h/IA comprises 232 In some ei'nbodintents MA com )iises ( ll’1iflfj Jets/cine 1.4“(seiine3 wherein: MA is 7 9 . . V ., . . . attached to A1 via the ine; MA is attached ta 'I" via the ; and MA is attached to LD via the serine.

O O *\N/\)k1N/WNH 41* H H 0 1-4 In some embodiments, MA comprises ifik [234} In some embodiments, MA ses (B—aiai‘iine)~(giycine)4~(serine), wherein: MA is attached to A1 via the nine; MA is attached to T1 via the serine; and MA is attached to LB via the serine, 0 O \N N N ** H H O *** [235} In some embodiments, MA comprises [23 6] In some embodiments, the peptide moiety comprises (fi—alanme)—(glycine)—(glutamic acid}, wherein: the peptide moiety is attached to L3 when present, or to L“ when L3 is absent, via the fi-alanine; the peptide moiety is attached to T1 when present, Via the glutamic acid; and the peptide moiety is attached to LD when present, Via the glutamic acid.

O 0 * /\/u\ \N NWN ** H H Q iii [23 7}_ . i _ . . .

In some embodiments, the e meiety ses . [23 8} It is understood that for ments of MA, * indicates attachment to A}, ** indicates attachment to "l”, and *** indicates attachment to LB.

Hydrophflz'c Group ("tartan/73 2’) [23 9} In some embodiments, the hydi'ophihc group included in the conjugates or scaffolds of the disclosure is a water—soluble and substantially tigenic polymer. Examples of the phihc group, include, but are not limited to, poiyaicohols, pciyethers, poiyanions, poiycations, poiyphosphoric acids, polyamines, polysaccharides, pciyhydroxy compounds, polyiysines, and derivatives thereof, In some embodiments, one end of the hydrophilic group can he functionalized so that it can be ccvaiently attached to the MA tinker (eg, to an amino acid in the MA tinker) by means of a non~cleavahle linkage or via a cieavable Iinkage, In some embodiments, t‘unctionaiization can be, for e, Via an amine, thioi, hfiiS ester, maleimide, alkyne, azide, carbonyl, or other functional group. In some embodiments, the other terminus (or i) of the liydrophilic group will be free and untethered. In some embodiments, by hered”, it is meant that the liydrophilic group will not be attached to another moiety, such as D or a Drug Unit, or other components of the conjugates or scaffolds of the disclosure. In some ments, the free and untethered end of the hydrophilic group may include a niethoxy, carboxylic acid, l or other suitable functional group. In some embodiments, the methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a cap for the us or i of the hydrophilic group.

In some embodiments, a cleavable linkage refers to a linkage that is not substantially sensitive to cleavage while circulating in the plasma but is sensitive to cleavage in an intracellular or intraturnoral nment. In some embodiments, a nonmcleavable linkage is one that is not substantially sensitive to cleavage in any biological environment. In some embodiments, chemical hydrolysis of a hydrazone, reduction of a ide, and enzymatic ge of a e bond or glycosidic linkage are examples of cleavable linkages, In some embodiments, exeinplaiy ments of the hydrophilic group are via amide linkages, ether linkages, ester linkages, hydrazone linkages, oxime linkages, disulfide linkages, peptide linkages, or triazole linkages. In some embodiments, the attachment of the hydrophilic group to the MA linker (eg, to an amino acid in the MA linker) is via an amide linkage, [241} In some embodiments wherein the conjugate or scaffold of the disclosure comprises more than one hydrophilie groups, the multiple hilic groups may be the same or different chemical moieties (e.g, hydi'ophilie groups of dillerent molecular weight, number of subunits, or chemical structure). In some embodiments, the multiple hydrophilic groups can be attached to the MA linker at a single attachment site or different sites, In some embodiments, the addition of the hydrophilie group may have two potential impacts upon the cokinetics of the resulting conjugate. In some embodiments, the desired impact is the decrease in nce (and consequent in increase in exposure) that arises from the reduction in non-specific interactions induced by the d hydrophobic elements of the drug or drug-linker. In some embodiments, the undesired impact is the se in volume and rate of distribution that may arise from the increase in the molecular weight of the conj ugate. In some embodiments, increasing the molecular weight of the hydrophilic group increases the hydrodynamic radius of a conjugate, resulting in decreased diffusivity that may diminish the ability of the coniugate to penetrate into a tumor. Because of these two competing pharmacokinetic WO 02984 effects, it may be desirable to use a liydi'ophilic group that is sufficiently large to decrease the ate clearance thus increasing plasma exposure, but not so large as to greatly diminish its ivity, which may reduce the ability of the conjugate to reach the intended target cell population. {243} In some embodiments, the liydrophilie group, includes, but is not limited to, a sugar alcohol (:also linown as polyalcohol, polyhydric alcohol, l or glyeitol, such as inositol, ol, erythritol, threitol, arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, and the like) or a derivative thereof (eg, amino polyalcohol), ydrateteg. a saccharide), a polyvinyl alcohol, a carbohydrate—based polymer (cg, dextrans), a hydroxypropylinethacrylamide (HPIVIA) a polyallrylene oxid 6, and/or a copolymer thereof. [244} In some embodiments, 'I'1 comprises a plurality of hydroxyl (“~OH”) , such as moieties that incorporate inonosaceharides, oligosaccharides, polysaccharides, and the like. [245} In some embodiments, Tl ses a plurality of {CRigOH} groups, wherein R53 is "H or Cj-S alkyl, [2465} In some embodiments, T1 is -OH or éNHMRGO""""(CRSEOH)H'1‘”R61 wherein: ni is an integer from G to about 6; each R53 is independently —H or Crs alkyl; R60 is a bend, a C16 alkyl linker, or - wherein R59 is It, (Ii—s alkyl, cyeloalkyl, or arylal kyl ; Rei is CIIgORsz, COORez, —(Cl:l2)iizCOORez, or a heterocycloalkyl substituted with one or more hydroxyl; R62 is ill or C14; alkyl; and nz is an ii'iteger from l to about 5.

In some embodiments, 'I'1 is ----()I-I. In some embodiments, T] is "lNH"'R‘5”_(‘3R580H)“‘WRN. [248} In some embodiments, R53 is —H; R60 is a bond or a {Ti—6 alkyl linker; n1 is an integer from 1 to about 6; and R61 is CI-leI-l or . [249} In some embodiments, R53 is ~I-l; R60 is ----CI-IR'~;9—; m is O, and Rel is a hetei'ocycloalkyl substituted with one or more liydroxyl, eg a monosaccharide. [250} In some embodiments, "Ifj comprises a glucosyl~amine, a di— amine, or a tri— amine.

In some embodiments, 'l‘l comprises one or more of the following fragments or a isomer thereof: (1) '(C'1OH;M“R59; OH OH ' OH . QH I’MNamEJYwOH E OH OH OH OH .

NH OH OH \Vhe1ern: R59rs H, €1-22, a1ky1, eV'e1oa11tyl or awlalkyl; n: 15 an integer f1o1‘h 1 to about 6.; n2 13 an integer from 1 to about 5; and 113 13 an 1hteger from about 1 to about 3 It is understood that all stereoehemieal forms of the hydrophihe groups are eontenrp1ated hererh, For examp1e, in the above fornru1a, the 111hc group may be derived from ribose, X§i1ose glucose, nrahhose, galactose, or other sugar and retain the ehernreal arrangements of pendant hydroxyl and alkyl groups present on those molecules. {253} It is to be understood that in the foregoing formulae, various deoxy compounds are also oontempiated. lllustratively, one or more of the ing features are contemplated for the hilie groups when applicable.

In some embodiments, ns is 2 or 3. {255} In some embodiments m is l or 3 In some embodiments, nz is l. {257} In some embodiments, R59 is hydrogen.

OH QH {258} In some embodiments, I,” is CH 0H . In some embodiments, 'I'1 is “E'blV/‘xx/JEN OH OH OH OH )w/S‘VOH; V‘kfi/WVOH OAT? OH OH EH 9H mng OH OH , OH O 1 Y/V N/z\/0\ . fl: ‘ OH OH . In some embodiments, 1* is H R63 Rea “ism——"R54”{m0------- (:2 ------o ----- R55 In some embodiments 'I‘1 is R63 R53 wherein n4 is an integer from I to about 25; each R63 is ndently —I—I or CHE alkyl; Rm is a bond or a CH; alkyl linker; Rio is "H, CH; alliyl, -(CH2}112C00R62, or “(CHZ>11LICOR66; Rm is H or CH; aikyl; 0 0H 0H «ES /’\¢ 1 4 N/“\r \EA/OH OH QH H OH 0H : O, NH oi—i OH A WDig, If \ fi/Y kp// OH : N O“ i. w on ‘5" V 2N\// R66 is H, OH OH 0H CH 9 5 :12?!N xJL NAV’OWO/‘x/Owo/ H /HN ,Ni-iz ‘7 HN / C) /\ / 9 N/‘v wo’w VKO/O V fie :55,” " Q ,, H 0 or 0‘ 0H and nz is an integer from i to about 5.

In some embodiments, I" is: O wherein R67 is: (I) "OH; OH OH K,1Y1VOH wherein m is an integer from about 2 to about 20, from about 4 to about to, from about 6 to about 12, i about 8 to about 12, a: Eh,0>\/\r0b é i: n4 In some embodiments, T1 is o [262} In some embodiments, n4 is an integer from about 2 to about 20, from about i-‘I to about to, from about 6 to about 12, from about 8 to about 12. [263} In some embodiments, n4 is 6, 7, 8, 9, IO, 1 i, or 12.

In some embodiments, m is 8 or 12, g“M” NH“--\{i;rf:/QH [”4 /\g [265} In some embodiments, T1 is 0“ Q“ or 0H OH Fiat/\fiE’V/ Qf/HN;:MNHWOHANH OH OH OH OH Y'YVH OH OH wherein m is an integer from about 2 to about 24, from about 4 to about 16., from about 6. to about 12, from about 8 to about 12.

In some embodiments, n4 is 6, 7, 8, 9, IO, 11, or 12.

In some embodiments, n4 is 8 or 12 In some embodiments, n4 is 8, Q on on 9":N{/WQWNY/HN' A/kN/‘d/jY’PVOH H "A g H , on on NH on on [268} In some embodiments, "l‘j is OH OH wherein r14 is 8. [269} In some embodiments, Tl comprises a polyether, e.0;., a lrylene glycol (PAC). PAO es but is not limited to, polymers of lower alkylene oxides, in particular polymers of ethylene oxide, such as, for example, propylene oxide, polypropylene glycols, polyethylene glycol (PEG), polyoxyethylenated poly ols, copolymers thereof, and block eopolymers thereof. [270} In some embodiments, the polyalkylene glycol is a polyethylene glycol (PEG) including, but not limited to, polydisperse PEG, nionodisperse PEG, and discrete PEG Polydisperse PEGs are a geneous mixture of sizes and molecular weights whereas monodisperse PEGs are typically purified from heterogeneous mixtures and are therefore provide a single chain length and molecular . in some embodiments, the PEG units are discrete PEGs provide a single molecule with defined and specified chain length ln some embodiments, the polyethylene glycol is mPEG. [271} In some embodiments, Tl comprises a PEG unit which ses one or multiple PEG chains. The PEG chains can he linked together, for e, in a , branched or star shaped uration The PEG unit, in addition to comprising repeating PEG subunits, may also comprise non-PEG material (eg, to facilitate coupling of multiple PEG chains to each other or to facilitate coupling to the amino acid): NornPEG material refers to the atoms in the PEG chain that are not part of the repeating —CH2Cl-l2()- subunits. In some embodiments, the PEG chain can comprise two monomeric PEG chains linlred to each other Via non—PEG elements. in some embodiments, the PEG Unit can comprise two linear PEG chains ed to a central core that is attached to the amino acid (i.e., the PEG unit itself is branched).

The PEG unit may be covalently bound to the MA linker (eg, to an amino acid in the MA linker) via a ve group. Reactive groups are those to which an activated PEG molecule may he bound (eg, a free amino or carbonyl group). in some embodiments, N—terminal amino acids and lysines (K) have a free amino group; and C—terminal amino acid es have a free carhoxyl group. Sullhydiyl groups (eg, as found on cysteine residues) may also be used as a reactive group for attaching PEG. [273} In some embodiments, the PEG unit may be ed to the MA linker (egg, to an amino acid in the MA linker} by using methexylated PEG G“) having different reactive moieties, including, but not limited to, succiniinidyl succinate (SS), succinimidyl carbunate (SC), mPEGu imidate, para—nitrophenylcarbonate (NFC), succininiidyl propionate (SPA), and cyanuric chloride.

Examples of ml’EGs include, but are not limited to, mPEG—suceininiidyl ate (mPEG—SS), mPEG2—succininiidyl ate (mPEG2-SS), inl’EG- succininiidyl carbcnate (mPEG-SC), niPEGz—succinimidyl carbonate (mPEG2-SC), mPEG—imidate, mPEGupara~nitrephenylcarbcnate (mPEGnNPC), inPEG—iinidate, mPEGz."parawnitrophenylcarbonate (inPEG2~NPC), n1PEGw succininiidyl prepionate (nil’EG~Sl’A), n1PEG2— succininiidyl prepionate (nil’EGz—SPA), inPEG—N—hydroxywsuecinimide (n1PEGvNHS), mPEG),nN~hydroxy—succiniinide (mPECb—NHS), nil’EG~cyanuric de, n1l’EG.z~cyanuric chloride, Lysinol—NPC, and mPEngLysn NHS. A wide variety of PEG species can be used, and substantially any suitable reactive PEG reagent can be used. in some embodiments, the reactive PEG reagent will result in formation cf a carbamate or amide bond upon attachment to the Multifunctional Linker or MA linker (eg, to an amino acid in the MA linker), The reactive PEG reagents include, but are not limited to, mPEGz— N—l‘iydroxy~suceinimide (mPEG2—NHS), bifunctienal PEG propionaldehyde (n‘iPEG2~ALD), multi—Arm PEG, maleimide—containing PEG (mPEGt'lVlAL); mPEGiMAlJ), mPEG—l‘fllz, mPEG— succinimidyl propienate (mPEG—SPA), succinimide of mPEG butanoate acid G~ SBA), mPEG—thioesters, inPEGwlouble Esters, mPEG—BTC, mPEG—ButyrALD, mPEG— acetaldehyde dietliyl acetal (mPEG~ACET/l, heterofunctional PEGs (eg, NH2~PEG—COOH, Boc— PEG—NHS, FIE’IOCePEG-NHS, G~vinylsull‘one {NHS-PEGJJS) or" GJWAL), PEG acrylates (ACRleEG—NHS), PEGsphospholipids (eg, mPEG—DSPE), rnulti—armed PEGs of the SUNBRITETM series including the glycerine—based PEGs activated by a chemistry chosen by those d in the art, any SlINBRlTE activated PEGs ding but not limited to carboxylw PEGs, p~NP~PEGs, Tresyl—PEGs, aldehyde PEGs, acetal~PEGs, amino~ PEGs, tliioleEGs, maleimidanEGs, ylsPEG-arnine, amino—PEG~COOK oxyhPEG— aldehyde, carboxylic anhydride type-PEG, functionalized PEG-phospholipid, and other similar and/or suitable reactive PEGs. [274} ln some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least ll subunits, at least l2 subunits, at least 13 subunits, at least l4 subunits, at least 15 subunits, at least 16 subunits, at least l7 subunits, at least 18 subunits, at least l9 subunits, at least 20 subunits, at least 21 subunits, at least 22 ts, at least 23 subunits, or at least 24 subunits. In some such embodiments, the PEG unit comprises no more than about 72 subunits. [275} In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least l0 subunits, at least ll ts, at least l2 ts, at least 13 subunits, at least l4 subunits, at least l5 subunits, at least 16 subunits, at least I? subunits, at least l8 subunits, at least 19 subunits, or at least 20 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least ll subunits, at least l2. subunits, at least 13 subunits, at least l4 subunits, at least 15 subunits, at least 16. subunits, at least l7 subunits, or at least its ts.

In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 ts, or at least 12 subunits.

In some embodiments, the PEG unit ses at least 8 subunits, at least 9 subunits, at least 10 subunits, at least l l subunits, or at least l2 subunits [279} In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, or at least 8 subunits, [280} In some embodiments, a linear PEG unit is: (I), ”%"Y7i""(CHzCHgD)d9”Y72; (it).. “Ewflmicugcuzoidmet-73m(cchuzommyn ; or “§”Y7i“(CH2CH2O)dio Y73“(CH20H20)diiJ/+Y72 (iii) an wherein; 3 indicates site of attachment to the MA linli'ei' (e.g to an amino acid in the MA ); YVI is a PEG attachment unit, “177:, is a PEG capping unit, Y7; is an PEG coupling unit tie, for coupling multiple PEG subunit cliains together); d9 is an r from 2 to 72; each die is independently an integer from 1 to 72; and {lit is an integer from 2 to 5. [281} In some embodiments, d9 is an integer from 2 to 24. In some embodiments, d) is an integer from 4 to 24. In some embodiments, dt) is an integer from 6 to 24, from 8 to 24, from 10 to 24, or from 12 to 24,.

In some embodiments, there are at least 6 PEG ts in the PEG unit. In some embodiments, there are at least 8 PEG subunits in the PEG unit. In some ments, there are at least 10 PEG sub units in the PEG unit. In some embodiments, there are at least 12 PEG subunits in the PEG unit.

In some embodiments, d9 is 8 or about 8, 12 or about 12, 24 or about 24. [284} In some ments, each Y7). is independently "Cl—l0 allsyl, €2.10 alkthOzH, {7240 alkylHOI-I, {7240 alkyl—NI—Iz, {7240 alkyl—NI—HCM alkyl), or C240 alltyletCia alkyl)2, [285} In some embodiments, Y7: is “C140 alkyl, "C240 alkylnCOfi-I, "C240 H, or — 7240 alkyl—NI-Iz. [286} In some embodiments, the PEG coupling unit is part of the PEG unit and is non—PEG material that acts to connect two or more chains of repeating 0— subunits. In some embodiments, the PEG ng unit Y73 is -C2-ic- alkyl—C(O)~NH—, —C2-io alkyl~Nl—I—C(O)~, —C2--io alkyl—l‘fllg «Cg-.10 alkyl—C(O)~, c- alkyl~O—, or —C2--10 a.lkyl—S~. [287} In some embodiments, each Y73 is independently - Ciao a,lkyl—C(O)~N — CLIC- alkyl~l\Tl,—I— ., —, C(O)~, ~C2-10 alkyl~NH—, ~ C240 O~ €1.10 alkyl—S~, or — Ciao l‘flla [288} In some embodiments, the PEG attachment unit is pan: of the PEG unit and acts to link the PEG unit to the MA linker (eg, to an amino acid in the MA linker). In some embodiments, the amino acid has a functional group that forms a bond with the PEG Unit. In some embodiments, the functional groups for attachment of the PEG unit to the amino acid include sulthydryl groups to form disulfide bonds or thioether bonds, aldehyde, ketciie, or hydrazine groups to form hydrazone bonds, hydro-xylarnine to form oxime bonds, carboxylic or amino groups to form peptide bonds, carboxylio or hydroxy groups to form ester bonds, suli‘onic acids to form suli‘onamide bonds, alcohols to form oarbama‘te bonds, and amines to form sulfonamide bonds or carbainate bonds or amide bonds. In some embodiments, the PEG unit can be attached to the amino acid, for example, via a distilfide, thioether, hydrazone, oxime, peptide, ester, sulfonamide, earbainate, or amide bond. In some embodiments, the reaction for attaching the PEG unit can be a cycloaddition, addition, on/elimination or substitution reaction, or a combination thereof when applicable. [289} Examples of linear PEG units include: H H (i),. ”EMNmt'CHZCHQOMQMCHZCHECDOH cugcugouemcug ; (“l i - t A , l1“) “gmcmlCHgCHzflhg‘mCl-lg , 4 ' I ""§""N"'“(CHacHZO)sQ“““CH2CHzC(O)-NHn(CH2CHgO}~CH2CH2COOl-lh ‘ 9 l“) and (V) “E“NWQHQCHzoldfimCHQCHg-NH-(CHgCHgO)-CH2CHzCOOHH wherein 3 indicates site of a‘ttacl'unent to the Multifunctional Linker or MA linker (eg to an amino acid in the MA linker), and each d9 is independently an integer from 4 to 24, 6 to 24, 8 to 24, 10 to 24, 12 to 24, 14 to 24, or l6 to 24, [2%] In some embodiments, d9 is about 8, about 12, or about 24. In some ments, d9 is about 8.

In some ments, the PEG unit is from about 300 Da to about 5 kDa; from about 3 00 Da to about 4 cha; from about 300 Da to about 3 cha; from about 300 Da to about 2 kDa; or from about 300 Da to about 1 kDa. In some ments, the PEG unit has at least 6 subunits or at least 8 10 or 12 subunits. In some embodiments, the PEG unit has at least 6 subunits or at least 8, 10 or 12 subunits but no more than 24 subunits. {292} In some embodiments, suitable hylene glycols may have a free hydroxy group at each end of the polymer molecule, or may have one hydroxy group etherified with a lower alkyl, e. g, a methyl group. In some embodiments suitable polyethylene glycols are derivatives of polyethylene glycols having esterifiable carboxy groups. In some embodiments, polyethylene glycols are commercially available under the trade name PEG, usually as mixtures of polymers terized by an average molecular weight. In some embodiments, polyethylene glycols having an average molecular weight from about 390 to about 5000. In some embodiments, polyethylene glycols having an average molecular weight from about 600 to about lOOO. [293} In some embodiments, examples of hilic groups that are suitable for the conjugates, scaffolds, and methods disclosed herein can be found in eg, US 8,367,065 column 13; US 8524696 column 6; Eli/702015,,"057699 and ‘WO 2014;062:697, the contents of each of which are hereby incorporated by reference in their entireties.

SZYNGAg0}?is: Drug [140i813/ (Varz’aE) ’e 1)) In some enilindiments, the STI NG agnnist drug moiety {D} is a nd eani'niula (A): REE sll ::::N iZ‘Y/ R10- MX4 2 C] WE'XZRW '15 (A), R or a predrug, sols/ate, pliannaeeutically acceptable salt, or tauteinei thereof, wherein: Yi, Y2, 2;, and 22 are each independently 0, S, C or N, Xi, X2 Wt and W2 are each independently C or N X3 and X4 are each independently S or NR“, X5 is N or CR“; X6 is N or CR“; R3 and R5 are each independently ~CON(Rd)(Rf), "CH7N(R5)(R} ~N(Rd)(R), ~ N'(R‘l)CO(Rf), "CH2N'(R‘l)CO(Rf) or one of R3 and R5 is "CON(R5)(Rf), “CH2N(R‘5)(Rf), _ N(Rd)(Rf), —N(Rd)CO(Rf) or —CH2N(Rd)CO(Rf), and the other of R3 and R5 is H, —COOH, or — COARC); RC is CH alkyl; RA2 and R“ are each independently H, n, hydroxy, amine, amine(C1-4 elkyl}, optionally substituted (Cl-6 , or optionally substituted ((31.6 all<yl)exy~, wherein Ci-e alkyl of said optionally substituted (Ci—6 , or optionally substituted (Ci—6 alkyl)exy— is optionally substituted with l~r-‘l substituents each ndently selected from the group comprising hydroxyl, CM alkexyl, —N(R?}(Rf), —C02(Rf), ~CON(R.e)(R.f), and neon; each Rd is ii’idependently H, hydroxy, or (Ii—.4 alkyl; R6 is selected from H, (Cm alkyl}, “COMM alkyl), —OCO(Ci—4 , and ~C()2(Ci—4 alkyl); each Rf is ndently H, hydrexy, or (Ci-r; alkyl); R14 and R“ are each independently absent or (31.4 all<yl, wherein (31.4 alkyl is optionally substituted by a substituent selected from halogen, —ORC, —NRCRG, £02K, R“, - SO2NRCRd, and -OC()NR“R“, RH" and R“ are each ndently absent, H or (31.4 alltyl, and R15, R17, R”, or R19 are each independently absent, H, or C14 alkyl, wherein C14 alkyl is optionally substituted by a substituent selected from halogen, —ORC, —NR"Rd, -C02RC, R‘l, sognacnd, and -oconncnd, wherein: (i) at least one of RA2 and RAj is present, and n at least one of RA2 and RA] is connected to LB via at least one functional group of the RA2 and/or R“; or (ii) at least one of RC2 and RC1 is present, and wherein at least one of R62 and RCj is ted to LD Via at least one functional group of the RC7: and/or R“.

In some embodiments, the STING agonist drug moiety is a compound of Formula (Ana): ‘ R19 O Ztiyf ._...

R sNLNfo : " l” 1‘ XS | reg s / ~ * >35“ R X4 2,13% 0 ZINE AR” (A—a) or a g, solvate, pharniaceutically acceptable salt, or tautonier thereof, wherein: Yr, Y2, Zr, Zz, Xi, X2, “’1, “’2, X3, X4, R3, R5, RC, Rd, Re, Rf, RM, RC2, R16, RC], R”, R17, R18, and R“) are as defined in Formula (A), X5 is CR“; and RA2 is halogen, hydroxyl, optionally substituted (Ci—6 alkyl), substituted (Ci—6 alkyl)oxyw, optionally substituted (Cm, alkyl)aniinom, or ally substituted (Cl-6 alkyl)(Ci~4 alkyl)aniinon, wherein €le alkyl of said optionally substituted (Cm alkyl) or substituted (Cm alkyl)oxy~ is optionally tuted with 1—4 tuents each ndently selected front the group comprising hydroxyl, {71-4 allroxyl, —N{R€)(R’), {702(Rf), -{TON{RG}{Rl), and —COOl—l; wherein: (i) RA2 is connected to LD Via a functional group ot‘RM, or (ii) at least one of R” and RC1 is present, and wherein at least one of R“ and RC] is connected to LB via at least one functional group of the R52 and/or Rm.

In some embodiments, the S'HNG agonist drug moiety is a compound is of Formula (A- \__ R19 / 1'IS>:::N :‘il‘Y: R QJQ s AN )N in X6 R61 9% N l : >er ZZ‘WR48 \ X4 2 )2" 0 p; 2~R17 or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, n: Yr, Ya, Zr, Z2, X1, X2, W'i, ‘Wz, X4, X5, X6, R3, R5, R“, R“, RA}, Rd, Re, Rf, R”, R52, R16, R“, R15, R17, R”, and R” are as defined in Formula (A); wherein: (i) at least one o’l‘RAZ and RA1 is present, and wherein at least one of RA? and R“ is connected to LB via at least one functional group of the R3"2 and/or R“; or (ii) at least one of RCZ and R“ is present, and wherein at least one of RC2 and RC1 is connected to LB Via at least one functional group of the RC2 and/or Rm. [297} In some embodiments, the STING agonist drug moiety is a compound is of Formula (An ‘1 we 0 Z1“ V/R / X3 RJ “I: ><®J<.44 _____ : N 1" t W 111 3 RC“: X5 N I 18 R“ l MN Z2‘Y’R \ X4 l2 I (“X 0 :{V2 1R1? (A~e) or a prodrug, solvate, pharmaceutically acceptable salt, 01" tautorner thereof, wherein: Y2, 22, X2, Wz, X3, X4, X5, X6, R3, R5, R“, R”, R“, Rd, Re, W, n“, R“, R16, R01, R15, R17, R13, and R19 are as defined in Formula (A), wherein one of W1, X1, Y1, and Z1 is N and the additional W1, X1, Y1, and 231 are 0, S, or whereii't: (i) at least one of RA2 and Rf“ is present, and wherein at least one of RM andRA1 is connected to LB Via, at least one functional group of the RM and/or R“, or (ii) at least one of RC2 and RC} is. present, and wherein at least one of RC2 and R“ is ted to LD Via at least one functional group of the RC2 and/or Rm. ln some embodiments, the S'HNG t drug moiety is a compound is ef Formula (A- O yvé “R17 (A-d) or a prodmg, solvate, pharmaceutically acceptable salt, or ner thereof, wherein: Y1, Y2, Zr, 2:2, X1, X2, Wt, W2, X3, R3, R5, R“, Rd, RE, R1", R”, Rel, R52, R16, R51, R15, R17, R13, and R19 are as defined in Formula (A); X5 is CR”; and wherein: (i) RA2 is ted to LB Via, a, functional group 0171?“; or (ii) at least one of R“ and R“ is present, and wherein at least one of R“ and RC] is connected to LB via at least one functional group of the R52 and/or Rm.

In some embodiments, the S'HNG agonist drug moiety is a. compound is of Formula (A- R3 X51 ”X3”:H®\lllfW1"\R«5 N> I; or a pi'orlrug, solvate, aceutically acceptable salt, or tautonier thereof, wherein: Yr, Y2, zi, Z2, xi, X2, XSNW, we at“, as, as, RC, Rd, Re, Rf, R”, n‘33, it”, n“, n”, n”, R13, and R19 are as defined in a (A); X6 is CR“; and wherein: (i) RM is connected t0 LD Via a functional group ot‘Rm; or (ii) at least one of R” and RC1 is present, and wherein at least one of R“ and RC] is connected to LB via at least one functional group of the RC2 and/or Rm.

In some embodiments, the S'I'lNG agonist drug moiety is a compound is of Formula (Anf): / Girl RBQ1X3 \,..—‘N X5 'N (A'fl or a prodrug, solvate, pharniaeeutieally acceptable salt, or taiitonier thereof, wherein: X2, X3, X4, X5, X6, Viz, Y2, Z2 R3, R5, RC, RAE, RM, Rd, Re, Rf, R16, R17, R13, R19, RC2, and R“, are as d in Formula (A), and wherein: (i) at least one of}?Al and R“ is present, and wherein at least one ofRA2 and RA} is connected to LID via at least one functional group of the RA2 and/or R“; or (ii) at least one of R“ and RC1 is present, and wherein at least one of RC2 and RC1 is connected to LB via at least one functional group of the R52 and/or RC1. [301} In some embodiments, the STING agonist drug moiety is a compound is of Formula (1» — fl ): L1; pu-J O T f—xN \ N MN Z‘Y/ 222 HQ)? 0 \Né”X2 (Anf1) or a prodi'ug, solvate, pharn'laceutlcally acceptable salt, or ‘ler thereof, wherein: X2, X X4, W2, Y2, 22., R3, R5, RC, Rd, R, Rf, R16, R“, R1 '7, R18, R”, R”, and R“, are as defined in Formula (A); X5 is CR“; and wherein: (i) R“2 is connected to LD via, a functional group of R“; or (ii) at least one of RC2 and RC1 is present, and wherein at least one of RC3 and RC1 is connected to LB Via at least one functional group of the RC: and/or RC]. [302} In some embodiments, the STING agonist drug, moiety is a compound is of a. (A,~ (A42) or a prodmg, solvate, pharrnaeeutically acceptable salt, or tautomer thereof, wherein: X2, X4, X5, X6, 3.7372, Y2, Z2 R3, R5, RC, RM, RA}, Rd, Re, R“, RC3“, R”, R17, R”, R”, and Rf are as defined in Formula (A), wherein: (i) at least one otTRA?‘ and R“ is present, and wherein at least one of RA? andRA1 is connected to LD via at least one functional group of the RA?” and/or R“; or (ii) at least one of R“ and RC1 is present, and n at least one of RC2 and R“ is connected to LD via at least one functional group of the R“ and/or RC1. [303} In some embodiments, the S'l'lNG agonist drug moiety is a compound is of a {A— O 0 R4: 1i “” \‘QN/L‘N' III X5 N I 0 W2 AR“ 7 or a piodrug, solvate, pharniaceutieally acceptable salt, or tautomer thereof, n: X2, X4, W2, Y2, 2:2, R3, R5, RC, Rd, 126, Rf, R16, R“, R“, R”, R”, R18, R19, and RC1 areas defined in Formula (A); X5 is CR“; and wherein: (i) RA2 is connected to LB via a functional group 0th“; or (ii) at least one ofRC2 and R51 is present, and wherein at least one of RC2 and RC1 is connected to LB Via at least one functional group of the R52 and/or RC1. [304} In some embodiments, the STING agonist drug moiety is a compound is of Formula (1» — U; U) O 0 / N T 3.. \ \ . NH N R E $er)L N (it—f4) or a prodrug, solvate, taceutically acceptable salt, or tautonter thereof, wherein: X2, X4, Wz, Y2, 2.2, R 3?, RC, Rd, Re, Rf, R16, lit/‘2, R92, R16, R”, R18, R19, and R“ are as defined in Formula (A); X5 is CR“; and. wherein: (i) R“2 is connected to LD via a functional group of R“; or (ii) at least one of RC2 and RM is present, and wherein at least one of RC“ and RC1 is connected to L” Via at least one functional group of the RC: and/or RC]. [305} In some embodiments, the STING agonist drug, moiety is a compound is of Formula. (A,~ O R tom / N I N/L‘~N /X5l N L R 8 R5 .:N My,” \ S 2 >22 0 V1,; Z‘R'ET (rift-f5) or a prodmg, sch/ate, pharrnaeeuticaliy acceptable salt, or tautomer thereof, wherein: L}; J; x2, ‘Wz, Va, 22, R3, R5, RC, R“ R9, N, R”, R“, it“, R16, R”, it“, R”, and R“ are as d in Formula (A); X5 is CR“; and wherein: (it RA?“ is connected to LD Via a functional group of R“; or (ii) at least one of RC2 and RC] is present, and wherein at least one of RC2 and RC1 is connected to L'D Via at least one functional group of the R“ antl/or RC1. [306} In some embodiments, the S'l'lNG agonist drug moiety is a compound is of Formula. (A— RBQ/ 1X3 \,..—‘N X5 'N or a. protlrug, solvate, pharniaoeutieally acceptable salt, or tautonter f; wherein: X3, X4, X5, X6t R3, n5, n2 RAE, n“, R“, n”, R13, R”, Rd, Re, Rf, REE and R“ are as d in Formula (A); Y2 and Z: are each independently 0, S, C or N; X2 and Wz are each independently C or N; wherein: (i) at least one oft?A2 and R“ is t, and wl'ierein at least one of RA2 and RA} is connected to LB via at least one functional group of the RA2 and/or R“; or (ii) at least one of R“ and RC1 is present, and wherein at least one of RC2 and RC is connected to LB via at least one functional group of the R52 and/or RC1. [307} In some embodiments, the STING agonist drug moiety is a compound is of Formula (1» — L}; ‘JT (Ael) or a piodiug, solvate, pharniaceutically acceptable salt, or tautonier thereof, wherein: X Wz, Y2, 2 X X4, X5, R3, R5, RC, Rd, Re, R, R“, R“, R”, R”, R”, R1“, and R“ are as defined in Formula (A);wiierein: (i) RA12 is connected to LB Via a functional group of R“; or (ii) at least one of R52 and R“ is present, and wherein at least one of R52 and R“ is conneeted to LD Via at least one functional group of the RC and/or R“.

In some embodiments, the STING agonist drug moiety is a compound is of Formula (Aw 0 “YR / S l 1 "N N X6 N X5 N / ' >1?“ R l8 R“ a \ ZZ‘Y: X; H@' O yug’ 2.R1-_, or a g, solvate, pharmaceutically acceptable salt, or tautomer f, wherein: X2, X4, X5, X6, wt, Y2, 22, R3, R5, RC, RAE, R_“‘1,RC2, R17, R18, R”, W, Re, Rf, R16, and RC1 are as defined in Formula (A); U; CF wherein: (i) at least one o’l‘RAZ and R“ is t, and wherein at least one of RA? and R“ is connected to LD via at least one functional group of the RA?" and/or R“; or (ii) at least one of R“ and RC1 is t, and wherein at least one of RC?“ and R“ is connected to LD via at least one functional group of the R“ and/or RC1. [309} In some embodiments, the S'l'lNG agonist drug moiety is a compound is of a {A— XE. N l o / V R Rf. fl/ >:.':l\l \ 72‘Y; 0' \i;>22$12 ,» 47 (Avail) or a. prodrug, solvate, pharniaceutieally acceptable salt, or tautomer thereof; wherein: X2, X4? ’Wz, Y2, Z2 R3, R5, R5, R33, R52, R17, R18, RWRM, and RC1 are as defined in Formula X5 is CR“; and wherein: (i) R"2 is connected to LB via a functional group ofRM; or (ii) at least one of RC3 and Rm is t, and wherein at least one of RC3 and RC1 is connected to LB Via at least one functional group of the RC2 and/or RC1; and optionally, wl'ierein R” is connected to LB via a functional group of RA? [31G] In some embodiments, the STING agonist drug moiety is a compound is of Formula (A~ L1; \1 (TA-g4) or a prodrug, solvate, pharn‘raceuticafly acceptabie sait, or tautonrer thereof, n: X2, X4, W2, Y2, 22 R3, R5 RC, Rd, Re, Rf, R”, R”, R17, R13, R19, R16, and R“ are as defined in Formula (A); X5 is CR“; and wherein: (i) RA2 is connected to LD via, a functional group of R“; or (ii) at {east one of RC2 and RM is present, and wherein at least one of RC“ and RC1 is connected to L” Via at least one functional group of the RC: and/or RC]. [311} In some embodiments, the STING agonist drug, moiety is a compound is of Formula. (A,~ Few“/ N I NH MN '\\ ing- N L {’R13 R5 /X5l mN \ LZ‘YZ S 2)E< O W; l’ (Are-‘5) or a prodnig, soivate, pharrnaeeuticaliy acceptable salt, or er thereof, wherein: L1; OZ} X2, Wz, Y2, Z2, R3, R5, RC, Rd, Re, Rf, RM, RC2, R”, R18, R19, R16, and RC1 are as defined in a (A); X5 is CR“; and n: (i: R“ is connected to LD Via a functional group of R“; or (ii) at least one of RC2 and RC] is present, and wherein at least one of RC2 and RC1 is connected to l1"D Via at least one functional group of the R“ and/or RC1. [312} In some embodiments, the S'l'lNG agonist drug moiety is a compound is of Formula (A— _l R19 0 Z’hy: ,/ «X3 @i R” l PM W?’ ‘\ LR” N l > RC] xxx/N R18 (A—h} or a prodrug, solvate, pharmaceutically acceptable salt, or tautoiner thereof, wherein: X1, W1, Y1, 21, X3, X4, X5, Xe, R3, R5, R0, RM, RA}, Rd, R9, Rf, R”, R”, R18, R”, R“, and R“ are as defined in Formula (A);wherein: (i) at least one of RM and R“ is present, and wherein at least one of RM and R“ is connected to LD via at least one functional group of the RM and/or R“, or (ii) at least one of RC2 and RC1 is present, and wherein at least one of R” and RC1 is ted to LD Via at least one functional group of the RCZ and/or RC}.

In some embodiments, the STING agonist drug moiety is a compound is of Formula (Aw L}; \O (A-hl) 01' a, pmdrug, te, pharmaceutically acceptable salt, or tautomer thereof, wherein: X1, X3, “H, Y}, 21, X5, X6 R3, R5, R5, RAZ, RA}, Rd, Re, Rf, R14, R15, R18, R19, R16, and RC1 are as defined in Formula (A), wherein: (i) at least one of RM and RA} is present, and wherein at: least one of RA2 and RA} is connected to LB via at least one. funetimal group of the RA?" mid/or RA}; or (ii) at least one of R“ and RC1 is present, and wherein at least one of RC2 and RC1 is connected to LD via at least one functional group of the R“ and/or RC1. {314} In some embodiments, the G agonlst clrug moiety is a compound is of Formula. (A— 112): O [AMY/R1 / X3 / fl (D; R" l FN w: 1, 6 \ ' \ , R N N ,XSVSN 18 Rb'“ _: l. %N \ ”S OYRl 0' .«N (A—hf/Z) or a prodrug, solvate, pharmaceutically acceptable salt, or tautoiner f, wherein: X1, X3, W1, Y1, Zi, R3, R5, R“, Rd, Re, Rf, R”, R”, R5, R18, R19, R”, and RC] are as defined in a (A); X5 is CR“; and wherein: {i} at least one of RA2 and R“ is present, antl wherein at least one of RM antl RA1 is connected to LB via at least one functional group of the RA2 and/or R“; or (ii) at least one of RC2 and RC1 is present, and wherein at least one of R62 and RCj is connected to LD Via at least one onal group of the RC2 and/or RC".

In some embodiments, the STING t drug, moiety (D) is a compound of Formula (A), wherein the compound is of Formula (Ani): i I HgN’ {Ext >m<72>l1'f"\\.”l or a prodrug, solvate, pharntaeeutieally acceptable salt, or tautonier thereof, wherein: Y1, ‘172,21,22,X1,X2,X3,X6,\Nl,\V2,RA1,RA2,Rial-{Lilac}Rf,R14,R,C2,R16,RC1,R15,R17, R18, and R19 are as defined in Formula (A), and wherein: (i) at least one ofRA2 and RA1 is present, and wherein at least one of RM and RA1 is eoi'ineeted to LB via at least one functional group ot‘the RA2 and/or R“; or (ii) at least one ‘9 " ~ - :\ F Cl . i ot R"- and RL1 is present, and wl'ierein at least one ot R‘2 and R\ is eonneeted to LB Via, at least one functional group of the RC2 and/or R“. [316} In some embodiments, each STING t drug nioiety (D) indepei'idently is: WO 02984 WO 02984 WO 02984 WO 02984 HZNW/K/fingH N~ 0 / ” ‘lé.....NH N CM H.4NYo N . g??? WO 02984 HéN \ \ E , ‘>—NH A’SLRQMQ WO 02984 O NNN ”» N H R2 \ s l ‘3)me \/J\ N N K’I‘Tr’N . NH HzN &}.‘F\f {TEN o 0’ O o ,,L, N 7K “ \ , Hi?” ’ N O O / \ \q \IT \ \ I >_N% yN / N x /’C ’> §1R2V\\O \ (y ,A: i >—NH o H NEA\/ N” 0):] 1 1/ its/”N U Q / th \(N \TF WI; Ma N r" N ,J x": N I ,«>_NH 0 f a» if o (L.

HZNM”N‘>....:\§H““ \ IN Nrfi‘N ! .J E‘RZWO j ”Wk "‘3‘I NH «f0 . VKEN/ 0/ "N JO O 0% H N Ri/T‘aflhi’ \ iN KNf’ N //j BENRZANAO \ ; N b N 0 3.. 3‘ \ ,ngmVAO R‘ ‘ N ‘ \ijN” “H i»:21\1\T f />-NH / HQN \Jj: ’ (E) ____ O \n/[r N A?” 0 U $3}:a ‘9 O o O ‘0. / HQN’. \ a I\.’ \ 'N E 2 I \>—NH, "Nil: / ~~ N’ ‘N N I. e < s‘\ 2N’\.

R o ( 1%” EN “N- , HQN /I\ S \11/ \I \ N k} l 0 ’ ’ /‘\ ”N W /NE:O\—(Wag; HAN ”T N :L f)me N», ”J \N N ,J fog‘V‘xO genzwfl K ( « h‘. 57‘ «N :1:>=NH ::::I\gI O HEN i\/3 “3}j)T W I o” \-—N <3 5HN'N O WO 02984 PCT/U82021/025556 o “*N HzN)1,» H / \ ,.~S E N“ N J \.

:' “(N / 'NHNWN ” NH ifiNI! ! - HnN / 1; ~ om! HQN E? \g/ \ N _____ if 0 PK iffy-x: f} E‘RZN/NQ HzNL13:’>“Ol ‘OTN or v» ; wherein: R2 is absent, -O— or —NR4-; R4 is H 01' (:1—3 aikyl; and "gm denntes attachment 10 LD. [317} In some embodiments, each STING agonist drug moiety (D) independently is: e O i42N)J\\:r’/‘\\EWN O QN/ / ii ‘ \ I “—sz N 3:57/ \ J“ / ' [I N ,4 HzN/IVENO\>‘NHKEN £5:RZx/AQ / f ./ ‘ \R2WO j“?IMN ! \—NH 0]“‘ / HgN ’>“NH i r? \ 'Z’KIINi 'N HgN /*‘\\\/ 071/ m («4' N\ ‘ \ \ :3ngm0 ”:RAN/NQ \ if“ ’” ‘N 1/ NH 03 HZM‘H/ \ «N ’Wf N4 H?“ QNQ- 5 0m/ O ,1” :51; \ (3 N ”,1 .

\ ,N x N u N" “N ,4 \N\ , .5 \\ A < \RZMO F‘RQMQ 8 WO 02984 WO 02984 wherein: R2i3é1bsetlt—(D or NR5,4R is H {31"(13 alkyl; and 5 denotes attachmentto LD ( In some embodiments, each STING agmist drug moiety (D) il'ldependently is: o o 0 [07K /U\r /\ O O‘Tf/ Hg»: / 'N \ HzN 5” N ‘ \ § N>~\ N ’N NH ‘ 5i V“‘NH \ ’4’, {K‘N/ \N ”J, 3:5: [QC-{W0 1/ M/A“ HQNT‘:I N t NH ov’ HNTEL/5N ’ E: E10>—-NHL" C) o s -” E HZN \ \ : >=:N' N ’ II N #4 «J ; or wherein: R2 is absent, AZ)- 01’ -NR4—; R4 is H or {31.3 alkyl; and _§“ denotes attachment to LD.

In some embodiments, each S'HNG agonist drug meiety (D) independently is: / Pa o 0 Han \ l ""fi/Ec / /’ N N ”—4 fiRg’mG wherein: R2 is absent, -O— or —NR4-; R4 is H or CL; allryl; and i“ denotes attachment to LB.

Pram/inflated Recogzm‘onnil/1016mm (PthM) [321} In some embodiments, proteinwbased recognition molecule directs the conjugates to specific tissues, cells, or locations in a cell. In some embodiments, the protein—based recognition molecule can direct the conjugate in culture or iii a whole organism, or hoth. In each case, the proteinnhased recognition molecule may have a ligand that is t on the cell surface of the targeted cell(s) to which it binds with an ef"eetive specificity, affinity, and avidity. In some embodiments, the protein~based ition molecule targets the conjugate to tissues other than the liver in some embodiments the n—based recognition le targets the conjugate to a specific tissue such as the liver, kidney, lung, or pancreas, The n—based recognition molecule can target the conjugate to a target cell such as a cancer cell, such as a receptor expressed on a cell such as a cancer cell, a matrix tissue, or a n associated with cancer such as tumor antigen.

Alternatively, cells comprising the tumor vasculature may be targeted. The protein~hased ition molecules can direct the conjugate to specific types of cells such as specific ing to hepatoeytes in the liver as opposed to Kupffer cells, In some embodiments, protein-based ition molecules can direct the conjugate to cells of the reticular endothelial oi' lymphatic system, or to professional phagocytic cells such as macrophages or eosinophils. in some embodiments, the conjugate itself may élSO be an effective delivery system, without the need for specific targeting. [322} In some embodiments, the protein-based recognition molecule can target the conjugate to a location within the cell, such as the nucleus, the cytoplasm, or the endosome, for example, In some embodiments, the protein—based recognition molecule can enhance cellular binding to receptors, or asmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles. {323} In some embodiments, the protein—based recognition le is an antibody, an antibody fragment, a protein, a peptide, or a peptide mimic. {324} In some embodiments, the protein-based recognition molecule is an antibody. In some ments, the protein~based recognition molecule is an antibody fragment. 111 some embodiments, the protein—based recognition molecule is a protein. In some embodiments, the protein—based recognition molecule is a peptide. In some embodiments, the proteinnbased recognition molecule is a peptide mimic. [3:25] In some embodiments, the antibody or antibody fragment is an antibody or dy fragment n one or more amino acids of the corresponding parent antibody or antibody fragment (eg, the corresponding wild type antibody or antibody fragment) are substituted with cysteines (e. g, ered cysteine). In some embodiments, the parent antibody or antibody fragment may be wild type or mutated.

In some embodiments, the antibody or antibody fragment may be a mutated antibody or antibody fragment, In some embodiments, a monoclonal antibody known in the art is engineered to form the antibody. In some embodiments, an antibody fragment (eg, a Fab antibody nt) known in the art is engineered to form the antibody fragment (e.g a cysteine engineered Fab antibody fragment). In some embodiments, a single site mutation of a Fab gives a single residue in a Fab whereas a single site on in an antibody yields two amino acids in the ing antibody due to the diineric nature of the lgG antibody. [327} In some embodiments, the antibody or antibody fragment retains the n binding capability of its corresponding wild type antibody or antibody fragment. In some embodiments, the antibody or antibody nt is capable of binding to the one or more antigens for its corresponding wild type antibody or antibody fragment. [328} In some embodiments, exemplary antibodies or antibodies derived from Fab, FabZ, scFv or camel antibody heavy~chain fragments specific to the cell surface markers, include, but are not d to, 5T4, ADC3, ALK, AXL, 1374114, C242, C4.4a, CA_125, CCL11, ma 5, CD2, Cos, CD4, CD5, CD15, CAi5—3, CD18, CD19, 9, CD116, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD44 yo, CD51, CD52, CD54, C1356, CD625, CD621: CDesL, CD70, CD74, CD79—B, CD80, {313125, CDi03, CD138, CD141, CDI47, CDISZ, CD 154, CD326, CEA, CEACAIVI—S, dumping factor, Clech, CSFRl, CTLAu 4, CXCRZ, DECZOS, EGFR til-1131211), ErbBl, 13113132, 13113133, EpCAM, 15131-11512, 15131-11352, 13131-1134, PAP, PGPR (1e. PGFRI, FGPRZ, FGFR3, PGFR4), FL'1'3, ectinuEDB, felate receptcr, (3132, GD3, CPNMB, GCC (GUCYZC), HGP, HERE, 11ER3, 113411.24, 1CAM, 1CDS-L, 1GP-1 recepter, VEGERI, EphAZ, 'I‘RPVI, CFTR, gpN’MB, CAQ, , c—K1T, c—ME'I', ACE, APP, rgic receptor—beta; Claudine 3, LEVI, LYSE, Mesotheiin, MUCI, MUC13, NaPiZb, N()"£‘C1-11, N()"£‘C1-12, N017{71-13, ND'17C1-14, RON, R()R1, I’D-L1 , PD—LZ, P'1'K7, 87—1-13, B’7-B4, 1L~2 receptor, IL—4 receptcr, IL—13 receptcr, TROP—Z, frizzled—7, integrins (including rm, ang, 01 v55, , , 414131, @4137, 01551, 014%, (1111783 integrins), IFNwa, IFNw, IgE, IgE, 1GF—1 receptor, IL— 1, 11342, 1Ln23, 1L—13, 1Lw22, 1L—4, 1Ln5, 1L~6, interfercri receptor, ITGBZ (CD18), LFAU1 (CD11a), CDMb, L—seleetin (CD62L), mucin, myostatin, N’CAéPO, N'GF, PDGFRCL, phesphatidylseririe, prcstatic carcinoma cell, Pseudomanas aerugmasa, rabies, RANKL, respiratory syncytial Virus, Rhesus factor, $LAN1F7, sphingcsinem1uphcsphate, TAGm72, '1'~ee11 receptor, tenascin C, TGF~1, 'I‘GP~ B 2, TGF"13, 'I'NFnrx, 'I‘RA1LnR1, '1'R41DR2, turner antigen 88, VEGDA, Z, Vimentirt, and the like. [329} In some embodiments the antibodies or antibody derived from Fab, Fab2, scFv or came} antibody 1ieavy~ehaiu fragments specific te the ee11 surface markers iuc1ude (EA—125, C242, CD3, CD111), CD'EQ, CD22, CD25, CD30, CD31, CD33, CD37, CD49, CD44, CD51, CD54, CD56, CD6213, CDESZP, CD62L, CD70, CD103, CD 1,38, CD141, CD326, CEA, CleeQA, CSPRI, CTLA— 4, DECZOS, EGFR (HERD, ErbBZ, ErbB3, PAP, DDTOIIGCUEPEDB, fclate receptor, 1017—1 receptor, GD3, GPNMB, HGF, 2, VEGRA, VEGFRZ, VEGFR1 , EphAZ, EpCANL 5T4, PTK7, TAGuZ, tenascin C, TRPVl, CPTR, gpNMB, CAQ, Crip‘tc, ACE, APP, PDGFR a, phesphatidy1serirae, presiatic carcinoma ceils, adrenergic receptcr—betaZ, Ciaudirie 3, mucin, MUC], NaPiZb, B7113, B7114, C443,, ~5, MUCI 3, TRIP—2, frizzledfl, etin, 1L— 2 receptor, 113-4 receptor, 111—13 receptor and integrins (inciuding av E3 3, (1y [3 5, (lv 1% 6, 111 S 4, (14 6 1, (1 5 B1, (16 134 intergins), terrascirt C, TRAIDRZ, and vii'ricritiri. [3301 In some embodiments, the pedies are directed to ceil surface markers for 534, (IA—125, CPA, CD116, CD3, CD111), CD19, CD20, CD22, CD30, CD33, CD40, CD44, CDSI, (ID—103, C'1‘LA-4, CEACAMS, CiecQA, CSFRl, DECZOS, EpCAIVI, 1113112, EGPR (HERD, FAP, ectin—EDB, fclate receptor, (ECC C), , integrin avBs, integrin 015131, 1(3Fu1 receptor, GD3, B, rnucin, LEVI, LYGE, irresctheiin, N111C1, MUC13, NaPiZb, PTK'], phosphatidylserine, prostatic oma cells, PDGFR u, 11516-72, tenascin {7, R2, VEG ‘_ A and VEGFRZ. In this embodiment the antibodies, include but are not limited to, aliagovomab, edecatumumeh, elacizumeh, altumoniab, anatumomah, arcitumomab, bavituximah, bevacizumab (LAXVASTINCE), bivatuzumab, blinatumomab, ximab, cantuzuniab, catumaxomab, ab, cetuximab, zumab, clivatuzumab, conatumumab, dacetuzurnah, edrecolomab, epratuzumab, ertumaxomab, cizumab, farletuzumab, figitumumab, gemtuzuma‘o, glem‘oatuniumab, momab, igovonia‘o, intetumumab, inotuzuniab, labetuzumah, lexatumumah, lintuzumab, lucatuniuniab, niatuzuniab, niitunioniab, napturnornalo: natox, necituniumab, oportuzumab, oregovoma‘o, panituinuniah, peniturnoinab, pertuzuniab, pritumuinab, rituxiniab (RITUXANQi), rilotuinuinab, robatuniui‘nab, satuinoniah, sibrotuzumab, taplitunioniab, tenatuinoinab, inoi‘nab, ticiliinuniah (tremehinuinab), tigatuzuinab, trastuzuinab (HERCEl"IlN®)), tosituniomab, ti'eineliinuniah, tucotuzuinab celi‘noleukin, ximab, and zalutuniuniab. [331} In some embodiments the antibodies directed to cell e markers for HERE are pertuzuinab or trastuzui‘nab and for EGFR (HERD the antibody is cetuxiinab or panitumuinah; and for C1320 the dy is ritnxiinab and for VEGEA is bevaciznniab and for CD—ZZ the dy is epratuzuinab or veltuzunial) and for CEA the dy is labetuzuineb. [332} Exemplary peptides or peptide mimics include integrin targeting es (RGD peptides), LHRH receptor targeting peptides, ErbB‘Z (HERZ) receptor targeting peptides, prostate specific membrane bound entigen (PSMA) targeting peptides, lipoprotein or LRPl targeting, ApoE protein derived peptides, ApoA protein peptides, sonietostatin receptor targeting peptides, chlorotoxin derived peptides, and bomhesin. [333} In some embodiments, the peptides or peptide niirnics are LHRH receptor targeting peptides and ErbBZ (HERE) receptor targeting peptides [334} Exemplary proteins comprise insulin, transferrin, fibrinogen-garnma lragrnent, thrornbospondin, claudin, apolipoprotein E, Al’l’ibody rnolecul es such as, for exan'iple, ABY—OZS, Ankyrin repeat proteins, ankyrirnlilte repeats proteins and synthetic peptides, [335} In some embodiments, the protein-drug conjugates comprise broad spectrum cytotoxins in combination with cell surface markers for HERZ, such as, for example, umab or trastuzuniab; for EGFR such as cetusirnab and panitumumeh; for CEA such as labetuzumah; for CDZO such as rituximab; for VEGF—A such as hevacizumeh; or for (ED—22 such as epratuzumah or veltuzumab. :33 l In some embodiments, the protein~drug conjugates or protein conjugates used in the disclosure comprise combinations of two or more protein~based recognition molecules, such as, for example, combination of bispecific antibodies directed to the EGF receptor (EGFR} on tumor cells and to CD3 and CD28 on T cells; combination of antibodies or antibody derived from li'ab, FabZ, scFv or camel antibody heavy—chain fragments and peptides or peptide miinetics; combination of dies or antibody derived from li'ab, FabZ, SCFV or camel antibody heavy- cliain fragments and ns; combination of two bispecit‘ic antibodies such as CDB~CDl9 plus CDZS—CDZZ ific antibodies. [337} In some embodiments, the proteinwdrug ates or protein coniugates used in the disclosure comprise proteinnbased recognition les are antibodies against antigens, such as, for example: 'I‘rastuzumab, mab, Rituxiniab, zuniab, Epratuzuniab, Veltuzuniab, Labetuzuniab, B7nH4l, B7—H3, CI‘illb, CD‘lOB, cams, cone, CD33, oxcnz, CEACAa/is, ClecQA, CSFRl, onczos, EGFR, PAP, fibronectinnEDB, FGFRI, FGFRZ, torus, Form, GCC (GUCYZC), HER2, LIVL LYbE, NaI’iZb, owl‘v‘let, mesothclin, NO'I‘CHl, NOTCHZ, NOTCH3, NOTCH-él, PIE—Ll, PTK'F, c—Kit, l‘vfilCl, ‘MUClS, and 5T4. [33 8} In some embodiments, the protein—drug ates or protein conjugates of the disclosure comprise proteinvbased recognition molecules which are CSRFl, CDllb, DECZOS, clecQA, CDl 03, B7H4, mesothelin, PTK7, 1.51613, PAP, fibronectin~EDB, Her—2 or NaPiZb antibodies, NaPi2b Azrzribodies In some embodiments, the NaPiZb antibodies suitable for conjugation bind to the extracellular region of SLCMAZ. In some embodiments, the present disclosure es NaPiZb— targeted monocloi'ial antibodies that specifically recognizes NaPiZb, also known as sodium- dependent phosphate transport protein 23 In some ments, the NaPiZb antibodies used in the conjugates disclosed herein are capable of and useful in modulating, erg blocking, ting, reducing, antagonizing, neutralizing or otherwise interfering with at least one biological activity of NaI’iZb. In some embodiments, dies disclosed herein also include antibodies that bind soluble b. In some ments, the Nal’iZb antibodies ically bind to an epitope on an extracellular domain (ECD) of the human NaPiZb. These antibodies are collectively referred to herein as “NaPiZb” antibodies.

WO 02984 {340} In some embodiments, the NaI’iZb antibody—drug conjugates provided herein include antibodies that bind to a Nal’iZb epitope with an equilibrium iation constant (Isa or Kn) of £1 uM (cg, :1 100 nl‘vl; : 10 nM, and g 1 nM). In some embodiments, the NaI’iZb antibodies used in the antibodbdrug conjugates disclosed herein exhibit a lid in the range imately between ; l rib/l, to about 1 pl’vl, [341} In some embodiments, the NaPiZb antibody—drug conjugates provided herein can include antibodies that seiye to modulate, block, t, , antagonize, neutralize, or otherwise interfere with the functional activity of NaPiZb, In some embodiments, onal activities of NaPiZb include for example, participating in the transcellular nic ate (Pi) absorption, thereby contributing to the maintenance of phosphate tasis in the body, In some embodiments, the NaPi‘Zb antibodies completely or partially inhibit NaPiZb functional activity by partially or completely modulating, blocking, inhibiting, reducing nizing, neutralizing, or otherwise interfering with transcellular inorganic phosphate absorption.

In some embodiments, the NaPiZlJ antibodies are considered to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPiZb functional activity when the level of NaPiZb functional activity in the presence of the NaPiZlJ antibody is decreased by at least ass/s, e.g;, by 96%, 97%, 98%, 9 %, or IOO‘Vis as compared to the level of NaPiZb functional activity in the e of binding with a NaPiZb antibody described herein. In some embodiments, the NaPiZb antibodies are considered to partially modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with NaPi2b functional activity when the level of NaPiZb activity in the presence of the NaPiZb antibody is sed by less than 95%, cg, %, 209/5, 25%, 30%, 40%, 50%, 60%, 75%, 809.4), 859/5 or 90% as compared to the level of NaPiZb activity in the absence of binding with a NaPiZb antibody described herein.

In some embodiments, exemplary antibodies disclosed herein include, the 35 antibody. These antibodies show specificity for human NaPiZb and they have been shown to inhibit NaI’iZb activity. [344} NaPiZb human or humanized monoclonal antibody, XM'I‘~l535, includes a heavy chain (HC), heavy chain variable region (VI-l), light chain (LC), and a light chain variable region (VL), as shown in the amino acid and corresponding nucleic acid sequences presented in Table l below.

The le heavy chain region and variable light chain region for each dy are shaded in the amino acid sequences below. The complementarity determining regions (CDRs) of the heavy 00 U) chain and the light chain are underlined in the amino acid sequences presented below. The amino acids encompassing the complementarity determining regions (CDRS) for the I‘m/[ll l53 5 antibody are disclosed in US Patent 8,603,474.

Table l: NaPiZb human or humanized monoclonal antibody XM'l'—1535 sequences Xh/l’l'nl535 Heavy Chain Amino , - , , = Acid Setuence Xh/l’l21535 lgGl Heavy chain e , ' “ ‘ constantregion : _ l535 Heavy chain variable re i011 Mir—1535 Heavy chainl variable region nucleic acidl setuence i XMT— 1535 Light chain variable rerion nucleic acid seuence Fullnlength human Nai’iZbl sequence )5thl535 (DRLZ Antibodies sed herein specifically bind to an enitope on an extracellular domain (ECD) of the human Nal’iZb.

In some embodiments, those skilled in the art will recognize that it is possible to determine, without undue experimentation if a monoclonal antibody has the same specificity as a monoclonal antibody disclosed herein (egg KNIT—1535, lOHl ,l l .43) by ascertaining whether the former prevents the latter from binding to a natural binding, partner or other molet.ule known to be associated with i lf the onal antibody being tested competes with the monoclonal antibody disclosed herein, as shown by a decrease in binding by the monoclonal antibody disclosed herein, then the two monoclonal dies bind to the same, or a closely related, epitope. [3-47] An alternative method for determining whether a monoclonal antibody has the icity of monoclonal antibody disclosed herein is to presincubate the monoclonal antibody sed herein with soluble NaPiZb (with which it is normally reactive), and then add the onal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind NaPiZb. If the onal antibody beii'ig tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody disclosed herein. [3483 ing of monoclonal antibodies disclosed herein, can also be carried out, .,by measuring h—niediated activity, and ining whether the test monoclonal antil):dy is able te te, block, inhibit, reduce, antagonize, neutralize or ise interfere with Nal‘iffib activity. [349} In some embodiments, the antibodies disclesed herein comprise a heavy chain variable region having an amino acid sequence at least 8596, 8696,8796 8896, 896, 90° 6,91%, 92%, 93%, 94%, 9596, 969/6, 97°6 98966, 99% or more identical to a sequence selected from SEQ ID NOS: 3 and a light chain variable region having an amino acid sequence at least 8596, 8696, 8796 88% 899/6, 969‘6 9 96,92/6, 93%, 9496, 9596, 969/6, 9796 989//6, 9996 or more identical to a sequence selected from SEQ II) NQs: 4.

In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence at least 859/6, 8696, 8796 88968996 9096,9l%, 929/6, .9396, 9496,9596, 96%, 9796 98% 999/6 or more identical to the amino acid sequence of SEQ ID N0: 1 and a light chain amino acid ce at least 859/x6, 86%, 8796 889/6, 89%, 9096, 9l9/6. 929/6, 9396,9496, 95%, 96%, 979/6 98% 999/6 or more identical to the amino acid sequence cl SEQ ID NO:2 [35l] In some embodiments, the antibodies disclosed herein comprise the heavy chain variable region amino acid sequence of SEQ ID NO: 3 and the light chain variable region amino acid sequence of SEQ ID N0: 4', [352} In some embodiments, the antibodies disclosed herein comprise the heavy chain amino acid sequence of SEQ ID NO: I and the light chain amino acid sequence of SEQ ID NO: 2. [353} In some embodiments, the antibodies disclosed herein comprise the CDRIIl amino acid ce of SEQ ID NO: 5, the CDRIIZ amino acid ce of SEQ ID NO: 6, the CDRII'B amino acid sequence of SEQ ID NO: 7, the CDRIJI amino acid sequence of SEQ ID NO: 8, the CDREZ amino acid sequence of SEQ ID NO: 9, and the CDRI3 amino acid sequence of SEQ ID NO: 10. [354} In some embodiments, the antibodies disclosed herein that comprises the amino acid sequence at least 85%, 8696, ’796 8896, 8 96, 90%, 9i '?/6, 9296, 9 96, 9496, 95%, ,9696, 9796 98'.3/6, 9996 or more identical to the amino acid sequence of SEQ ID NO: 5; a CDRI-IZ that comprises the amino acid ce at least 85%, 869-6, 8796 88%, 89'.3/6, 9096, 919-6, 9296, 93°6 9496,9596, 969/6, 97% 9896, 99% or more identical to the amino acid sequence of SEQ ID NO: 6, a CDEIB that con1p11ses the a111in0 acid sequence at least 85%,86‘3/13, 87% 88%, 89%, 90%, 91%, 92%, 9313/11, 94%, 95°,0, 96%, 97% 98%, 99% or m01e identicai to the an11n0 acid sequence of SEQ 113 NO: 7; a CDRL1 that comprises the an11n0 acid sequence at 85%, 86%, 87% 88%, 89%, 90%, 91%, 9213/11, 93%, 94%,0333/“'1'i, 96%, 97% 98%, 99% or m0re identicai t0 the an11n0 acid sequence of SEQ 1D NO:8;a (DRLZ that ses the amino acid sequence at ieast 85%, 8613/11, 87% 88%, 89%, 9013/11, 91%, 92%, 9313/11, 94%, 95%, 96%, 97% 9" 0,99% 01 more identical to the amino acid sequence 01‘ SEQ 11) NO: 9, and 21 CDRL3 that comprises the amino acid sequence at least 85%, 86%, 8 % 88%, 89%, 90%, 91%, 92%, 938/13,, 94%,95%, 96%, 97% 98%, 99% 0r more 1dentica1 to the amino acid sequence of SEQ 11') N0: 10.

In some embodiments, the antibodies disclosed herein include one or more conseivative amino acid substitutions in a variable domain sequence such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1'1, 12, 13, 14, 15, or more conservative tutions in a variable domain sequence. in some embodiments, these conservative amino acid substitutions are in a CDR region,e ,,1 2,3 , 4,“ o, , 89_, 10, 11, '12, 13, 14, 15, or more conservative substitutions are made cu1nu1ative1y across aii CDRs and in some particular embodiments, up to 1, 2, '3, or 4 conservative amino acid substitutions may be present in each CDR sequence, eg,S1§Q ID NOs: 5~1O In some embodiments, those skiiied in the art wili recognize that it is possibie to determine, without undue experimentation, it‘a, onal antibody has the same specificity as a monocionai dy XIX/114535, by aining whether the former prevents the iatter from binding to a naturai binding partner or other ie known to be associated with NaPiZb, 1f the monocionai antibody being tested competes with the rn0noc10na1 antibody disc10sed , as shown by a decrease in binding by the 1nonoc10i1a1 ai'1tibody discl0sed herein, then the two monoclonal dies bind to the same, or a c10sely related, epit0pe. [357} In some embodiments, an alternative method for determining whether a inon0clona1 antibody has the specificity of a rn0noc1ona1 dy disclosed herein is to cubate the 1nonoclona1 antibody sed herein with soluble NaPiZb (with which it is normaliy reactive), and then add the monoclonal dy being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind NaPiZb. 111 some emb0din1ents, 11‘ the monocional antibody being tested is inhibited then it has the same, or functionally equivalent, epitopic specificity as the nionocionai antibody disclosed herein. {358} Screening of monoclonal antibodies disclosed herein, can be also carried out, cg, by measuring NaI’iZb —niediated activity, and determining whether the test monoclonal antibody is able to modulate, block, inhibit, reduce, antagonize, lize or otherwise interfere with NaI’iZb activity. {359} In some embodiments, the NaI’iZb antibodies suitable for conjugation can be generated and purified by well—lmmvn techniques eg, each of which is incorporated herein in its entirety by reference.

HER2 Antibodies In some embodiments, the HER2 antibodies suitable conjugation bind the human IIE'RZZ in soluble form, or membrane bound (1'. e, , when expressed on a cell surface). In some embodiments, the present sure provides monoclonal antibodies that bind HER2 and are humanized or fully human. In some embodiments, the present disclosure provides monoclonal antibodies that bind HER2 ically. 'l'hese antibodies are collectively referred to herein as HE“R2” antibodies. [36,] In some embodiments, the HER2 antibodies suitable for conjugation bind to a HER2 e with an equilibrium dissociation nt (Kd or KB) of :El uM (tag, :3 100 nM; :3 l0 nM; :1 l nM) In some ments, the present disclosure es monoclonal antibodies that bind HER2 and are humanized or fully human. for example, the HER2 antibodies provided herein exhibit a Kd in the range approximately between is; l nM to about l pM. [362} In some ments, the HER2 dies disclosed herein serve to modulate, block, inhibit, reduce, antagonize, lize, or otherwise interfere with the functional ty of I‘IERZ.

HER2. In some embodiments, functional activities of HER2 include for example, modulation of Pl3l<l~Al<t pathway activity. In some embodiments, the HER2 antibodies completely or partially inhibit HER2 onal activity by partially or completely modulating, blocking, inhibiting, ng antagonizing, neutralizing, or otherwise interfering with PI3K“,Ak‘t pathway activity.

PI3K—Akt patl'iway activity is assessed using any arterecognized method for detecting PI3K—Akt pathway activity, including, but not limited to ing levels of phosphorylated Akt in the presence and absence of an antibody or antigen binding fragment disclosed herein. [363} In some embodiments, the HER2 antibodies are considered to tely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity when the level of I-IERZ functional activity in the presence of the IIER2 antibody is decreased by at least DC \1 80%, 8.33,by 81"/o, 87% 83% 84%, 85%, 86%, 87%, 88%, 895343, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9706 95'”9Ar, 99%, or 100% as compared to the level of HER2 functional activity in the absence of binding with a HER2 antibody described herein. in some embodiments, the HER2 antibodies are considered to lly inodulate, block, inhibit, reduce, nize, neutralize, or otherwise interfere with HER2 functional activity when the level ot‘HER2 activity in the presence of the HER2 dy is decreased by less than 95%, erg}, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75/o, 800//,o 85%, or 90% as compared to the level of HER2 ty in the absence of gwith a HER2 dy described herein. [364} In some ments, exemplary antibodies disclosed herein include, the m/l’l'nl5l9 antibody. This antibody show specificity for human HER2 and they have been shown to inhibit the functional activity of HER2 m virro. [365i HERn2 monoclonal antibody Xl‘v/l’l'nl5l9 includes a heavy chain (HC), heavy chain variable region (VH), light chain (LC), and a light chain variable region (VL), as shown in the amino acid and corresponding nucleic acid sequences presented in Table H below, The variable heavyt:hiain iegion and vaiiable light chain region tor each antibody are shadedin the amino acid sequences below The complementarity determining regions (CDRs) of the heavy chain and the light chain are underlined in the amino acid sequences presented below, Table ll: HERZ human or humanized monoclonal antibody KNIT—1519 ces sco siio H} Ni): Sequence Description {D N0: Sequence ption Fullelength hurnan HER2 XMTJ 5l 9 Light chain variable 16 molar_____________________________________________________________ 24 new__________________________________________________________________ XMTJ 5 l 9 Heavy chain XM’I'nl 5 l 9 Light chain constant variable region '25 re Iion XMTl5l9 lgGl Heavy chain . “9115193919019“ Xl‘vlTn 15 l 9 HeaW Chain Aininoi Acid Se uence Xhil—l5l9 CDlH—ll Xl‘vl'll—l 5 l 9 CDlH—lfl XVlT— l5l9 (_DRH3 5liliiiii )Cvlll5l9Heau Extracellular doinaiii (ECD) of variable region nucleic acidl L ' the human HER2 receotor [366i Antibodies and antigen binding fragments thereof disclosed herein specifically bind to an WO 02984 2021/025556 epitope on the full-length human HER2 receptor comprising the amino acid sequence of SEQ ID NO: 16. [367} Antibodies and antigen binding fragments thereof disclosed herein specifically bind to an epitope on the extracellular domain (ECD) of the human HER2 receptor comprising the amino acid sequence of SEQ ll) NO: 3l.

In some embodiments, the antibodies of the present disclosure exhibit HER2 binding characteristics that differ from antibodies described in the art. ln some embodiments, the antibodies disclosed herein bind to a different epitope of HER2 in that they cross—block each other but not trastuzumab, pertuzumab, Fab37, or chA2l from binding to HER2 Further, as opposed to the known antibodies, the antibodies disclosed herein can alize efficiently into HER2meXpressing cells without promoting cell proliferation.

In some embodiments, the antibodies disclosed herein are fully human monoclonal antibodies that bind to novel epitopes and/or have other favorable properties for eutic use.

In some embodiments, exemplary properties include, but are not d to, favorable binding characteristics to cancer cells expressing human HER2 at high or low levels, specific g to inant human and cynomolgus monkey HER2, efficient internalization upon binding to HER2, high capacity for killing cancer cells expressing high or low levels of HERZ when administered as an antibody drug conjugate (ADC), no substantial agonistic effect on the proliferation of HERZ—expressing cancer cells, and/or provide for effective antibody—dependent cellular cytotoxicity —mediated killing of lilERZ—expressing cells, as well as any combination of the foregoing ties. [370} In some embodiments, the antibodies sed herein also include an antibody or n binding fragment thereof that ically binds to an epitope of the human HER2 or that includes residues 452 to 531 of the extracellular domain of the human l‘lERZ receptor, residues 474 to 553 of SEQ ID NO: l6 or residues 452 to 531 of SEQ ID NO: 31. [371} In some embodiments, the antibodies disclosed herein include an antibody or an antigen binding fragment thereof that binds at least a portion of the N—terminus of domain IV of human HER2 receptor but does not cross—compete with an antibody that binds to epitope 4D5 of the human HER2 receptor. In some embodiments, the antibodies or antigen binding fragments thereof described herein do not cross~compete with trastuzunrab for binding to the human HER2 receptor, as trastuzuinab is known to bind epitope 4D5 of the human HER2 or. As used herein, the term epitope 4135 of the human HER2 receptor refers to amino acid residues 529 to 627 of the extracellular domain of the human HERZ receptor, residues 551 to 649 of SEQ H) NO: 16 or residues 529 to 627 of SEQ ll) NO: 3l. in some embodiments, the antibody or antigen binding fragment thereof also binds at least one epitope on cynoniolgus monltey l-lERZ or. 372} In some embodiments, the antibodies disclosed herein also include an antibody or n g fragment thereof that specifically binds to an epitope of the human HERZ receptor that includes residues 452 to 500 of the extracellular domain of the human HER2 receptor, residues 474 to 522 of SEQ ID NO: lb or residues 452 to 500 of SEQ ID NO: 31. [373} In some embodiments, the antibodies disclosed herein also include an antibody or antigen binding fragment thereof that specifically binds to an epitope of the human HERE receptor that includes at least one of amino acid e selected from amino acid residues E52l, L525 and R530 of the extracellular domain of the human HER2 receptor, cg, residues 543, 547, and 552 of SEQ ID NO: 16., and residues 52l, 525, and 530 of SEQ ID NO: 31. In some embodiments, the dies disclosed herein include an antibody or antigen binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HERZ or that includes at least two amino acid residues selected from amino acid es E52l 1.525 and R530 of the extracellular domain of the human HERZ receptor. in some embodiments, the antibodies sed herein also include an antibody or antigen binding fragment thereof that specifically binds to an epitope of the human HER‘Z receptor that includes at least amino acid residues E52l, L525 and R530 of the extracellular domain of the human l‘lERZ receptor. In some embodiments, any or all of these antibodies or antigen binding fragments thereof also bind at least one epitope on Gyl’IOITlOlngS monlrey HERE receptor In some embodiments, antibodies disclosed herein also include an antibody or an antigen g fragment thereof that binds to at least a, n of domain HE and at least a portion of the N—terinimis of domain 1V of human HERZ receptor but does not cross-compete with Fab37 monoclonal antibody or an antibody that binds to epitope 4135 of the human HERZ receptor. ln some embodiments, the antibodies or antigen binding fragments thereof described herein do not cross—compete with the Eab37 monoclonal antibody and/or trastuzumab for binding to the human HER2 receptor. in some ments, the antibody or antigen binding nt thereofalso binds at least one epitope on lgus monkey HERE receptor. {375} In some embodiments, the antibodies disclosed herein also include an antibody or antigen binding nt thereof that specifically binds to an epitope of the human HERZ receptor that es residues 520 to 531 of the extracellular domain of the human l-lERf/Z receptor, residues 542 to 553 of SEQ lD NO: 16 or residues 520 to 53l of SEQ ID NO: 31. 376} In some embodiments, the antibodies disclosed herein also include an antibody or antigen binding fragment thereof that specifically binds to an epitope of the human HERZ or that includes at least one amino acid residue selected from residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HERZ receptor, e.g residues 475, 478, 495, 498, 517, 518, 519, and 521 of SEQ in no: is or residues 455, 456, 475,, 476, 495,, 496, 497 and 499 of SEQ ll) N0: 31. In some embodiments, the antibodies disclosed herein include an antibody or antigen binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human HERZ receptor that includes at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid es, or at least six amino acid residues selected from amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HERZ receptor, In some embodiments, the antibodies disclosed herein include an antibody or antigen binding nt thereof that specifically binds to an e of the extracellular domain of the human HERZ receptor that es at least amino acid es C453, H456, Hr—‘l73, N476, R495, G496, H497, and “7499 of the extracellular domain of the human HER2 receptor, In some embodiments, any or all of these antibodies or n binding fragments thereof also bind at least one epitope on eynomolgus monkey HER/’1 receptor. [37' } In some embodiments, the antibodies disclosed herein also include an antibody or antigen binding fragment thereof that specifically binds to an e of the human HER/’1 receptor that includes at least one amino acid residue selected from residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HERE or, eg, residues 475, 495, 498, 517, 519, and 52l of SEQ H) NO: l6 or es 453, 473, 476, 495, 497 and 499 of SEQ ID NQ: 3l, In some embodiments, the antibodies disclosed herein include an an tihody or antigen binding fragment thereof that specifically binds to an epitope of the extracellular domain of the human l-lERZ receptor that includes at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, or at least six amino acid es selected from amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human I-IEIQ receptor. In some embodiments, the antibodies disclosed herein include an antibody or antigen binding fragment f that specifically binds to an epitope of the extracellular domain of the human I-IERZ receptor that includes at least amino acid residues C453, I-l473, N476, R495, H497, and W499 of the extracellular domain of the human l-IERZ receptor. In some embodiments, any or all of these antibodies or antigen binding fragments thereof also bind at least one epitope on cynornolgus monkey I-IERZ receptor. [378} In some embodiments, these antibodies show specificity for human , and they have been shown to te, e,g block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the Pl3K~Akt pathway which promotes cell survival by reducing levels of phosphorylated AKI In some embodiments, these dies internalize from the cell surface ofHERZ—expressing cells at a rate that is the same or substantially similar to the rate at which trastuzuinab or a biosiniilar thereof internalizes. In some embodiments, these antibodies and antigen binding fragments have a rate of internalization that is about 50% of the total surface bound at time 0 being internalized by 4 hours.

In some embodiments the antibodies disclosed herein comprise a heavy chain variable region having an amino acid sequence at least 89%, ), 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 9l%, 92%, 93%, 949/8, 95%, 96%, 97% 98%, 99% or more identical to a sequence selected from SEQ ID NQs: l7 and a light chain variable region having an amino acid ce at least 80%, 8l%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 9l%, 92%, 93%, 94%, 95%, 969/8, 97% 98%, 999%: or more identical to a sequence selected from SEQ ID NQs: 24, In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence at least 8094:, 8l %, 82%, 83%, 84%, 859/8, 86%, 87% 889/8, 89%, 98%, 9l%, 92%, 93%, 9' %, 95%, 96%, 979%: 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: l9 and a light chain amino acid ce at least 80%, 8I £343, 82%, 83%, 84%, 85%, 869/8, 87% 889/8, 8994:, 90%, 91%, 92%, 93%, 949/8, 9594:, 96%, 97% 9894:, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, In some embodiments, the dies disclosed herein comprise the heavy chain variable region amino acid sequence of SEQ ID NO: l7 and the light chain variable region amino acid sequence of SEQ ID NO: 24. [382} In some embodiments, the dies disclosed herein comprise the heavy chain amino acid sequence of SEQ ID NO: l9 and the light chain amino acid sequence of SEQ ID N0: 26. [383} in some embodiments, the antibodies disclosed herein comprise the 111 amino acid sequence of SEQ ll) NO: 20, the CDRHZ amino acid sequence of SEQ 11) NO: 21, the CDRHB amino acid sequence of SEQ 11) NO: 22, the CDRLl amino acid sequence of SEQ 11) NO: 27, the CDRL2 amino acid sequence of SEQ ll) NO: 28, and the CDRL3 amino acid sequence of SEQ 11) NO: 29. [384} ln some embodiments, the antibodies disclosed herein include one or more conservative amino acid substitutions in a variable domain ce such as 1, 2, 3, 4, 5, t3, 7, 8, 9, 10, ll, 12, 13, 14, 15, or more conservative substitutions in a variable domain sequence. 1n some embodiments, these conservative amino acid substitutions are in a CDR region, eg, 1, 2, 3, Al, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conservative substitutions are made cumulatively across all CDRs. In some embodiments, up to 1, 2, 3, or 4 conservative amino acid substitutions may be present in each CDR sequence, e.g., SEQ ID NOs: 2022 and 2729. [385} Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a monoclonal antibody has the same specificity as a onal antibody 19, by aining wheth =1“ the former prevents the latter from binding to a natural binding partner or other molecule known to be associated with HERZ. in some embodiments, it‘ the monoclonal antibody being tested competes with the monoclonal antibody disclosed herein, as shown by a se in binding by the monoclonal antibody disclosed herein, then the two monoclonal antibodies bind to the same, or a closely related, epitope. [386} In some embodiments, an alternative method for determining whether a monoclonal dy has the specificity of monoclonal dy disclosed herein is to pre~incubate the monoclonal antibody disclosed herein with soluble HERE {with which it is normally reactive), and then add the monoclonal dy being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind 1113122, 1f the monoclonal dy being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody disclosed herein. [387} ln some embodiments, screening of monoclonal antibodies sed herein, can be also carried out, eg, by measuring HEM—mediated PlSK-Akt pathway activity, and determining whether the test onal antibody is able to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with P13K—Akt pathway activity. in some embodiments, the 1113112 antibodies suitable for conjugation can be generated and purified by nown techniques eg, W0 959l7 and PCT/US$2018/019873, each of which is incorporated herein in its entirety by reference.

Conjugates {388} In some embodiments, conjugates of the disclosure comprise one or more occurrences of D, wherein D is a STING agonist, wherein the one or more occurrences of B may be the same or In some embodiments, one or more occurrences of PBRl‘v‘l is attached to the Linker—Drug moiety, wherein the one or more occurrences of PBREVI may be the same or different. in some embodiments, one or more Linker—Drug moieties that comprises one or more occurrences of D are connected to one PERM (eg, a antibody).

In some embodiments, the conjugate of the disciosure comprise a PBRi‘vi that has a nrolecuiar weight ofabout 40 kDa or greater (e. g. about 6.0 RDa or r; about 80 RDa or r; about 100 kDa or greater; about 120 kha or greater; about 140 kDa or greater; about 160 kDa or greater; about 180 kDa or greater; or about 200 kDa or greater, or about 40—200 kDa, about 40— 180 kDa, about 40—l40 kDa, about 60—200 kDa, about 60—180 kDa, about 604 40 kDa, about 80— 200 kDa, about 80—180 kDa, about 80—140 kDa, about 100—200 kDa, about “30—180 kDa, or about 0 kDa) and has a sulfhydryi (ite,,—SH or thiol) group, In some embodiments, the total number of sulfide bonds formed between the Linker—drug moieties and the PERM (or total number of attachment points) is ”30 or less (eg 8, 6, 4, or 2,), In some ernbodin'ierits, for conjugation until one or more Linker—Drug moieties, the PBRM has a molecular weight of about 40 ltDa or greater (eg about 60 k’Da or greater, about 80 kDa or greater, about 100 kDa or greater, about 120 kDa or r, about 140 era or greater, about 160 kDa or greater, or about 180 kDa or greater; or about 40—200 kDa, about 40—180 kDa, about 40— l40 kDa, about 60—200 era, about 60—180 kDa, about 60—140 kDa, about 80—200 lgDa, about 80— l80 k’Da, about 80440 kDa, about 100—200 kDa, about 100—180 kDa, or about 100—} 40 kDa). in some ments, for conjugation with one or more Linker—Drug rnoieties, the PERM has a molecular weight of about 40 kDa to about 200 era. in some embodiments, for conjugation with one or more Linker—Drug rnoieties, the PERM has a molecular weight of about 40 kDa to about 80 kDa. [394} In some embodiments, for conjugation with one or more LinkeruDrug es, the PBRI‘x/l has a molecular weight of 46 kDa to 200 cha. in some embodiments, for conjugation with one or more Linker—Drug es, the PERM, has a molecular weight of 40 kDa to 80 hDa.

In some embodiments, PERMS in this molecular weight range include, but are not limited to, for example, antibody fragments, such as, for example, Fabs.

In some embodiments, for conjugation with one or more Linker-Drug es, the PERM has a lar weight of about 60 cha to about 120 kDa.

In some embodiments, for ation with one or more Linlteerrug moieties, the PERM has a molecular weight of 60 kDa to 129 kDa.

In some embodiments, PERMs in this molecular weight range include, but are not limited to, for example, camelids, FabZ, sclE'ch, and the like.

In some embodiments, for conjugation with one or more Linlteerrug moieties, the PERM has a molecular weight of about I40 kDa to about lSO kDa.

In some embodiments, for conjugation with one or more Linlteerrug moieties, the PERM has a, molecular weight of Ir—‘lO kDa to 180 kDa. [401} In some embodiments, PERMs in this molecular weight range include, but are not limited to, for example, full length antibodies, such as, IgG, IgM, [402} In some embodiments, the targeting ligands, the linkers and the drug or prodrug fragments described herein can be assembled into the ate or ld of the disclosure, for example according to the disclosed techniques and methods, Therapeutic and targeting conjugates of the disclosure, and methods for ing them, are described below by way of non—limiting example, [403} In some embodiments the total number or“ sulfide bonds formed between the LinkeruDrug moiety and the PERM (or total number of attachment points) is 8 or less, [404} In some embodiments the total number or“ sulfide bonds formed between the LinkeruDrug moiety and the PBRh/l (or total number of ment points) is 8. In some embodiments, the total number of sulfide bonds formed between the Linkeerrug moiety and the PERM (or total number of attachment points) is 6. in some embodiments, the total number of sulfide bonds formed between the LinkeruDrug moiety and the PERM (or total number of attachment points) is 5. in some embodiments, the total number of sulfide bonds formed between the Linker—Drug moiety and the PERM (or total number of attachment points) is 4. In some embodiments, the total number of e bonds formed between the Linker-Drug moiety and the PERM (or total number of attachment points) is 3. In some embodiments, the tota1 number of sulfide bonds formed between the —Drug moiety and the PERM (or tota1 number of attachment points) is 2. [405} In some embodiments, the ratio between Linker—Drug moiety and the PBRi‘x/ft is between about 1:1 and about 8:1. In some embodiments, the ratio between Linker-Drug moiety and the PEER/1 is between about 1:1 and about 6: 1. in some embodiments, the ratio between Linker-Drug moiety and the PBRNI is between about 1:1 and about 4:1. in some embodiments, the ratio between Linker—Drug moiety and the PERM is between about 2:1 and about 2: 1.

In some embodiments, the ratio between Linker~Drug moiety and the PERM is between about 6:1 and about 8:1.

In some embodiments, the ratio n Linker—Drug moiety and the PERM is about 8:1. [408} In some embodiments, the ratio between —Drug moiety and the PERM is about 6: 1.

In some embodiments, the disciosure aiso relates to a nDrug moiety comprising at 1east two es, wherein each moiety is capabie of conjugation to a thioi group in \/1 so as to form a protein—LinkenDrug conjugate. {-410} In some embodiments, one or more thiol groups of a PERM are produced by reducing a protein. The one or more thioi groups of the PERM may then react with one or more Linker—Drug moieties that are capable of conjugation to a thiol group from the PERM with the LinkeoDrug moiety. In some embodiments, the at teast two moieties connected to the PERM are maleirnide groups. [41 1} In some embodiments, the antibodies may be activated for coniugation with Linker'~Drug moiety by ent with a reducing agent such as DTT (Cie1and‘s reagent, dithiothreitol) or TCEP (trist'Z—carboxyethyi)phosphine hydrochioride}. In some embodiments, t‘u11 1ength, monoclonal dies can be reduced with an excess of TCEP to reduce disu1fide bonds (egg, between the cysteine present in the corresponding parent antibodies) to yield a reduced form of the antibody.

The newly introduced and unpaired cysteine may remain bie for reaction with LinkerDrug moiety to form the antibody conjugates of the present disc1osure, In some embodiments, an excess of Linker-drug moiety is added to effect conjugation and form the antibody—drug conjugate, and the ation mixture is purified to remove excess Linker—drug intermediate and other impurities.. [412} In some ments, for conjugating of the Linker-Drug moiety, a PERM has a molecuiar weight of 40 kDa or greater (eg, 60 cha or greater, 80 kDa or greater; or 100 him. or greater; 120 1:32; or r; 140 1:32; or greater; 160 1:32; or greater or 180 kDa or greater). in some embodiments, the ratio of PERM per Linker—Drug moiety is between about 1:1 and about 1:8; about 1:1 and about 1:6; between about 1:1 and about 1:5; n about 1:1 and about 1:4; between about 1:1 and about 1:3; or between about 1:1 and about 1:2. [413} PBRb/Is in this molecular weight range include, but are not limited to, for example, full length antibodies, such as, IgG, IgI‘x/I. [4141 In some embodiments, for coniugation with one or more Linker—Drug moieties a PERIVI has a molecular weight of 60 lgDa to 120 kDa. In some embodiments, the ratio of PERM per LinkenDrug moiety is about 1:1 and about 1:8; between about 1:1 and about 1:6; between about 1:1 and about 1:5; between about 1:1 and about 1:4; between about 1:1 and about 1:3; or between about 1:1 and about 1:2.

PBRMs in this molecular weight range e, but are not limited to, for example, antibody fragments such as, for example Fab2, schFV and caniehds.

In some embodiments, for ation with one or more LinkenDrug moieties a PERM has a molecular weight of 40 kDa to 80 kDa: In some ments, the ratio ofPBRM per Linker— Drug moiety is about 1:1 and about 1 :8; between about 1:1 and about 1:6; between about 1:1 and about 1 :5; between 1:1 and about 1:41; between about 1 :1 and about 1 :3 or n about l:1 and about 1:2: [-417] In some embodiments, PBRMs in this ntolecu1ar weight range include, but are not limited to, for example, antibody fragments, such as Fabs. [41 8] In some embodiments, the disclosure features a scaffold useful to conjugate with either or both of a proteiiubased itionqnolecule (PERM) and a STING agonist moiety (D): In some en'ibodiinents, the drug»carrying scaffolds (ie, without linking to a PERM), described herein each typically have a polydispersity index (P131) of 1. [42G] Conjugates and scaffolds disclosed herein can be purified tie, removal of any starting als) by extensive diafiltration, 1f necessary, onal purification by size exclusion chromatography can be conducted to remove any aggregated conjugates. In general, the conjugates as purified typically contain less than 59/5 (e.g <12% w/w) aggregated conjugates as determined by SEC; less than 0.5% (e.g ?/b w/w) free (unconjugated} drug as determined by 1U?-I-1PLC; less than 1% drug carrying—peptideucontaining scaffolds as determined by SEC and less than % 1:e.g., <1% w/w) unconjugated PERM as ined by IIIQT-I-HPLC. [421} In some embodiments, the scaffold is selected from the lds bed in 'l'sble A1. {422i In some embodiments, the scaffold is selected from the scaffolds described in Table AZ. [423} In sbme embodiments, the conjugate is selected fl‘Om the conjugates described in 'l'sble B 1.

In some embodiments, the conjugate is selected from the cbniugates described in Table BIZ.

Table A1 Structure H E) H NV/\O’A\/U\’/\Ofl E 0 OH QH ()cx/moAx/OVAn/Nf\/J\N/fififi/OlrlH I ,, OH OH Hg ’Xrifil WO 02984 Structure Structure WO 02984 Structure Structure WO 02984 Structure Structure /' r " ’7 " ‘ wherem R“, R“, R10, Rl’, R13, R19, R“, Rt“, X3, X4,X6, XL, Wl, Y1, ZR, X2, Wg,~ ‘7' .- v . , r ‘12,,. 2'32? are as d herein, Tabfle A2 '1 '1 7:2. 55 (NHH) 1 1 92. 5 3 (M-+~2H} Cmpd Structure LCMS 1 206, 5 8 (NHZH) ()H 1113.G4 HUM/v" Harm (_M+-2H) r OH O NH 9 H ‘3) H (I) NVAN/\,O\/»\Omv.0\/\Ox\,!3(v’\O/~\’.CL/\O/\V’LN. "(NV/\f/HAOHd I.) (I) ' ¥ ’ 1’ H, (TD-Li 9H \ \NAWN?A?\:/\H’N\/|LN’ 0 2‘ “I o H o H H 0 6H OH LCNIS 1220.58 0 (NHZH) \/ ’ ‘x/ OH 9H 3V“ ’A\ro\ /\ ’N‘~ \f‘3 ”N /”:\ r0“ 0 , 701/ I. N EH ~ er OH HO fl/\’OH 1 19953 ;’\o'”\/’O\/J (Ix/mm' H 9 OH QH GVAO/\/o\/\Y,N\ N/iLNAE/H/xxm C1 H OH OH . m4 , 9“ HQ “pm-i WO 02984 Cmpd Structure LCMS 118556 (NHQH) 117354 (hflgfij 1929.85 Q 0 “\f’i‘N/AVH i Vikki/\F/N\/Ji——Nv/\o/\VA.O\V/\\Oii H H :Hii. H 0 O. J (\G/VQV/ f b QN/NQ/NV/O‘x/fill/ NVJ WO 02984 Structure LCMS 1 21 8, 60 (”R/1+2) (Mali) WO 02984 101 8. 3 9 (M+2H) WO 02984 Ex Cmpd Simflute LCMS “W N0 "1'55",5736“ (NHH) "ET?223)2 (NHZH) WO 02984 Cmpd Structure LCMS 1 148.43 (NHZH) 1 143.33 (M+-2H} Cmpd Structure LCMS 1 1 83 , 63 (NHZH) 1 121 .06 1 193.48 (M+-2H} Cmpd Structure LCMS 11”"931 (Ki-i-ZH) 0 OH 9H x/LLEAYKE/l-‘V’OH OH OH HO )S’xmi '1 '1 88. 46 (NHH) 1 466. 64 (NH—H) ”a ”a 9547 (M+—H) (iM-i-I—i} 1 1 97. so (M+—H) 13 4. 40 (NHH) 1 243.24 1 @0522 (M111) 1 134.24 (1111-11) Cmpd Structure LCMS 129mb 1049. 15 1049.38 (M+H) 1 1 20. 41 Cmpd Structure LCMS 1 1 20. 3 8 (NH—H) 1 1 20. 3 7 (M411) 1 120.1 1 (Ix/14H) 1 13-4 19 (Ix/HE) Cmpd Structure LCMS 1 177. 40 (NHH) 1 177.41 (M-+—E-I) 1 1 77. 43 1 1 77.44 (M+H) WO 02984 1 106. 17 (M+H> ”a 091 ( 17 (NH-H) WO 02984 114517 (111%) 1108.12 (M+H) 1137.15 1 1 08. 14 (111-111) WO 02984 Cmpd Structure LCMS Structure LCMS WO 02984 Structure LCMS WO 02984 Cmpd Structure LCMS Cmpd Structure LCMS Cmpd Structure LCMS Structure RLB‘ / W’G‘ Zr-RC? ‘Kclx‘ WO 02984 Structure WO 02984 Structure WO 02984 Structure Structure Structure /' n O Q C H v \ H H , I - [L Li g h PERM—7L5\ A€/\{,N\;/ NV NV b.\//\O/A\,.CJ\//\O‘ N‘ . N‘ .

. : ,; H H c": c": i ’0 \ {I 0 > I \ V/ r 9 _‘ ‘3 (AU H - o ,0 N CH \ OHM vN ,.\/ v , C 2:: Y 3% 5. w? a) 0/ 0a OH . NH \ KY Kr OH “OH ’I \ HO \«OH / é. “0 \ 1&0 / I [Nta/ / §LH V “‘ HNAr “\K' \ Y,VI§§‘/\w/\Nk L «TN 9 \N’ \ NHZ Structure 9 E.) . L ax/E 9H ‘\ H 9 9H O\fxo/AVOVATrszw/1'\.fi/YEX/\/.OH\.

O (fix OH SH Structure , N L JCS 0H gm a . A NY I V 0H 0H . J— i Q . 0 G . 0 PBRMfLSVkNxxr/AE/ifi’z . Hi 5 ’“ngfirhvH 5-; L.“ “‘v’w ’O\/“o \ - g \....’\O a E \ .I' G O 0 \1 r/xO/VOV/ \‘ (fix i: ! : a) A / a ,N EH : GH' E 0" NH \/ ‘0 \/ \x” / I] i R T W/ N/\ /2\ H ({H K 1 0 NH i: \\ a 01-: i x] 8/8! 5.3+: ,/ ,0 HO “~on /’ \ H0 .-_ / HR“? / {S15 “a W?“- 0,45% 0 mix (3’ 3 C r": V .1 76%f WO 02984 Sf;Tu :1 uTn» Structure N‘VAo/V’O‘VAQ \ (AU/\VKJV/ G \ »"\/O\/’~ /N‘W‘VJLN/\V’OV/\O/\V’OV/\O/ E | H 0 DAN"VON/\GAVD‘V/‘o/ WO 02984 Sf;Tu :1 uTn» Structure . H ‘ N\AQNG\/\Q (”‘eflv0‘V” a CH 9H U\/\O/AVO\/\F/,N\ VJkN/*\§/LT/\\/GH r3 H ‘ , 0“ OH Structure N\AQNG\/\Q xv/i H p U\/\O/AV0\/\r,xN\ 1' .””‘x LT/ V AVG-H (3 x OH OH H0 f\—-€3H WO 02984 Structure WO 02984 Structure H :3»: OH ‘.

N vAVAE/\OH } CH CH i / d15 Structure ////’ 0 GH 0H 1/1”“ OH OH 0 0H 9H 0’.:Vka’“£zfi i} OH HO ’\»OH Structure OH (31-: L fAVfiH WO 02984 Structure H :0 H NV N\,/\0/\\/C'\v/\O /\QAVG‘V O‘vf\Q/\\/ON/ ,/N WO 02984 Structure 0H GH \ WO 02984 Structure Structure WO 02984 Structure Structure Structure Structure Structure WO 02984 Structure WO 02984 Sf;Tu :1 uTn» WO 02984 Structure wherein (115, R”, R”, R16, R17, R18, R”, R“, RC2, [3, X4.X6, X1, W1, Y}, Z}, X2, W2, Y2, 22, are as defined herein.

Tabie BE Structure H 9 O H H 9 H \ XMTH151§WES\L/J\NAT/N“: 5.3] NX.‘ . E”YN\/L“N\/’\L\/\/0\/\I3 \ : .v . \\\“<\O 'n \ O ‘2 O (A00N0V OH OH ‘\\ 0" (ix/N0/V'kk”:1,—rim/u\\‘x\Ek{NOH\ HNx 'EH OH L NH \ E 8/3. gm \\ 7 HO ’\,-0H Q 1 m¥ i 0:;(M12 (3‘ \J=/ I, x x w ,9 \ .I “‘__ HN II NEKNVWAN‘AN / NH ‘5: Ln. \ /O‘ Lg f‘vfi/ )Lfg // \ ‘ ‘i ‘r IVE-12 . Cmpd Structure N9. N0. la / Trastuzumab—{fixfiiN/‘fNEAR/Wv. o .9 P 1 9 \\ Jaw/x“, 1V3 NA AVON/N \ F H \ \ O /’\0~v\/"f. C 0L. 9. \ o U% H \\ “A‘O’N'CK /\_}"’N\/’\/ " /\va ‘ 7 ‘ NH \\ \G {T,. OH J 3 \ \f L10 4 \-0H 1. ”‘9. 1 L?‘ E ...( I 0‘3NHZ I! 1 \9—N “1/ ./ ‘r” HI \‘N \ ‘0 HW§ [,1 hf ' r vwwwfll / n ‘ 1‘ . " NH ,/ L Y W \q‘ / ’3‘" ‘i // AN< / fv'HZJO d1!) lb E/ 9 '3 O 0 H H ,- H \\ N‘A“E/‘}r"vk"“NWO/xrcVAC XMT-1 5354‘s{ N‘A . 'NY’ELNi’b \\\ Lug) C!) ‘ O FANGA\’OVE OH OH 0/ VOVYNH\,¢\j\tmALT mu \ HN\ OH wU I\ OIKNH W' \ i" i 9* \ \! HO V :FXOH 1 1'69 : ””1: i 950 I] NHZ \ 0‘ \fllewfl/ \ ll! (’ ‘\ \>// I ‘ l \~ Q 2“ 119-140 N‘;/NVMN/§N 3%,NH / \w’g O ’1 “Na / 1 / N“ NHVO I 16 $01 1/ \ p H g9 H q o H H DEXR zumab—fms\(:NrNT/NE/i‘gj”‘n'l\l\/L‘N“TNvfi—“NVA:/\/O\/\C \\ i z ' o c \ o Q OH 9H 7.5 \ 0 (HomeI x u -N i L 5p .3 \/ OAV \x"xi/ \/ Nfi/ VN/ - HN H A r. Oh, 7 “1 OH NH \ \, i OH 8C“2 O 0/»‘r‘kwQH \\ DAR 5 \1 s. 8 L?" NH i In”;‘0 i 0&2,L > ,N=.~.~*’ I] X \-«N 9’ \L Y) II 3“ ‘. I r) MV§~O / NV“\//\‘\~/ \ 4,4,4 I/ 1 I” WO 02984 Ex Cmpd Structure N9. N0. f/ «2 qvfi nL n \ Paiwuzumab m!gG2£§--f-—S- J‘WY \"*H’\fi".. h. i \ a 5- HMy v “\/*~o/\/“~v/\g \ é . x., C. \ \\ O 0 OH OH d («MC/\gkv/ L1 ' \ U 0).. . 0‘ vaoxxJO\/YN\ /\!“§;\§/K{‘\’/ ‘ 0 (X / 0. H XMTn‘ES35 hagm«nigcazaw-Jrsfikmf \ x .5 \ .

O Q XMT-1535 AAG-- "Is. 1k. / xNY‘KN/aH ‘ T 1431?: 75 \ C", Structure 0 o .

J1 H =L I.

NV \14NI\\t --»I\\/\O;\,O\/\C \ H ,l; K. 0 j : 0N V 0. L2H 9H 0VACAVOWIL ~,fixN/xATAV/KVAJH " \ / OH OH \ S1WOH \ 0 I Target (3 higG’ia—- NH \ I FL‘.1. 9H \ m ”\fo'I \ HO 1 \ 911/ I \_—N\I4&1 III n“é. ’§o , \ /‘3‘ / NHé / (L6 9 n o. H \ Target E mEgGEan “v E NxAQ/x/Ox/x? \ O (NC/«V'O‘v/ OH QH OVA/\./U\/\fi/:\:rVimA/L}/\,3H H \ OH OH \ NH \ I 0.: \ I-o OH \ ’41»er 1 C» I] \ Fer" I \--N I IN’g. / O I Structure Ill 0 H 9 'ri j; H E H Trastuzumab AAG»-l--s\\*,f 1 \ _ “g yyk ‘ * ~N\/""“\/‘w’\vbv\g ‘ “$0 . c o 0 r o\/ _/ \v/V” O onH H (3:4. \ 0., OV/\1:‘/NV¢D\/\71/N\NJL~N/Y%/-\/OH \ ‘ ”N3 55 {k H OH OH 1 M! \\ “c if"? 9H \ ) " Hg]. 5545’ \ur‘ofi,. \ T .0 i IVU< 1 Ox‘L‘o , NH? 1 w: \rwwt/ i K 1 / NF¥~L, ”3&9 V”\/‘\r"\’\\\ O\,J£H ‘ g I, \ .> , “175 L‘: ll Nfi ‘.

\ U NH2 I (115 / 0 E a E n \‘ \ArrN'o\/\g, Trastuzumab migGfiaT-SY NA "5 ‘11” 1/ ‘ *u/fir v \ \_.(\Q o ‘1 o o r"r’\-’O\/ On 0H N ‘3 \\ ‘ éV/\»/\~,Cvx ,H‘ JE‘N/ Nu” \ 73F f/ H fit; L \ NujH 11H 1\ 8k 145 2"on \ 1k DAR 8‘1 1 1/ L115 11 8E \ -N‘~’/\O AVzo\/’\O \\ (”\o/\/°VJ 9 0H gr- \1 D 5:? O\/"-3”’\V”°‘~/\r”N‘WJ‘W’\ k AVQHH \ A i i H ‘ S o 44‘ ' OH OH 4 7 O \INH (‘H \\ ‘ ’ 0‘» 3, I?” \\ H? HO “\«fii' HO 1 L .

.\ : NH” Q/\0 O: .1 ., \ > NS I! \“N1 {J \/ \“0 V II 54);. “Fl—k0 I, . . I §,NV/\(/\N.\N [I Lo§mh o. 1):“: I/ N’g , “\L/ I fling >z0 / dab Structure 0 (“o/‘V’c‘1\r’J 0 6H 9H VOVNF/WN\f‘VJKNaNIJNN/Vf-‘H. : H o ,,2 OH or: OA‘NH P10 ‘\,.GH \pmlr:M.

HN"§~O 0‘ \L%f“ 2 1/ \ .n o 0 o I ‘-’ R : {i 1L 3 : H \\ XMT-1519’fSKxN/x a \r EATK” WT V "NV”‘C’AVwAS’ \ . .J o b \ \1 r“°"\”"“ a . 0 i:' 0% NH \/‘o’\/ \/\.r’ \WVo .N A ii" CH H }/\’ ’\ 0 0H OF: u_/,€\ E NH “\L 'i 4' 9H 740 HO T’\,Gh L‘o \K’N‘Nw O’KO L‘-‘(’ \ \3 )r—o . L W2 N )1“ 0%r ,6 ~N [fig/r 3/? ,x ' 1 IN#/ 9r’\,/ 0 M‘x w Structure Trastuzumab{-S~‘/’1\Nm\( wk: “Fr| h ' Nx/J‘'~ fiATf \/N , NVAO/\,‘3 V/\C \ \___’ K 0 0 ,J \ «\0 O r\ /\ ,O\ f 0 A ‘ ’ H '9 OH {3H \ 0/ VOV/\ /N:[AV‘)L\N’ /LT/\‘/OH \ NH H -.>fl 0 / (JP- CH \ 0 NM“ 0% \ . OH _ \ [NH 3/ 9:1 \ “.4 \ >20 a’C'H i Ho HN‘ \p— 1.

”HZ /) ‘\./N\'(/ E omi \ )b-o s 13’ \ ’ “‘4' \/ 0 I / )\ >171 -f‘“ \f“ I N\ mfi— \_4 ’ Y H4204 ,\ I 0“ ,NH ' \___x/ 1 \ [I H2’ N"\ NH: ,1] f / / c115 / ' a: Paiivizumab—Ik - \/\Q/\, VACo \ {\ (Ae/VuV/J O OH \ \\ aux/\G,A\/O\/\ ,,«N\A/J\Nx\v/ »\/ on . : H \\ O qé OH OH \ 0 N“ \ ; 39H 1 hi) ”\WOH i HO 5 / difi 4; 3L XMT—‘ifi‘i “A A ow v o r \ Jo \ fN‘ONMV 0 ’JH ONI F ‘\ OVA. ”AN/”\x/ ."k J'LN,»~.\/ AVG} \ \ :3 H \ O? 0‘1 DAR I K ”\m I ’/ d15 Ex Cmpd Structure N9. N0. 28 I Q C‘ O H H H '73 XMT-1519‘IZS\ N/xY/N:’4‘kafi’jg'”VJLNEll/Am,Nx/‘J"""NNfQA’QVH \ g L__ \/J OH \\ ‘0 H Q 9H \ 0/ ON/KO/NXR/W/N‘ «iNAl/Kr'xfxm \ DL‘XR ‘ ”N\ b fir H CH CH NH \\ 60 kO‘K/X‘ 0 ,L 8/0” :GH 1. (rm/LN“\' NC”?. » m \»CH “VD JXEC' ] O‘WK‘ “3mm ,i ,N i “2“ o f \/ \\ ' ,2. (,4 )ngx ,/ 0 \kg‘\ .J NH2 / “I N 0; Nr",\ / NH / or ./ d1: I!”o* \/*I\ /)\~ 6 29 ,/? k;. o 0 o XMT~1519TI~Sx \ D XR N/ V/\O/\V’ V/\o L g “if \q’ v' ' \ : I3’ \F’ V \ j , { 0-") H 0 O 0 («0,x/QVJ C? OH ,— H 9" 3,3 \‘ in 0/ ON’A‘Q’AVOV“H"N\.”VI N/NYJR/V'OH \ \ H' HN\ o och ” OH OH Structure , '9 EL EL g I? ‘ IMF-153', 5*. MY Y EA!" v fiflrf V \/«O.Avk\/\o . . \\ E\ \W,, _ U \i o o {/‘O’NV‘O'V/I 0 OH OH \ u . H I \ \ 0A; QVA0/\/0‘v"\II/r‘\l/“\.

\ Nr"T/l\E/\,-0H \ Mk 5 ' H OH 0“: \ L gIA‘NH \ 0N 0 ’l 1 53H \% «m S ”‘0’ka .

‘NH '\ - m3 )4#0}- L "' o HG y N? ___J :0 b HN . \ x_. \‘x Nrfifik jf h m L f Y?" LJ \\ J x. O >.\. I, NV} \( 0 \‘| \~ 1'” \ -/ NH2 \ / / "’ I In“' / 0-,. I/ 0 / d ~ ‘ . 1 5 ------------------------E;- ------------------------------------------------------------------------------------------------------- / o pahwz”mabrs‘~\/L\M"\4/N\:’»\fi”W'N\‘A§éfln"d“J' H ,. H - H H ' .. . L.“ ‘ . .I P \ NV'\O/'\_/0\/’\c L"<\ I. -. - a \ ll ‘5 \ 0 O /»\0/~Vo\/J O OH OH o = H .; ‘\ \ ; , \ O, OVAOAVOVWN “RWY .\,.oH \ \ HN\ E: H r/ OH DH \ k 4 w \. 0 DH \’"‘0’ (3 9L“ \1’ \ \r’KMH “\ )* H0 \.0H ho- \ q -: _.»'\<r«‘ _/ =0 “ ‘0 Q? , \‘\ \FN . 3‘ I] Jam \6 {‘N‘f/ \\ o / AAK ,2 My; 9 \l (XNJ NH, / “:4. I [NH / aux. / [FR / (‘15 \V”‘N’//\' / *‘ n° ti 2‘31 \ F‘ralivizumabmilgx‘szafsNi NAT" 'v’ V4) NAG/xA V/‘c-3 \ . H“(will V “fi/‘xflx .. , £ \‘ O 0 a ”\0’\’°\’J J 0 0H 0H \\ 0 j o R )5 v .3“ ' - \\ 0,1 Jv’\O./\,- ‘VAW’f ”N, x N/‘\/’\§/’\_/ \ H“ " j c- 0H 0H 0‘ \ \ 0H .\ 0 . . —{’r0H .

Vx, ‘3 A. 0 \"\‘NH N\/ 9 his I \—-OH k L ,0 "-‘r- Ho : “w ~f/ ._,_/ F10 0‘ H I \ ' ~\ N " I] HEN /\\\ ‘ f) J . I3 N§/\f I] cffifi’xfi‘ ,9— ‘K/ N": N. / Nf—k / NH / l O\ / dii \/&.N«:‘\ Structure ll 1) 0 O \ . H \ XMT1535 «:ngst*kaYNi/u‘g’\r’H‘k/RN’YNVj—RLA«Niki? E \ » H 0' \\ l \\ 0 \ ”‘0”\’0 OH 9H \ \ o : f \ or”: RAD/weVf:53¢»kaA ,L ,m. \ \ Hi! EH EH\ \ I ‘7" 0“,“ ‘ 4+- .3 ‘1 M41 Niki) ’\_(~H i K? _/ .50 j: a“ HN\I=N i 1 Kw c / % (“VA {é fifi/kf’ I L/ NH; 11' / N I NH // c¥\ mo / x i A 1/ Q o o o H k“ L1 A n : L, XMT~151Q MG-":LSSV’KNm/V \ HN"\I_/"\— ”ATNVHV\/\Q/\, Vac : Lil E, “\1 o o (\O/N—LK) 9 rm OP \\ \\ I :51 \ /‘\;VW’IVVAVNVAOH\{NOH j 0’}\NH m \\ RD S {W. ow \ f I ;' \ HO «WI/MW , \ (2 ‘ L. 1 “ : 71. ‘J I " i Li‘\ 8 \..§“~{~ / /N\‘\ . \T’O / Lj-o’ - / .’k E 1"?"\o / _ VNV\/\N’\N - ,Nn E N)" / W0 ‘ég" /’ N?"i\ 2H“) O: I/dlr ‘5‘, Q \ N n \ . /\\ ‘st w‘ I, 4:”,qu \" A f \\ H liq/N ‘ \ ,/ “#1 0‘ ~ E i’ ‘3 i i E )k Aer\/’\ xx AJ'OVA /"V:i /\/\0 L5 Trastuzumab‘-s~r’ N 0 0 <3 fl K: ( I! \W )\/ 3/ [>43 N~N I ‘5 \ // 0\ I 0 o l i I V e .

Paiivizumabéwsxg'?’ ”\x(3‘ O ,0 A K , ' A x VAG/\/ V/\o/\’ ,.w\ 0 \/ ‘01/x E \ $3 < fl” H I; \ ’“\0 2%,“: £31}; I \2‘N / \Xx//I’\\ / \ HINm/\ 0 N: Cmpd Structure _ ,§ /\c€ “”2 MN“(~ij7 \E \ II (3‘ E f ‘? “f2 ‘ :3 i 3/ V ; 23’ Y E—NAO \ XMT-‘isw s(kwn’ 1’ \.—J\\0 0 AA! L J H i >N. I‘M 04 / OH HzNw/ N/‘N *4: JR / o 0' / dfis N4: .0 i N--/\x j: /\o’ waxff ”H2 \ f V7/ 8/ ‘1 I 0\. q E XMT—‘ifi‘igis_N<‘:‘YET Q/V‘xi?E \, I I; E a w ; a, ma (”V N“ \ am 0 O //d15 (3 \\ N! \----:l N //\T’A \ \. / \“H N“) o HN—uq‘ l ,i I IN \ / o -' i 2 ii anww-E-s< NY Y O’mo 0 (5/ DA\// 1/ C315 N’\ 9 1 JL F :\ NH: \ I ‘ G “N “I? “ck 50 \\ mmm {5“, j \ / K agrim/Nxiv/«VAC? o\ C.‘ ‘ 0' I-i :/ 0 ESPN>__ / NH L [1 HZN \ KN" /‘ N \g/ dHG’kI/ I] C115 3:1 Cmpd Structure w U) \, I Q ,N ‘NH2\\ xwwm/ “° ~" ”HM / I \ n ’3 (Hp? gfl K» ‘3“ \‘S’vaJ‘xN W,N\:/j\0/‘\¢/\_D 1! x : \0 i) 3' / .N ( ,l Ham kj hag—NH r!“ // E: 0/ O”\/ .-’l G15 N5 0 o \\ ,N‘~:”\’J\NP2 \ mV0 ”Magi A- XMT-151Q7/W*ij \\ 0 I» ox } JL{4‘ ' \T’flE/kOAx/A‘O j ./]:N>_ ( I'I “EMT/Rf! NI: NI;</’\F ’I «K/15 " ‘3 0 \ DAR h} \l ,N f \ J‘NHZ 6‘ 4 0 XMT'amw/s ”W" ”MN-jg \ £31:ng ’ \ ,N.\ x ,5 m,N ONAO } ‘5 / ,I’ no ,0 N» / \ XM?~1519‘TS . 0 1 ' c2 _/ o \ E E H I '\ N “N ’ , 5&0»on l \o' H 0 A.“ \: / ii ,}\—NH HgN \ N 1: ~N ) {,1 / 9/] OJ 1/ I C115 IIl/ ($ )Nfl/fi/E gird/YE: CE, xx O/AVNO {‘1 } XMT=151§TS ‘wk of H 1’ O fikfié I \ O / F NHNHW 1' \\ 04L0H WNW/irks? 0, {3: 0 ll," Structure \,0 0 “L4 HIM—(Pa i/KTJLNHZ \ LN Njfie/ \ "I, 1 0 O 0 [\' 0‘ XMT—stQW’ ‘ H ’l / N K N\/LLH A ] ‘2” m ow 1*» " , o o = X A; \ O :3ng Hz“ ’g/“Ij ’II/ E o’Mg?!”o/K/ [1 U15 0 a) $3 N ’/ gym—“(Nfl/IKNH? \ ~~ ESV ‘\ XMTu151ai—s}N/fir:\ffl‘ Egg/\n’"\E’L‘Q’Nflfi/ O 0 Q‘\ H II 1i I : \, i \’/%O O 0 \\ r5" x N I, \ .

E‘ L ,>—NH k,” / HZNNKN/ N i ’ \ 0 OH I I / C” O/K/ d \r—0 p 9 \ g “4 N . A “‘1 “WNI? ””2 \ / ' 1 I/ O“ 0 0“ H ([1 K7/ ', NE/N”; 0M0 \I } XMT~1513TS~4 INT m r ry . ‘ '\ \Ovizx 0 ‘ é}? ris>_ “\ /’ og‘on HzNjf/N/ENix / NH )5: , ! fig /, 0 0’ I/d 15 o \ \ x/Lo9.4%: N _ §N~<’N TA“\ \ HOVO N / XM?~151§~%S \ ,. i O 1 I L Q _/ 0 0\ E H i 'NNANN’NN/H‘O’AV’\0 I I \OI H i! : 5' O 5"“ ”N < I WNW/RH; ">45” “N 1/ Structure Hoar» (“z/‘oxi (AW \ L‘x NH T \ r: fk ? 9H 9H \Tr.\' ~ 0 0 , 0 , , 0 z z t \4 N” \/. \/”\Q/‘\/ \/"\.:r \/ \/ \0” \/ \/ \G' \‘x 4 ] N . VAVA A0”.V : L,_ H 5 i I w I 0 on 0H / (515 DAR Ms C~ o "f o 0‘ ’ {\ 'N""\N _, 0’) EN iJL0/\‘/\Q <u< : IAIVN‘"i ; NH 0. -' HA1 ' \ 5 \E xfim 0/}“§/IN 22' 8‘33p. i / ‘9 H i v; i H h) H \ Pafivizumabfigfi?» .N\;, EATY’N\' i fifW’N‘Y/ OA/0\/\?' \\ I \ \ 0 0 (“D’V'OV/ 0 cm \ H 9H 0 ' \ \ A A \ 0A, C\/’\O/‘\,0\,A ,,»N\ N \{ /\\/OH \ \ HN TE 54 I - \ ‘7 0 OH or: NH .‘ DA_ 7 [‘0 I OH OH I \xq Q r - *71.4 ' - D \rkmg i no NW0“ : ' H N I0 U 2 ‘ HO 5 '0‘“ \N .\\\Q [I K\ W Mk I? I' ”EN/X“ "” “’“ro I' “‘ “:7. i /\\ I, o \L / /‘ C \/\N~ / Nh , . 1/515 If \ \/\N//“~ Structure XMTm’ES’EQ N/O N // HN—(t’ a * 1/ /0 > fl/‘J\N{y Paiivizumaby/ N x0 0 Q A» \ /u\ H : 7’ \ E! 8’“ "NV ,N fi/ 0/\"/\0 : 1: : : ,I i K O O - C 1 ""NH I “ ”Wk"‘ir’fiN’ />-----</ / \ ca 0/\ / 0 ' (£15 \,0 Q H H N ’\ N ’ I‘M-i HN~—<’ T 7 2 , Have «a 4' i N \ CTLx48132___mEgGZ .1’ P170 0 Q S \ S’X, N J g 1L ,/ \ O o = ékflé ‘ ii ,b—NH . / \ “2N \/~-N’N >, - /< L5‘ ‘1: I ,, , \ c’: 0 G’\/ ___________________________________________________________________________________________________________________________________________________________________________________________________________________________‘3 \fo ”0 :1: 1}“ [“er MFPsmsgmg/ W39 0 i 0 \, \ ’9“\J‘N H 1k If] (3 v 0 x’\ /\ {:1 H / ' = B 1 : , ., / v xw--1519/ ,. 30 o o /o \\ «W WWa i H 2 9<3 \ O a 1 (fix R /'\\/E ‘mi w p l/A 'l \ H:N T \ n / a3- 0 M / d15 WO 02984 Structure I, Ho‘fio W?“x: ,J \ Paiivizuma'q/ \ P O /O \ i: L H \ \ M’9", NV ‘ i , I, . n H [f'N\-’j{\0’\/\o‘ \ V O ‘1 %J\§”N\,ymNH / I ‘ i i o i ‘ kw A, : “ WW N .1 \EE / \ n " O V / a” / d4 G o / A /‘ C x s HPKl \ N ' I z / / xT5\i:u\j’N ,, \ / O .3 \ i “ x ”‘ 3“ \ Q ~<KNW V O\/\/’\O / 1‘... i! E H u ‘\ ’ 0 Xi‘v’iT—‘iS‘ig o \: ‘ : (fix ME / /---‘ I} 0V ‘ g} “0%, HN2 A\ \ Nr H nW}; / \ O "J, 1/ 5-1:) O r PalivizumaL \ Q O {7 E HoA o\ H»N L I 3PM 07/ I \ 4 \H/‘k N O ‘D \\ 1/ ’— I d r, I O xfi‘i:N / HN “INJE’N lLNH: xmmms / ,0 H0 0 ‘K N \ y “3/ E“ N ”xix T I’ r’ 0 \ I g ? H A / \ OAN/H’hk- O’N‘Ao . o ,1 l 1 1 {4/ N i i \ A») H“N ,>—x,, N / \e[ ,u\ ,1 \ e) o 0' / 1 '74 N8: Cmpd Structure N/\l\\ JG E I‘m-K N ’Kvj) HN1N1“? 4‘ i 2 / CE ° , \ E8{43 E N/xxfl',—N\:’ l " / L E XMT-1519' /\./N 0 i : />_N}" i E I N 0 0H H?N~ /‘§/”“NL/ i l I] "</ ~ 0 0J\ 0 / J1 o\/ HQN ELEM)“ }’\ 3m.“"\E;O?‘ H0\fo XMT~1519?/ ,0 k N J; E CV .3 H. n ; \2 A ‘\. \.

./ «N i H, N E " O‘m/ I I Exf / ° ,/“‘E7 0 o \ KNIT-1519* )k H [L l' \ . l S’k \V N/ , \r’ TBA/«(‘3 \ 0' “ a = 2. x ( \\ ’V'N” H N ; E A» \Or N (3/m \\ <0‘;L\: // ' : H _ i O _ '4‘; M N If \ H7NV/ikv/l N)“NH ___/S“n/ / \ 0/ \ N I O / ’I G 1 r.

N: Cmpd Structure H ._ I" D ”- \ ./L\IT»'N\> \ H :N ‘ ll Hsz \ AS’ ’/ I \ >~ if / 0’“ " \ Q ‘ Q / XMT1519 1 1 - 0 / HO 0 gig—NH 07/ 273/ N ,/ \fN O ‘3' ,' 2/, /d45 Hwy/\ws k Q ’ 3/ - I; >==N’ M x 21.M (2' L N10:? (.J i3 "%k\ I2 <M ,u. " O / HO,“ J 1/ \ Hr. L III “H I “T: ./ CH.

; OVNH \ a 9 » 1S N'Nl‘ 0 \H H H 9H V 5.” N\r/iol‘.”x Nvi“ F i ¥ "NP ./\ N\/”‘\ A A A j >f fi’V If N VV/\3/‘\JV 0 V’V/\3/\/V AOrv/x?/\n’‘~/\,A /\ xm‘r-1s1e\ V OH, , H .. \\ f; o r; 0 5H Ur / \ mg” *0 II “is )0\ (“Y3- Cy —u’O‘fl __.__ \\,.N , . f \Nr/xN‘ ”J k' UN\_/ \ /\/’\O . (‘ f’Kr'N Hp! /K/LN/>—M1 (071/ o CVi rm WO 02984 / r) (2 o O ’ ““‘mr‘Nfz’i’E/ E“Mm“wk-“mo/wwo\' H H H,, i E i": PERM—‘95* m { x o O <3 1 r/\G/\V.O\/J QH \ "4 OVA(x/‘\/Q‘\/ ,—- N \ I HN\ «E INN/3LE,/\{/i\A/):\\/OHOH OH M,- / \KA/NVTFA .r . 0"“ N774) R U “w?K R; V */\ ’9‘ 2.. U x i'\§>j'2 R \Q/Z X2~Y2 ,X1NY‘1 R1 ‘R13 R15 ‘Rm 9 OH )2 / 0 )2“? “RC/ £115 9? O /\Z“‘Y?’R19 ”YMMHO' \R” o W?“ /1 ,, 5 R02 0 0 9 Vi ‘9 a I \ 7 R9 IANY) WNW? \ x" 4 2 I< /' II 0 "a, 2 O >12 R!/// dR 5 R 2'. \ '19 O G ;Y?R \ H)\E“Jme / g ”- i (\:\)‘f,/ 4 \ 1% ’LNf-‘h R15 \ XL :{M DC \ ,/ 0 O H i 2 :‘ 1 .r E SQN H/‘xr/ fig/’“\ \._/ x/NQ \ Rn: Ii \‘ I z 0 ,’ 0 . N H: \ O A :47 ..’ V ‘ E 3' "I?“ \ y Z?‘\/{ o (23H *9sz” \ ”L“ 2 i‘ 0 W} kWh/€115 R19 ; or 0 O \11“Y"R1§ / : HeNA / ("X3 “HQ!“ ii WT" '~ 1 \ R HO .0 \Xe/NN’ 5’“ i mm,/»2. 0 j 1 ’ N? o 2 o \ ;2 JL S 1k I v - \x‘ 0«Av/“ti r fifir R02 ‘3 i Rm wherein R”, R15, R96, R”, R“: R9, R“, R“, 2 3, X4.X62X1, W2, Y2, 22, X2, W2, Y2, 22, are as defined herein. {426} In some embodiments, the conjugate is: WO 02984 wherein (115, R14, R15, R16, R17, R13, R19, RC}, RC3, X3, X4. X6, X1, W1, Yl, Z1, XL W721, Y2, 22, are as defined herein. [427} In some embodiments, the conj agate is: 9 O H ‘3 PERMMFN H H 9 H \ ' 1\‘i/le/Nx/ ”firN\/L\§/\\:rNV/L-N\/’\0/\/o\/\Q, \ 1 . I \ “ " \ .

L 8‘__ \ o Q .- x O i/A'O'AV 0v, CH H C," QH \\\ a I \ leNO/N.C)‘V/\Tr/VN, k r . 0,; ’JKN/\‘E/ 1‘A/O?!\ HNW "' 0 f OH OH \ K 0% \\ 93 K K 9H \ \1 Hfg l ’x‘k,0‘- \. _ HO u 1 , - o NHA ’ CZ" ,5 $24-$40 I, {fix > V’KNT'I 1/ >1?“ 0 l»!5\l’\K,) 0 \x}, kW/‘~N/§\i‘ ' VM! £3“fig // L ,4 \f / mp ‘ I «‘x NH:O / d15 I/ \ l ‘ o O H H H H Sw’kw’fi/ *2/wa \H‘J‘Ork”‘. .~ = ‘ L n PBRM A : l \\ \“JAVOV‘Q \ ' E \ «O g .: I x c. O O C \V/ 0:».

H (I; (:3H \ CI, ‘ wa\,)/’\\/Q\/"\n ”N \_/h\§/AT1\{’;VOH ‘\ “'ij ' c (f OH OH . NH 'UH \ \? {‘2' " pH \ do ’X—GH \.

Ck 1 \g .5 \ I he I , U Mm,3 i ‘ ’ 0.3“ Ngk \ 1“ / >c- ’ N ,g HF 0 I, ' \\~’N‘V’A‘§/AN’ \N' 1/ C) E 1' \ NA: // Yak? ‘\’/ ~r‘3 / (J15 HN uwkh , ...V 0.. PM 0.... HM... v.1“ \ H .v AV\L.2Aa»v .C.I \\\ ..O :3 /Am o v K/V ....0 H.

Vf....A / /VxHN/flu 0H,“ WA o, N A Ar:V40 .../aw\ \\\\\H\\\ \...L n. H \O:\ Ni\r..m m/ 0...\H 7.0 ,W ....m. \4..V\a - ..h/\L., ) I ; NL.x o \\L. V . W\I\fl wrxN. \AA .N-«\ G N / v.ILM wk ./1 In,“ N-L A..... rm //// , d: 5 n.1>/,.,.\FH.

Ni: A\ NH\/,\\ xx DNAw. Gnu“ / \deawam a H.N Hav0 NJ\Ni / -\ 0 0, I.V Sx /... \I]! PBR 0 O\ /:W.WH\/.»W.I\mv\ a V.: A o \ x \N i.\ OH Vmix /.(..KI M0\A ,WYN A V {A HM.p H\ 0 x ,0 V NJ\N/\/I\.

I0\. .NH (-0 .

. W0 __xi. 0:, I . N. / Km”....mv \ HH um. Wx NV: /\#M\O Ill, #2 “ml /..:\/ “mo N H/ ...o. .flG ..o WA». \\.::.://, WI/Aw.

.LJ \l/ . d. 1|! 00 1 » o \ N.’\ I0 l \ N’\Z ‘9 [L \\ /L (Sw/\ NH? \ ,J\ / :l ‘ «7/ NH; \ , ___/S o N% ‘ \ / ‘ f o N E : '{0V0 N/KN/ FDVL‘ / \ 93R \ 1’ N N/,/ \ g E ‘ i l )\\i/ . S‘ o 1 / | (I: G { Ni? 0 [\3 a, 9 \\\;r—N\/Jlxfii \TlINN/lLQ/VAOj/ in [7 t ‘ fi’fil/N‘Yko/V'xoH [1/ ; , I ! ~ : l,’ \ \0 \o : i o O _ (x, r” W *LT’NFN { [I HzN /'L\\/' N” / EN ,’ H7N ~.\/L_s " \ ’Tr’ /\§N III O" \Q/K // Q: 0.? Q" 0 N [I 'I d”? , i ' C115 , 9 wherein dis is as defined herein. acentieal Compositions {428} In some aspects, the present disclosure provides pharmaceutical compositions comprising a conjugate described herein and one or more pliarmaceutically acceptable carriers or excipients. {429} The pharmaceutical compositions containing ates of the present disclosure may be manufactured in a manner that is generally known, eg, by means of conventional , dissolving, granulating, drageeuniaking, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate sing of the conjugates into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen. [430} Pharmaceutical itions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers e physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippanv, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. it must be stable under the conditions of cture and storage and must be preserved against the inating action of microorganisms such as bacteria and fungi. The carrier” can be a solvent or dispersion medium containinO‘, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for e, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of rganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, clilorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, cohols such as rnannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum inonostear'ate and gelatin. {431} Sterile injectahle solutions can be prepared by incorporating the conjugates in the required amount in an appropriate solvent with one or a combination of ients enumerated above, as required, followed by filtered sterilization. lly, dispersions are prepared by incorporating the conjugates into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. in the case of sterile powders for the preparation of sterile inj ectahle solutions, methods of preparation are vacuum drying and fr‘eezendr‘ying that yields a powder of the active ient plus any additional desired ient from a previously sterilewfiltered solution thereof. [-432] Oral itions lly include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets For the purpose of oral therapeutic administration, the conjugates can be incorporated with ents and used in the form of tablets, troches, or capsules, Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, n conjugates in the fluid r is applied orally and swished and expectora‘ted or swallowed ceutically compatible binding agents, and/or adju‘vant materials can be included as part of the composition, The tablets, pills, capsules, troches and the like can contain any of the following ients, or nds of a similar : a binder such as rnicrocrystalline cellulose, gum tragacanth or gelatin; an ent such as starch or e, a disintegrating agent such as alginic acid, Pr‘irnogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [433} For administration by inhalation, the conjugates are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. [434} Systemic administration can also he by transmucosal or transdermal means. For ucosal or transdermal administration, penetrants appropriate to the barrier to he permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and c acid derivatives. 'l‘ransmucosal administration can be accomplished through the use of nasal sprays or itories.

For transdermal administration, the conjugates are formulated into ointments, sal'ves, gels, or crearns as generally known in the art.

The conjugates can be prepared with pharmaceutically acceptable rs that will protect the conjugates against rapid ation from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable hioconipatible polymers can he used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

It may be especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to ally discrete units suited as unitary dosages for the subject to be treated; each unit containing a. predetermined quantity of conjugates can be calculated to p ‘oduce the d therapeutic effect in association with the required pharmaceutical c‘ rrier. The speeifi cation for the dosage unit forms of the disclosure are dictated by and ly dependent on the unique teristics of the conjugates and the particular therapeutic effect to he achieved. [43 7] ln therapeutic applications, the dosages of the pharmaceutical compositions used in accordance with the disclosure vary ing on the agent, the age, weight, and clinical condition of the recipient patient, and the ence and judgment of the clinician or tioner administering the therapy, among other factors ing the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the ms of the disease and also preferably causing complete regression of the disease, [43 8:} lt is understood that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Methods of Use [43 9] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need f, comprising administering to the subject a therapeutically effective amount of a ate sed herein. {440} In some embodiments, the present disclosure provides a method of treating a disease or disorder in a t in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein.

In some embodiments, the t disclosure es a method of activating or enhancing an activity of a STING in a subject, comprising administering to the subject a conjugate disclosed herein. [442} In some embodiments, the present disclosure relates to a method of treating a cancer in a t in need thereof, comprising administering to the subject an effective amount of a conjugate disclosed .

In some embodiments, the present disclosure provides a conjugate disclosed herein for use in treating or preventing a disease or disorder in a subject in need thereof, [444} In some embodiments, the present sure provides a conjugate disclosed herein for use in treating a disease or disorder in a subject in need thereof, [445} In some embodiments, the present disclosure provides a ate disclosed herein for treating a STING—mediated disease or disorder in a subject, [446} In some embodiments, the present disclosure provides use of a conjugate disclosed herein for treating a cancer in a subject in need f. [447} In some embodiments, the present disclosure provides use of a conjugate disclosed herein in the manufacture of a medicament for treating a disease or disorder in a t in need thereof. [448} In some ments, the present disclosure provides use of a conjugate disclosed herein in the cture of a medicament for treating or preventing a disease or disorder in a subject in need thereof. [4493 In some embodiments, the present disclosure provides use of a conjugate disclosed herein in the manufacture ofa medicament for treating a STING—mediated disease or disorder in a subject. [4503 In some embodiments, the present sure provides use of a conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject in need thereof. {451} In some embodiments, the present disclosure es use of a conjugate disclosed herein for the treatment or tion of a disease or disorder in a subject in need thereof. {452} In some embodiments, the present disclosure es use of a conjugate disclosed herein for the treatment of a disease or disorder in a subject in need f. {453} In some embodiments, the present disclosure provides use of a conjugate disclosed herein for treating a STING—mediated disease or disorder in a subject. {454} In some embodiments, the present disclosure provides use of a conjugate disclosed herein for treatment of a cancer in a subject in need thereof. [455} In some embodiments, the conjugate disclosed herein is administered to the t.

In some embodiments, the present disclosure provides a method of treating or ting a disease or disorder in a subject in need f, comprising administering to the subject an efficient amount of at least one ate of the disclosure; wherein said conjugate releases one or more therapeutic agent upon biodegradation.

In some embodiments, the t sure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an efficient amount of at least one conjugate ofthe disclosure; wherein said ate releases one or more therapeutic agent upon biodegradation. [458} In some embodiments, the present disclosure the conjugate is an antibody~STlNG agonist conjugate. In some embodiments, the disease or disorder is cancer. [459} In some embodiments, the disclosure provides methods of treatment or prevention of STING mediated diseases and disorders Exemplary diseases/disorders include, but are not limited to, cancer, infectious e (eg HIV, HBV, IICV, HPV, and influenza), and e adjuvant. [46G] In some embodiments, the STING path 'ay may induce anti-tumor immunity by upregulating IFNE and interferon (IFN)—stiniulated genes (18st in many cell types Within tumors in response to agonistic, cy‘tosolic nucleic acids [461} In some embodiments, the present disclosure provides a conj ugate disclosed herein for use as a vaccine adiuvant. There is also therefore provided an genic composition or vaccine adjuvant comprising a conjugate disclosed herein. [462} In some embodiments, there is provided a composition comprising a conjugate disclosed , and one or more immunostirnulatory agents. {463} In some embodiments, the present disclosure provides the use of a conjugate disclosed , in the manufacture of a vaccine. In some embodiments, the present disclosure provides use of a conjugate disclosed herein, for the manufacture of an inirnunogenic composition or a vaccine composition sing an antigen or antigenic composition, for the treatment or tion of disease.

In some embodiments, the disclosure is directed to a method of treating or ting disease comprising the administration to a human subject suffering from or susceptible to disease, an iminunogenic composition or a vaccine ition comprising an antigen or antigenic composition and a conjugate disclosed herein.

In some embodiments, the disease or disorder is inflammation, an mune disease, an allergic disease, an infectious disease, an HIV infection, an AIDS ion, an HCV infection, influenza or a human papillomavirus (HPV) infection. The scope of the diseases would be y recognized by a skilled n in the field. In some embodiments, these diseases are as described in PCT Application No. PC'I‘x’USZi’JZi’Jx’OMS38, the contents of which is hereby incorporated by reference in its entirety. [466} As used herein, the terms "cancer, H H neoplasm," and "tumor" are used interchangeably and, in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host sm. Primary cancer cells can be readily guished from non—cancerous cells by well—established ques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes rnetastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells, When referring to a type of cancer that normally manifests as a solid tumor, a "clinically able“ tumor is one that is detectable on the basis of tumor mass; eg, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), Xeray, ultrasound or palpation on physical examination, arid/or which is detectable because of the expression of one or more cancer~specitic antigens in a sample obtainable from a patient. Tumors may be a heinatopoietic (or hematologic or hematological or bloodurelated) cancer, for e, cancers derived from blood cells or immune cells, which may be ed to as d tumors." Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute inyelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia, plasma cell malignancies such as multiple myelorna, MGUS and Waldenstromls macroglobulinernia; lymphomas such as non— l-lodgkinls lymphoma, Hodgkin’s ma; and the lilre. {467} In some embodiments, the e or disorder is a pre~cancerous syndrome. [468} The conjugate of the present disclosure may he used to treat inflammation of any tissue and organs of the body, including niusculoskeletal inflammation, ar ation, neural ation, digestive system inflammation, ocular mation, inflammation of the reproductive system, and other inflammation. The scope of the diseases would be readily recognized lay a skilled artisan in the field. ln some embodiments, these diseases are as described in PCT Application No. PCT/’USZOZO/OMSSS, the contents of which is hereby incorporated by reference in its entirety. [469} es of cancer diseases and conditions wherein the conjugate of the present disclosure may have potentially beneficial antitumor effects include, but are not limited to, cancers of the lung, bone, pancreas, skin, head, neck, , ovaries, stomach, colon, hreast, esophagus, hiliary, small intestine, howel, endocrine system, thyroid gland, parathyroid gland, adrenal gland, urethra, prostate, penis, testes, ureter or urothelial, bladder, kidney or liver; rectal cancer; cancer of the anal region; omas of the fallopian tubes, endoinetrium, ceivix, vagina, vulva, renal pelvis, renal cell; sarcoma of soft tissue; inyxorna, rhabdomyoma; fibronia; lipoma; teratoina; cholangiocarcinoma; hepatoblastoma; angiosarcoma; lieinangioina; hepatoina; fibrosarcorna; chondrosarcoma; myelorna; chronic or acute leukemia; lymphocytic lymphomas; primary CNS lymphoma; neoplasms of the CNS; spinal axis tumors; squamous cell carcinomas; synovial sarcoma; malignant pleural mcsotheliomas; brain stem gliorna; pituitary na; bronchial adenorna; omatous hamartoma; niesotheliorna; n's Disease or a combination of one or more of the foregoing cancers, [470} In some embodiments, the disease or disorder is a solid tumor. in one aspect the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal , biliary cancer, nonesrnall cell lung carcinoma (NSCLC), te cancer, colorectal cancer (CRC), colon cancer, ovarian cancer, trial cancer, urothelial cancer, cervical cancer, bladder cancer, papillary thyroid cancer, papillary renal cell cancer, cholangiocarcinorna, salivary duct cancer, kidney cancer, cervical cancer and pancreatic . in some ments, the human has a liquid tumor such as diffuse large B cell lymphoma }, multiple myeloma, chronic lymphohlastic leukemia {ELL}, follicular lymphoma, acute WO 02984 myeloid leukemia, and chronic myelogenous leukemia. In some embodiments, the e or disorder is a skin cancer (: e.g non—melanoma skin , squamous cell carcinoma, basal cell carcinoma) or actinic keratosis. In addition to a field effect for clearing superficial skin cancers, the conjugate of the present disclosure may prevent the development of subsequent skin cancers and pro—malignant actinic sis in treated subj ects. [471} In some embodiments, the e or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endornetrial cancer, gastric cancer, esophageal , biliary cancer, urothelial cancer, head and neck squamous oma, melanoma, nonwsmall cell lung cancer, n cancer, or pancreatic cancer. In some embodiments, the disease or disorder is breast cancer, gastric , colorectal cancer, colon cancer, esophageal cancer, biliary cancer, endometrial cancer, urotl'relial cancer or nonnsmall cell lung cancer.

In some embodiments the breast cancer is HERE amplified/overexpressed breast cancer or HERZ low breast cancer.

In some embodiments, the endometrial cancer is serous endometrial cancer. [-474] The conjugate of the present disclosure may also be useful in the treatment of one or more diseases afflicting mammals which are characterized by cellular eration in the area of disorders associated with neo~vascularization and/or vascular permeability, fibrotic disorders, and metabolic disorders, The scope of the diseases would be readily recognized by a skilled artisan in the field, In some embodiments, these diseases are as described in PCT Application No.

PCT/US2G2G/O44538, the contents of which is hereby orated by reference in its entirety, In some embodiments, the disease or disorder is a, neurodegenerative diseases, Exemplary neurodegenerative diseases includes, but are not limited to, multiple sclerosis, Huntington‘s disease, mer's e, Parkinson's disease, amyotr‘ophic lateral sclerosis (ALS). The scope of the diseases would be readily recognized by a skilled artisan in the field. In some ernbodim ents, these diseases are as described in US Provisional Application Nos. 62/882,081, 62/944,643, and 62/982,935, the contents of which are hereby incorporated by reference in their entirety, [476} In some embodiments, the disease or disorder is an infectious disease, which is any disease instigated by or coincident with an infection from a pathogen, derived from bacteria, derived from the DNA virus families, or RNA virus es. The scope of the diseases would be readily recognized by a skilled n in the field. In some embodiments, these es are as described in PCT Application No. PCT/U 0/044538, the contents of which is hereby incorporated by reference in its entirety. {477} The conjugates of the present disclosure may be employed alone or in ation with other therapeutic agents. As modulators of the immune response, the conjugates of the present disclosure may also be used in monotherapy or in combination with another therapeutic agent in the treatment of diseases and conditions wherein modulation of S'l‘lNG is beneficial. Combination therapies according to the present disclosure thus se the administration of a ate of the present disclosure or a phar‘rnaceutically acceptable salt thereof, and at least one other therapeutically active agent. In some embodiments, combination therapies according to the present disclosure comprise the administration of at least one conjugate of the present disclosure or a phar‘rna ceutically able salt thereof, and at least one other therapeutic agent. The conjugatets) of the present disclosure and pharmaceutically able salts thereof, and the other therapeutic agent(s) may be administered together in a single pharmaceutical composition or tely and, when administered separately this may occur simultaneously or sequentially in any order. The amounts of the conjugate(s) of the present disclosure and pharmaceutically acceptable salts thereof, and the other therapeutic agent(_s) and the relative timings of administration will be selected in order to achieve the d combined therapeutic effect, Thus, in a further aspect, there is ed a combination sing a conjugate of the present disclosure or a pharrnaceutically acceptable salt thereof, together with one or more other therapeutic . [478} The conjugate of the present disclosure and plrarniaceutically acceptable salts f may be used in combination with one or more other therapeutic agents which may be useful in the prevention or treatment of allergic disease, matory disease, or autoimmune disease, for example, antigen unotherapy, anti-histamines, steroids, NSAle, bronchodilators, rnetlrotrexate, leukotriene modulators, monoclonal antibody therapy, receptor therapies, or antigen non-specific iminunotherapies. [479} The conjugate of the present disclosure and pharmaceutically acceptable salts thereof may he used in combination with radiotherapy and/or surgery and/or at least one other therapeutic agent which may be useful in the treatment of cancer and pre~cancerous syndromes. Any anti—neoplastic agent, anti—microtubule, anti—mitotic agent, hormone, hormonal analogues signal transduction pathway tor, protein tyrosine kinase, or anti-angiogenic therapeutic agent, may be utilized in the combination. The scope of the other eutic agents would be readily recognized by a skilled artisan in the field. In some embodiments, the other therapeutic agent is as described in PCT Application No. PC'l‘fllSZDZO/{l44538, the contents of which is hereby orated by reference in its entirety. [480} Agents used in therapeutic regimens, therapeutic agents used in ptotic regimens, or cell cycle signaling inhibitors may also be useful in combination with the ate of the present disclosure. {481} In some embodiments, the combination of the present disclosure ses a conjugate of the present disclosure or a salt, particularly a ceutically acceptable salt thereof, and at least one anti—neoplastic agent, anti—microtubule, antiwmitotic agent, e, hormonal analogues signal transduction pathway inhibitor, protein tyrosine kinase, or antinangiogenic therapeutic agent, or a combination thereof, Additional examples of other therapeutic agents (eg, anti—neoplastic agent) for use in ation or co—administered with a conjugate of the t disclosure or a pharniaceutically acceptable salt thereof are immune—modulators. [-483] In some embodiments, the combination of the present disclosure comprises a conjugate of the present disclosure or a salt, particularly a pharmaceutically acceptable salt thereof, and at least one inimunovmodulator or at least one inimunostimulatory agent, [484} As used herein ”ininiuno—modulators" refer to any substance including monoclonal antibodies that affects the immune system lmniuno~niodulators can be used as anti~ stic agents for the treatment of cancer. For example, e-modulators include, but are not limited to, anti-CTLA~4 antibodies and anti~Ple antibodies ()ther imniuno-niodulators e, but are not limited to, [COS antibodies, 0X40 antibodies, PD—Ll antibodies, LAG?) antibodies, TIM—3 antibodies, 4] BB antibodies and GlTR antibodies [485} Additional examples of other therapeutic agents {anti-neoplastic agent) for use in combination or co~administered witl'i a coi'ijugate of the present disclosure are antinD—Ll agents (ie, anll*?D—Ll antibodies) or PD-l antagonists, [486} Thus, in some embodiments, methods of treating a human in need thereof are provided comprising administering a coniugate of the present disclosure or a salt thereof and at least one immuno—modulator. in some embodiments, the immune-modulator is selected from an lCOS agonist antibody, an (DC—40 antibody, and a hill antibody. in some embodiments, the human has cancer. Also provided herein is the use ofa conjugate of the present disclosure or a salt thereof in combination with at least one immune—modulator for the treatment of a human in need thereof. {487} As used herein nostimulatory agent” refers to any agent that can stimulate the immune system. As used herein immunostimulatory agents e, but are not limited to, vaccine adjuvants, such as Toll—like receptor agonists, T-cell checkpoint bloclcers, such as mAbs to PDul and C'l‘L4 and T—cell checkpoint agonist, such as agonist mAbs to (ix-40 and 10383. As used herein ”immunostiinulatory agent“ refers to any agent that can stimulate the immune system. As used herein immunostimulatory agents include, but are not limited to, e adiuvants. [488} The term "Tolhlike or” (or "'I'LR“) as used herein refers to a member of the ike receptor family of proteins or a fragment thereof that senses a microbial product and/or initiates an adaptive immune response. ln some embodiments, a TLR activates a dendritic cell (DC). Toll— like ors (TLRs) are a family of pattern recognition receptors that were initially identified as sensors of the innate immune system that recognize microbial ens. TLRs recognize distinct structures in microbes, often referred to as “PAMPs” (pathogen associated molecular patterns).

Ligand binding to TLRs invokes a cascade of intracellular signaling pathways that induce the production effectors involved in inflammation and immunity [489} In some embodiments. the immunostimulatory agent for use in combination with the conjugate of the present disclosure is a T134 agonist. [490} Thus, in some embodiments, methods of ng a human in need thereof are provided comprising administering a conjugate of the present disclosure or a salt f and at least one immunostimulatoiy agent. In some ments, the iminui'iostimulatory agent is a TLR4 agonist. in some embodiments, the immunostimulatory agent is an AGP, In some embodiments, the human has cancer. Also provided herein is the use a conjugate of the present disclosure or a salt thereof in combination with at least one irnmunostimulatow agent for the treatment of a human in need [491} In on to the iniinunostirnulatory agents described above, the compositions of the present disclosure may further comprise other therapeutic agents which, because of their adjuvant nature, can act to stimulate the immune system to d to the cancer antigens present on the inactivated tumor cellis). Such adjuvants include, but are not limited to, lipids, liposomes, inactivated bacteria which induce innate ty {e.g., inactivated or attenuated Listeriamonocytogenes), compositions which mediate innate immune activation via, (NOD)-like receptors (NLRs), Retinoic acid inducible gene-based (RIGil—l—like receptors {RLRs}, and/or Cu type lectin receptors (CLRs). [492} Because of their adjuvant qualities, 'I'LR agonists may be used in ations with other vaccines, adiuvants and/or imrnune modulators, and may be combined in various combinations. In some embodiments, the conjugate of the present disclosure bind to STING and induce S'I'ING— dependent 'I‘BKI activation and an vated tumor cell which expresses and secretes one or more cytokines which stimulate DC induction, recruitment and/or maturation, as described herein can be administered together with one or more 'I'LR agonists for therapeutic purposes. [493} Further active ingredients (antineoplastic agents) for use in ation or rte—administered with the conjugate of the present disclosure are IDO inhibitors. [494} In some embodiments, the conjugate of the disclosure may be employed in combination with at least one other therapeutic agent useful in the prevention or treatment of infectious diseases bacterial infections, Viral infections, Kaposi's sarcomawassociated lierpesvirus infections, 'IB infections, Chlamydia, Plasmodium infection, lococcus infections, ophic lateral sis (ALS), multiple sclerosis, systemic lupus erytheinatosus and related lupus ers, psoriasis, or Sj ogren‘s me, {-495} The conjugates of the disclosure may be administered by any suitable route of administration, including both ic administration and topical administration, Systemic administration includes oral administration, parenteral administration, transderinal administration, rectal administration, and administration by inhalation. Parenteral administration refers to routes of administration other than en teral, trarisderinal, or by inhalation, and is typically by injection or infusion, Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion, tion refers to administration into the patient‘s lungs whether inhaled h the mouth or through the nasal passages. l administration es application to the skin, [496} In addition to the above described routes of administration suitable for treatment of oncology, the pharmaceutical itions may be adapted for administration by intratumoral or periturnoral injection. The intratumorally or peritumoral ion of a conjugate of the present disclosure directly into or adjacent to a single solid tumor is expected to elicit an immune response that can attacli: and destroy cancer cells throughout the body, ntially reducing and in some cases permanently eliminating the tumor from the diseased subject. The activation of the immune system in this manner to lrill tumors at a remote site is commonly known as the abscopal effect and has been demonstrated in animals with multiple therapeutic modalities,. A further age of local or intratumoral or peritumoral stration is the ability to achieve equivalent efficacy at much lower doses, thus zing or eliminating adverse events that may be observed at much higher systemic doses [497} The conjugates of the disclosure may be administered once or ing to a dosing regimen wherein a number of doses are administered at varying als oftinre for a given period of time. For example, doses may be stered one, two, three, or four times per day, Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing ns for a conjugate of the disclosure depend on the plrarmaookinetic properties of that conjugate, such as absorption, distribution, and halfwlife, which can be determined by the skilled artisan, In on, suitable dosing regimens, including the duration such ns are stered, for a conjugate of the disclosure depend on the disease or disorder being treated, the ty of the disease or disorder being treated, the age and physical condition of the patient being treated, the in edical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such d artisans that suitable dosing ns may require adjustment given an individual patients response to the dosing regimen or over time as individual patient needs change Total daily dosages range from l mg to ZOGG mg, preferably, total daily dosages range from 1 mg to 250 mg.

For use in therapy, the conjugates of the disclosure will be normally, but not necessarily, formulated into a pharmaceutical composition prior to administration to a patient. Accordingly, the disclosure also is directed to pl’iarmaceutical con'ipositions comprising a conjugate of the disclosure and at least one pharrnaceutically acceptable excipient.

The pharmaceutical compositioi'is of the disclosure may he prepared and packaged in bulk form or in unit dosage form. For oral application, for example, one or more s or capsules may be administered. A dose of the pharmaceutical composition contains at least a therapeutically effective amount ofa conjugate of the present disclosure (i.e., a conjugate of the present sure or a salt, particularly a phar'maceutically acceptable salt, f}. When prepared in unit dosage form, the pharmaceutical compositions may contain from l mg to 1000 mg of a conjugate of the present disclosure. [500} As provided herein, unit dosage forms (pharmaceutical compositions) comprising from l mg to 1000 mg of a conjugate of the disclosure may be stered one, two, three, or four times per day, preferably one, two, or three times per day, and more preferably, one or two times per day, to effect treatment of a STING—mediated e or disorder. {501} The pharmaceutical compositions of the disclosure typically contain one conjugate of the sure. r, in n embodiments, the pharmaceutical compositions of the disclosure contain more than one conjugate of the disclosure. In addition, the pharmaceutical compositions of the disclosure may optionally further comprise one or more additional therapeutic agents, (eg, ceutically active conjugates).

As used herein, "pharmaceutically acceptable excipient” refers to a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the conjugate of the disclosure when administered to a patient and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be of sufficiently high purity to render it na ceutically acceptable. [503} The conjugates of the disclosure and the pharmaceutically acceptable excipient or excipients will typically be formulated into a dosage form adapted for administration to the t by the desired route of administration. Conventional dosage forms include those adapted for ( l) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, ons, sachets, and cachets; (2) parenteral stration such as sterile solutions, suspensions, and powders for reconstitution, (3) transderrnal administration such as transdermal patches, (4) rectal administration such as suppositories; (5) inhalation such as ls and SOlUthl’lS; and (6) topical administration such as creams, ents, lotions, solutions, pastes, sprays, foams, and gels.

Suitable pharmaceuticé lly acceptable excipients will vary depending upon the particular dosage form chosen. in addition, suitable ceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain ceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharrnaceutically able excipients may be chosen for their ability to facilitate the carrying or orting the ate or coniugates of the disclosure once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharniaceutically acceptable excipients may be chosen for their ability to enhance patient ance. [505} Suitable pharmaceutically able excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating , coating agents, wetting agents, solvents, vents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, antincalcing agents, liuinectants, clielating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and ing agents. The skilled artisan will appreciate that certain pliarinaceutically acceptable excipients may serve more than one function and may serve alternative ons depending on how much of tlie excipient is present in the formulation and what other ingredients are present in the formulation. [5,06] Skilled artisans possess the dge and skill in the art to enable them to select le pharmaceutically acceptable excipients in appropriate amounts for use in the disclosure in addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically able ents Examples include ton's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The l‘landhoolr of Pharmaceutical Excipients (the American Pl'iarrnaceutical Association and the Pharmaceutical Press), In one aspect, the disclosure is directed to a solid oral dosage form such as a tablet or' e comprising an effective amount ofa conjugate of the disclosure and a diluent or filler. The oral solid dosage form may further comprise a disintegrant or a lubricant. [508} It will he understood that the conjugates of the present disclosure may also be formulated with vaccines as adiuvants to modulate their activity. Such compositions may contain antibody (antibodies) or antibody fragnientts) or an antigenic coniponent, optionally together with one or more other ents with adjuvant activity. {509} Certain compounds and/or coniugates of the disclosure may be potent inininnomodulators and accordingly, care should be exercised in their handling. The disclosure having been bed, the following examples are offered by way of illustration and not limitation. in some embodiments, the conjugate is Formula BB Q 0 gs~< )r‘u ' . é a > .

- HN 7+ , XMT~1519 ’\ ”S Y \ r 0 '3: HT,I“ if V \\ x” C _1 \. 1y: lg "LCi 9; f L ng‘kfi’ v3 / “’2 Hi“ /\ E 8“ ,4; ,N HZN \l/Kxg/‘L3"“? fi“37/4 0, j,“ n the conjugate comprises the XMT—l Si 9 antibody comprises a. variable heavy chain complementarity determining region l (CDRHI) comprising the amino acid sequence FTFSSYSVIN (SEQ ID NO: 29); a. variable heavy chain complementarity determining region 2 (CDRHZ) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 21); a variable heavy chain complementarity determining region 3 (CDREB) eomprising the amino acid sequence {KSHGYFDL (SEQ ID NO: 22); and a variable light chain mentarity determining region l (CDRLl) sing the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 27); a variable light chain con'ipiementarity determining region 2 (CDRLZ) con'iprising the amino acid sequence GAS‘SRAT (SEQ ED N0: 23); and a variable light chain complementarity ining region 3 (CDRL3) comprising the amino acid sequence QQYHI-{SPLT (SEQ ll) N0: 29), and dis is about 8. {:51 1} In some embodiments, the conjugate is Formula CC / ? H i H i H 3 H sT’s in” /N\/ *N’”\{ “\f N”\.{'N\/ ”NV"\0’“V'°\/\o : H l I 0 C‘ C‘ f/\O /\\/0\) O OH OH \\ O j : H : 0‘: Avon \ HN o\/\Q,\,o\/\T,NY»¢\§= \\ ‘7. c ngNH on on ’\ I OH 0 on 1 \/\O Q I; Ff)”: - \A‘N‘H :M ”‘0 \/.QH HO \ Yo --~)J\F—° O\\ Hi“V - _:N ,l NH H r" ’éfi III \2 O 1 \ O I aw ,2 N\ .

O \ \\ x J NH kw” 1 ‘\ g.- / N»—\ ' i ’ j 0\ '/(l15 \'/L\N/ \ wherein the conjugate comprises the XMT~l 535 antibody con'iprising: a CDRII] comprising the amine acid sequence GYTFTGYNIH (SEQ ID NO: 5), a CDRIIZ comprising the amino acid sequence AIYPGNGDTSWQQKFRG (SEQ ID NO: 6), a CDRIB ccrnprising the amino acid sequence GETARATFAY (SEQ ID NO: 7}; a CDRLI ccrnprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8,); a CERF/’1 cc-rnprising the amino acid sequence Y'I'SSLYS (SEQ ID NO: 9:); a CDRL3 comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: l0) and dis is about 8. {512} In scme embodiments, a ccnj ugate of Fermula BB or Formula CC is useful for treating a disease or er in a t in need thereof, sing stering to the subject a therapeutically effective amount cf the conjugate of Formula BB or Formula CC. In some embcdiments, the disease or discrder is cancer. {513} In senie embodiments, for the conjugate ochrniula BB, the cancer is breast cancer, gastric cancer, cclcrectal cancer, escphageal cancer, biliary cancer, endornetrial cancer, urcthelial cancer or nonwsmall cell lung cancer. In some embodiments the breast cancer is HERZ aniplified/cverexpressed breast cancer or HERE low breast cancer. In seine ments, the trial cancer is serous endornetrial cancer. [5M] In some iments, for the conjugate of Forrnula CC, is useful for treating a NaPiwa expressing tumor in a subject in need th erecf. In some embodiments, the NaPi2h~eXpressing turner is ovarian cancer, non—small cell lung cancer (NSCLC), ary thyroid cancer, endornetrial cancer, cholangiocarcinoma, papillary renal cell cancer, clear cell renal cancer, breast cancer, lridney cancer, cervical cancer or salivary duct cancer. {515} In some embodiments, the subject has epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, platinum resistant ovarian cancer, uamous NSCLC cancer, progressive, radioactive iodine—refractory, loco—regional recurrent or metastatic disease papillary thyroid cancer or epithelial endometrial cancer. {516} In some embodiments, the conjugate of Formula BB is useful for treating a e or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ate of Formula BB in combination with one or more therapeutic agents. ln some embodiments, the therapeutic agent is an iinmunowmodulator agent or an ostimulatoijvr agent. in some ments, the ~modulator agent is an anti—Crime" 4 antibody, an anti—PDnl antibody, an lCOS antibody, an 0X~4O antibody, a PDnLl antibody, a LAG3 antibody, a 'l‘an3 antibody, a 4188 antibody or a Gl'l'R antibody.

[Sl’i’] In some ments the conjugate of Formula BB is useful for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ate of Formula BB in combination with one or more HERZ antibodies that bind to a different epitope of HERE than the HERZ antibody XMT—l Sl 9. in some ments the HERE antibody that binds to a different ot‘HERZ than the HERZ antibody Xblll lSl 9 is trastuzumab, umab, Fall? 7, or chAZl.

EXAh/IPLES [518} The following examples illustrate the sure. These examples are not intended to limit the scope of the present disclosure, but rather to provide guidance to the skilled artisan to prepare and use the Compounds, compositions, and s of the present disclosure. While particular embodiments of the present disclosure are described, the d artisan will appreciate that various changes and modifications can be made t departing from the spirit and scope of the disclosure. {519} It will be understood that certain Compounds of the disclosure may be potent irnniunomodulators and accordingly, care should be exercised in their handling. {520} The reactions described herein are applicable for producing Compounds of the disclosure having a variety of different substituent groups (e.g R‘, R etc), as defined herein. The slrilled artisan will appreciate that if a particular suhstituent is not compatihle with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. Suitable protecting groups and the methods for protecting and de— protecting different substituents using such suitable protecting groups are well known to those d in the art; examples of which may be found in T. W. Greene *Protective Groups in Organic Synthesis‘ (4th edition, 3. Wiley and Sons, 2006}. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification.

Abbreviations [5:21] The following abbreviations are used in the reaction schemes and synthetic examples, which follow. This list is not meant to be an allninclusive list of abbreviations used in the application as onal standard iations, which are readily understood by those skilled in the art of organic synthesis, can also he used in the synthetic schemes and examples.

ACN Acetonitrile CDl l l '~Carbonyldi imidazole DCC icyclohexylcarhodi imide DCM romethane DIPEA, N,N~Diisopropylethylamine DMA ylacetainide Dh/lF Dimethylformamide DMPA Dimethylolpropionic acid 581 Electrospray ionization HATU 24 l Hr‘rzabenzon‘iazohl ~yl}~l l 3 ,3 —tetraniethyl ur‘onoi urn hexalluorphosphate HlC Hydrophobic interaction chromatograpl'ry HOBt Hydroxybenzotriazole lrlPl_,C High re liquid chrornatography Mel)H ol SEC Size exclusion tography 'l‘FA ’l‘rifluoroacetic acid THE 'l'etrahy drofuran l’yBOP (Benzotriazole— l —y lorry)tripyrrolidinopliosphoniurn hexafluorophosphate General ation {522} All reagents were purchased from relevant providers unless otherwise stated. [523} The diABZl S'l'lNG agonist was prepared as described in julu et al (Nature, 564(773 6):439—443 (2018)).

XM'l‘-1535 (anti—NaPiZb antibody} is disclosed in co-pending application US 15/457,574, filed March 13, 2017, the entire contents of which are incorporated herein by reference. XlVl'lTu 1519 (antinHer2 antibody) is disclosed in US 9,555,112, issued January 31, 2017 and US 9,738,720, issued August 22, 2017, the entire contents of which are incorporated herein by reference. [525} 14535 AFUHPA ADC, DAR 5.9 and Rituxiniab AFnHPA ADC, DAR 5.5, were prepared as described in conpending application US 62/958,916, filed January 9, 2020, and LS 63/941173 5, filed June 18, 2020, the entire contents of which are incorporated herein by reference HPLC purification was performed on a Phenomenex Gemini 5 pin C18 110 A, 251.1 x 10 min, semi—preparative column. [527} When applicable, the drug content of the conjugates was determined spectrophotometrically, otherwise RP—HPLC or 1...C/1\/1$ as med for quantitative ination of the drug content. [528} The protein t of the antibody—drug conjugates was determined ophotornetrically or by EMSA.

Antibody-drug conjugates, drug carrying Scat‘t‘olds, or antibody Scaffolds were purified (ie, removal of residual unreacted drug, unconiugated antibody, enzymes or starting als) by ive diafiltration, (THT chromatography or 1:116, as required if necessary, onal purification by SFC or 111C were conducted to remove aggregated antibody-drug conjugates. in general, the antibody-drug conjugates, as purified, contained <5% (w/w) (e,g <Z% (:w/wD aggregated antibodysdrug conjugates as determined by SFC; <O.5% (w/w) (eg, (401% (MM) free (unconiugated} drug as determined by lU’—11PLC and/or LC—MS/MS; <1% (w/w) of free drug conjugate as determined by SEC and/or Rl’ulslPLC; and <10% (w/w) (eg, <1% {XV/12V)) unconjugated antibody or dy fragments as determined by HPLC and/or REP-HPLC.

Reduced or partially reduced antibodies were prepared using procedures described in the literature, see, for example, sco et al., Blood l02 ('4): 1458—1465 . The total drug (conjugated and unconjugated) concentration was determined by UV—Vis spectrophotometry or RPuHPLC. [530} To determine the concentration of the free drug in a ical , an acidified sample was treated with acetonitrile. The free drug was extracted, and the itrile supernatant was analyzed. To ine the concentration of conjugated STTNG agonist in a non—clinical , the sample was subjected to immunocapture using anti-human Fc antibody magnetic beads followed by exhaustive hasic hydrolysis. The acetonitrile supernatant containing the released drug was analyzed by LCnMS/MS. The total antibody in nonmclinical samples was measured using an MST) ECL immunoassay.

Analysis of free drug was conducted by RP~HPLC using a 0—4 column and an acetonitrile gradient. The l‘v‘ftRM peak areas on the tandem mass spectrometry were integrated and ed to auristatin F (AF) and auristatin F hydroxypropyl amide (AFnHPA) standards. The method is quantitative for AFwHPA and All in plasma and tissue homogenates and linear over the concentration ranges of 0.1 to 150 ng/mL The total drug released after hydrolysis with NaOH was measured under the same condition with the dynamic range from l ng/inl, to 5000 ng/rnL. The total antibody rds range from 0.009 ug/mL to 20 ug/inL [5,32] The drug to antibody ratio (DAR) was determined by measuring the absorption of the conjugates. The DAR value was calculated using the appropriate molar extinction coefficients of the antibody and the STING agonist payload. [533} Tumors were measured twice weekly using digital rs and tumor volumes were calculated using the formula: tumor volume (1111113)) (width2 x length/V2, Body weights were recorded daily for the first weel: and twice weekly thereatter. Animals remained on study until weight was or lculated using the formula: body weight change (9'6) ((weight Stud-V W X weight Stud-V day ght Stud-V day 1} *lGO. Tumor volumes are ed as mean :i: standard error of the mean (SEM). Tumor growth inhibition (%TGl) was defined as the percent difference in mean tumor volumes (MTVs) between treated and control groups. Tumor size was measured throughout each efficacy study to determine tumor growth inhibition (Till). Percent tumor regression was calculated using the formula: % sion (l—(mean tumor volume final).1" (mean tumor volume day 1);) * l 00. A partial response (PR) is defined as a tumor volume of 50% or less for day l volume for three consecutive measurements and equal to or greater than l 3.5 mm3 for at least one of these three measurements. A complete se (CR) is defined as a tumor volume iess than 13.5 mm3 £01 three censeeuflve measurements A ‘Lumor——1ree su1viv01 (IES1cEa351fied as 1111111115; 3 (R at the end of study.

Exampie ,1: Synthesis 0f KNEE-1519 (Eenjugate 8 ”.1! g) “‘1? c 2L1 ‘1’\ “1..

: N r . <9 M ’~\_ '1: NI I .N \‘ u—(Nj. \ «.11, we,“ HN"<';.J,E\[,/r 1112 14' /J 11111 (‘5' I s "N 0‘ a "\ P \2/ Pan B Part C 1/ ”””””””””5" Ho’\~’\o g \ C‘r"\/1LD"‘VA ~A\/\.O 3 ...................x» r/in .N'\_ 'a1 3 (I; ’11) ,ALy—H (, : /. NH 3 ‘1-‘0': ./ H1 "" , 5: u, ‘1 /’\\ ‘ b {I_/ H111 “‘24 A“ \/\N -. T \/ N L N L 7/ , \fix O V v C! v o 1 2 _ 1-1 ’N C! U NJ” 5) v I -~€ 11. 1" 1L 1! N. 1 ,1 ,1 H‘N—(‘I Ij’ NH; /L\/ 11-1 .1111 , f; 1”’ ' 1“ \1 Ck Pan 0 P1 AI 1k 1. P """"""""""3’ ’N/\"OV"YM\/K"\/\ 2 HyuvfldAVQN/\n,fi\r}\o,VE I» I E I 1, / .-N (l $11M»!.1 xe‘ H”V1,: 31“ 0" \’J\ I 1: "K D S) 0 U C IrrVNy/KNAYNVKNWSVM,H H " ll_,‘H 13 H H 'n' 5:3 H 11/13/\,5VAC (k’l/ /NY nu"11”.“ kl4"ijV””"\/‘~)/\,U¢~C, L‘ E, K H lo n ‘5f/\13/V:Ivj\/12H R N 5 H a x/o\/‘ o v11 :11: \. 0 (“or c CVJ‘OH : UVNC1/\"\x" 5‘15 <\/\/\,1-’/~>V ”hum/k“-vi '\./DH Hi1 T {1 a Y '3 in"?1“?va D j 0A)“ :H OH SJ; 9“ ‘0 1% p11 6 HO “’3“ H9 S H! H?’\,QH 1. 1.51.

Part E 11111L ’ND‘Z o_3» f 111:1k ,‘ Hu ‘1: h?v“1._,/\\\/’\' J‘N E V /O$3\ 1) fl'?’\ \‘( M“ «*0 \ NH2 U D ‘ VH “- :I h; \ xmfine—FRJkN/Y-1. ‘gvl‘u’fi”"""‘“”flew“? \ 0 ./~1, A (-11 PEI-3 F \ .1 v‘ 1/ E_ H E 13h 0-4 11 \ _«<« a, 13VAo,-\,o.\/\n/NIVKWAT FA/J, \‘ ‘ ""2 H \L 11 . 1. 1,1 (111 N1- \ 1" 1311 k \ If 1"j; 1911 \ ‘7 no "111‘“1.5.”.1. \ ‘ ‘ L10 1 1.1., I NHZ ” ‘ ,NJ [I \“N .J r 1 i \LC, Y [I IE1" HN/(XO "ex/SAW ,’ i - 2:4 /' I\N \ ‘l ‘3 a) // 1’1: / fl :1 "”2 /d11 [534} Part A: '17o a mixture of nd 1 (prepared as bed in W02017175147Al, 0.5 g, 0.64 mrnol), Boe-L-fi‘ilanine (0.242 g, 1.28 nirnol), DNIAP (7.8 mg, 0.064 nirnol) and ECG (0.264 g, 1.28 rnmol) was added EMF (2 mL). The sion was stirred overnight at room temperature, then the solution was concentrated. and the residue was purified on silica gel (ti—40% MeOl-l in l) to give Compound 2 as a light-yellow solid (0.52 g, 85% yield). ESI-MS m/CZ Caled for Cal-lssN 13010 [1‘vl111‘3 9524; found 9524. [5351 Part B: To a sion of nd 2 (0.52 g, 0.55 mniol) in dioxane ('10 niL) was added 4 N HCl (2 mL, 8.19 niinol). The reaction mixture was stirred at room temperature for 2 h, then the suspension was concentrated and used without further purification in the next step.

Compound 3 was obtained as a white solid. ESLMS m/e Calcd for Col-1501413022, {NEH}: 852.4; found 852.3. [536} Part C: To a solution of Compound 3 (0.586 g, 0.6.6.1 mniol) in 131%? (5 mL) was added 2,2ndimethyl~4~oxom3,8,l l"trioxanSnazatetradecann14—oie acid (0.202 g. 0.727 mmol), followed by 11111313131 (0.230 11114., 1.322 mniol). The reaction e was stirred at room temperature for 5 min, then HATU (01376 g, 0,992 niinol) and HOBt (0,153 g, 0.992 niinol) were added. The reaction mixture was stirred at room temperature for 2 b. An additional aliquot of DIPEA (0460 nil”, 2.6 niinol) was added. After another 1 hour, the reaction mixture was concentrated to an oil. The residue was purified over silica gel (040% MeOH in DCM) to afford Compound 4 (0.9 g, >959?) yield) as a white so1idi ESLMS m/C: Calcd for CseriNmOisU‘s/HHT: 1111.5; found 1 1 1 1.5. [5371 Part 1): To a suspension of Compound 41 (0.9 g, 0.810 mmol) in dioxane (10 m1.) was added 4 N HCl (3.04 rnL, 12. l 5 nimol). The reaction mixture was stirred at room temperature for 1.5 h. The suspension was concentrated to afford Compound 5 as a colorless solid (0.56 g 66.0% yield): ESI—MS Calcd for 630114011 [Mi-1:111: 101 1.5; found 10114. [5381 Part E: To a solution of Scaffold 6 (100 mg, 0.072 mrnol, prepared as described in PCT/1182018/06719) and Compound 5 (72.7 mg, 0.072 inniol) in DIVE? (2 ml) were added PyBOP (37.5 mg, 0.072 mrnol) and DlPEA (0.075 mL, 0.431 mmol). The reactioi'i mixture was stirred at room temperature 2 h, then the on was concentrated, and the residue was purified by preparative HPLC 10—80% ACN in water) to afford Compound '7 (109 mg. 64% yield). ESl—MS mi? Caled for Ciriilli56N24O4i {M-l112+: 1192.5; found 11925. 5391 Part E: To a solution of —1519 (1 0 mg, 0.069 umol) in 50 rim/111154171559, l nib/113131141, pH '7 buffer was added TCEP (0.059 mg, 0.207 uniol) and the mixture was shaken at 37 "C for 90 minutes. Te the reduced antibody was added Cempound '7 (0.987 mg, 0.414 tirnol in 200 uL DMA). The i'esuiting mixture was shaken at 37 ”C for 60 minutes. The reaction was ed with cysteine (15 equiyaierits, 0.125 mg, 1.035 tirnol in 125 ML of 50 mM 1-1E1?ES, 1rnMED'1'A, p11 7:) and d at ream temperature fcr 1 h. The resniting Conjugate 8 was purified by uitrafiitration or CHT chromatography. The details of the antibody—drug conjugates 8—1 and 8—2 are given heiow. (Inningates 8—1 and 8—2 were prepared as described except that a higher ratie cf '1‘CEPJnAb was used in the synthesis of 8—2, compared to 8-1 (4:1 ES 3:1) as welE as a higher ratie of Compound '7 to rnAb (8:1 vs 6:1).

Exampfle 1a: Synthesis of stiiznmah Cenjngate 3a [1/ .3 L4 1:) H Q O " 5 ': ' ‘1' 1 F \\ Trastuzumah—fx/LNATKKSAE \WNVJKE, . NJ“ MAGIN.»\_AQ_. : ,‘l \ \ 2 . , .fi : ‘ O a. .\ ”£0 0 (ACIA¢.O\,/ C CH 9H H \ \\ C, OVAOm’G‘V/fir/N\AVXNKfoA/\“OH \ Y WV .3 (lug H OH OH \ \ 9H OH \ k3 3»: \ HO ,Z’XCH \ r'~ 110 ( 1 "M, I NHz 0‘: ['1 0:1 / \,. {Nb/ I . \\ av) I ” \ / \ I ~o / '11“ H\I’\O ,’l NYNNAVN‘Y/hrv I/ §,HH 0&1fig / \‘N/l\\ / E \EN’i N-z-J / \ IxH / d [5-40] Conjugate 8a was prepared and terized as described in Example 1 except that zumab was used instead of XM'T—1519. The details of the antibody—drug conjugates 83ml, 8&2. and 83:3 are given be1cw_ Exampie 1b: Synthesis of KIWI—1535 Cenjugate 8h, BAR 5.7 f O 1 :1 H 1 XMT—“ESES’rSj’kN/‘Y' N ' z' VT: v.N 1: ,/\ 1L :1“~11 A x ‘o x; a \ g’ v v Cr 3 75:? "§,‘ 0 \_ ‘ “? 0 OF on H \‘ \ J of}; “VNOAV VAT/EL qufl i ,AVQH \‘ ‘ H13 L, i 1;» \ ‘9‘ K OH 0L] \ 3 H6, (\r J 1 \ V0 3 (UH/.333]?!v (3&0 ! M42 - 0‘ ‘ 3 “‘1 1': I (’ \..N”j./ I y \ 2 \f/ . ‘\ 3 II 1‘ ”Viki: *\\/‘r~"r§r, / u Q \I ,/ i 0st o \ -.\k1:3 1:51; / I\='\ 12¢,» / NH; ‘ / [541} Conjugate 8b was prepared and terized as described in Example 1 except that )GMT» 1 535 was used instead of KEVIN-1519 The details of the an‘tibmiyedrug conjugates Sip-1, 823—2 and C16 b~3 are given below Exampfle 11:: Synthesis ei‘l’aiivimmab Csmjugate 8c / 9 O \ . . II :1 R i g fl .»__E;}. \/’\,3/\\x VmoO \\ Paisvmumab—FSY "NAT“ 2/ g 1v : \ I : .—. .SAW" x 0 o , o. r"~g”\/c‘\/’j (3 OH 9H \ \ ‘mfix L: F \ 0/ CV/\O/A\/C)\’)\F'”MW~/:I\N/\v/ "-\“,'3H \\ HN‘ ‘3 ,4:\ H 5H CH \ I V’ NH L \ .3 9H \‘ Ki Ho ”KL-OH \ O 1 o>¢s I NHz i .1- I) N; I \\ \--N' / / \ x / x / MI HAN/\OV .. k ll "Elk/”w . \ _ ,’ 0 ’U 3“ I L ly“ "‘2‘: / /J {x / l N \ - ‘ / N=:( ‘0 / \ Nd; / G16 WO 02984 {542} Conjugates St was prepared and characterized as described in Example 1 except that Pahvizurnab was used instead efXMT—1519. The details of the antihedy—drug conjugates Sc—‘i and Sc—Z are given beiew.

/ C: H |-' L h if i \\ Paiivizumab migGEa—f—5\(KN/\W,ngx ”(NF/MI} :YWKV *****AVAfi/x/ON/VI \\ R '\ “‘<\ 3 O ./\o"\/O‘v’J 0 OH OH \ \ O : Af I : \\ c’ (.LN/\O/\._/OV/\ri\lc ’JKN,»\E‘}\E/;\/QH \‘ HN in H I, c x, x OH OH. \ NH \ I OH »§ \ .0 :0” \ H HO ’Kfloya HO \ e N 1” II \"N, =_ I / \ g Y” / >7]‘0 HW§O ”9/“ Mel" N45 (3de R 3~ \K /I \N/S / \ / a: t / d \ ' MHZ 3 [5-43] Conjugates 8d was prepared and characterized as bed in e 1 except, that Paiivizumab niigG‘Za was used instead of X94115 i 9 The details of the antibody—drug conjugates 8d-1, 8d—2 and 8d-3 are given below.

Exampie 1e: Synthesis 0f Xi‘vi'i‘4535 higG’imtIgGZa Cenjugate 8e, BAR 7.9 II \ H \ hEgG1 -mEgGZa—J—FV\1NYNE:L§*\FNVJxH/Nr/INVJ-NVACK‘M-Ot/Tj) j‘ «.130 («NO/‘x/‘J‘V C) 01' H g! \ 9:: VON/\OAVOV/fif/Nr/JLQIS 'kI/K/OHr o’ mi OH k \111 \\ K, ‘ N 115’ §"\,m . ‘1 ‘1 NH 1 LOW ~ *1” 1’ Q /J~\ / / \. NH2 ' ‘31 {544} ate 813 was prepared and characterized as described in Exampie 1 except that XM'IL 1535 a was used instead of XMJ 1519 ”Ihe ptuified Conjugate Re bad a(”ENG aoomst to KNIT-153 5 hIgGl mflgGZa ratio 0f 7.0.

Exampie 1f: Synthesis ei‘XMT—ESSS AAG Cenjugate 8f, BAR 7.4 “g H CH: D 1' 1 1+ XMT~1535 AAG-{-S-( 'J‘Vgirl/“V .4\/\o’\rn~/\I \ NANY/NY EAR” \ 0 \ ”‘6 1;) N\ ("1%‘VO\/ L 1H 011 \ \ \ 3'\/’\O/\¢O\,/\ ,—N\r;0\V/'\EAT -\§/\/Orl, V? \‘ H\, F o A on Or! 1' 0 \ F11 M \ x? 3‘4; 914 \‘ ‘\j HQ 4 X014. \' Q 1 7. o.

\NH ’ :i-.,( #0 i \1112 O- I k 2 \ '11.: I 'N ,4 / ‘1 ) Y / hf” 4» ' N, 1 H? NO 3: «V \r“ §NN , .0 / “t“ I L r,‘ . *g ‘1 1 / v \ L4? , ‘V L / ,-~‘o / - 11112 I 0'1“ {545} Conjugate 8f was prepared and characterized as described 111 Exampie 1 except that XM'I'— 1535 AAG was used instead of Rik/[111519. The purified Conjugate 8f had a STENG t to KNIT—1535 AAG ratio of 7.4. e 1g: Synthesis of Target [3 migGZa Conjugate 8g DAR 794 / Q O 0 L: r't: P : ‘3: H \\ ‘ : , . target 0 mégGZa "vexemrxfldm"*%%“v” __“verve/Ne \ \\ \”*§P ('3 \I O 0 FAG/\XK/ U wt an \ J H \ \4, 3v’\n/\vo\/‘\\r’-M x/kNr ”x; C” “3'1 T: H ‘\ OH OH 0” ' ' I NH \\ \§ . Oh 9H \ ‘\; 10 ’\/‘()H \ HOV 1 ..... ( f .MHZ (é/L‘O I] \ /‘ \ aMK I AC; “v / N Hit—”1% ‘1 kame‘f’fi‘w L 0?: /, /,. \‘ P‘J \ ~\// MNfl 11—, “ I d' , [546} Conjugate 8g was prepared and characterized as described in e 1 except that Target D mlgGZa was used instead of $431519. The purified Conjugate 8g had a STING agenis‘t to Target D_m1gG2a ratio of 7.4.

Exampie 131: sis of Target C higGla Cenjugate Sh? BAR 6.9 / 0 O H H ? O ' H l . ' . -»_i- Target C higG1a'f‘SfixN/V'Ngka’R'IV‘Ji‘fi'afi’N‘v’L -4Vm0/\,~0\AG \ \ \‘“ "\ ‘5 —\ . '3 O \Q /“ WNO‘V'JL 'N. CH L3H \ i : H E \ 0,4 ‘3\/‘\rf’\\/o\//\ N fix/LkN/‘ j\r/I‘x/OH: \ ‘ HF; T/ l H T 5 > \_ 0/ 3+ OH ..... ( \ I .‘f‘TO I NHZ R . c .3 \.—N’,/.§/N: I i ( ,’ (5’; ~\\~ /‘ V F/‘L‘ Fifi/£0 I Reva/em“; / L I ,1"h~21 1/ NW x / N l/ {547} Conjugate SE} was prepared and characterized as described in Exampie 1 except that Target C TfigGla was used instead of XMT—1519. The purified ate 8g had a STING agonrst to Target C higGla ratio of 6.9.

Exampie 1i: sis ofTarget E migGZa Cenj agate 811, BAR 1013 'll \ 0 O 0 H H H Li H \\ Target E migfiza—LRaw/\fNYJLfirxj/Nvim/WWMum/sow» \ K ‘--\’\O 33 '\ '3 O (“o/"\r'ov‘l \‘ 0 OH QH \ 5/1 0Vr\()/\/O\,/\\‘/N~ ,JKwa »‘-‘H \\ HN Er TN H E x: u OH uh \ of; x I\ M.- OH \\ 9 Efls’ :70" \ \r HO flavor I STDu I NHZ I or. a } Mfi/ I 1‘ (I \_!\‘I ’1 I ;~’ \\‘~O, Y I 1Vffi/ qur§m q IWKNVQ/NN/§N L LI?,N;» [[1 \\ _;§ 11* \\."’ \< u0 , ‘t 5 [548} Conjugate 8% was prepared and characterized as described in Example 1 except that Target E inlgGZa was used instead of XTVT'ILTSTQ. The purified Conjugate 8g had a S'HNG agonist to Target E mlgGZa ratio of 19.0.

Exampie lj: Synthesis at"l‘rastuzumab AAG Conjugate Sj, BEAR 7.0 / O O E... \\ Trastuzumab AAG—LSY‘A:‘AYRAE!\Evfl~:E/\ENJ'NVAO’N’L’VAO \\ E ,5 \ U ”M WQVJ \ /\om\/‘3\/‘0’ \?« OH. 9%, \ v r ”WkN/N Xxx/5"“ \H m1 \5/ : H T 3, \ 1K“ (J h¢\ OH OH d \ \ g: OH ”H \\ HQF 28;\»OH \ .1 HO A? 1 Mn).. , 0 Org” 3 ’N:I/ L ( \er ,J . i / I \ ,2 7’ ~»‘ I ~\ WIN/KO y M\/\/’\N’J\‘t~r 0 Km L "I / #r‘ O-(j I w / v / IN:K ZHN/EO__ / d [5-49] Conjugate 83 was prepared and characterized as described in Example 1 except that Tras‘ruzumab s used mstead of YMT— 15} 9 [55G] The details 0f Cortiugetes 83 and 8j~1 are given below Exampte 1k: Synthesis efTrastuzumab migfl 2a Conjugate 8k, BAR 8.1 /O E \ zumabmEgGZaA-se‘il‘x wk;\Ei/E'LvAE/k}----- \ o«(Y \ \4 "Vo‘r’l (”‘0 H 9 OH 9H ' \ O \ “Lt-, V’\. ”Va A ”oN‘V m" “CH 0 r a Y r ' \ 0 nH w \ L 1‘ .NH M \ o Err 9* \\ Hr: ’\.—Cr \ 1 HO \KH 5 we \ [I OL‘ N\/ ‘1“. (" I \—.N: N) I $1 \9’ I “ ’2’ ’ ” / f“ HN'AK‘O ’, d / {551} Conjugate 811 was ed and characterized as described in Example 1 except that 'I‘rastuzumah n11gGZa was used instead of Xh/1T-1519. The purified Conjugate 81;; had a S’ETING agenrst t0 'I'rastuzumab mIgGZa ratio of 8 1 e 11: Synthesis 0f Xl‘vi'i‘4535 Conjugate 8L DAR 497 / 0 i H """ \/V'ND XMT~1535------s\/\:/~\r/NH\_)LNAx” N\,/ORE/vi!»V/i[2:401 . O (”W/\fgvj (3H \ QM 04 ONAO’N‘OV/ ,/Nwli AV,OH\\\ 2 d >4; 9H0H 0H ‘3A\NH \\ / OH \ \ HO ”\VOH 1 : 1 fee I NH: Q‘ 0‘ N I; L ( \«N x, ,’ [552} Cdnjugate 81 was prepared and characterized as described in Example 1 except that IKE/1T- 1535 was used instead 01' X\1T- 1519 and 2(2—(2(2aminoethe-xv )e‘dro‘wkthexwautrc acid was ineerperated into the eempeuhd strueture instead df (3-(2-(2—amiheethoxy)ethexy}pmpaney1)—1_,- e The purified Cenjugate 81 had a STING agonist to Xi‘V/{T']535 ratio 017417.

Exampie 1m: Synthesis afi’aiivizmnab Cenjugate 8m, BAR 4.5 PaiivizuanabTSN/JNINm/gfif fi’fi/NvS3 E: H E H 9 H . xOVNQ \ \ “fir ! \ ‘ a a \ OH OH \ fig MO/' H- i \\ \ / N fl\/'(J: ./\ \ c'v’\ NQV0 r 0 NH Y 1 t: t)Y;\/GH r a OH OH ‘3 \ \ r“ ‘ 0\ (kg, 9H \‘ \0 HO \aOH ‘ : 1 HH. C5 I OLJ \ N§{ I, La ‘Q/ \«N , '7' ‘ \ V) / F0") ’ / 17’ HH’§ / N ‘ / \7/N \,/\//\N'/(\\N /’ OVNH N, / L ,,I f) \\ ,x N \ / N:& / *0 {515 [553} Conjugate 8m was prepared and characterized as described in Example 1 except that Paiivrzumab was used instead 9f m/I'Tn1519 and 2~(2'3mrnoethoxy)ethoxy)ethoxy)aeetie acid was incorporated into the eempourtd structure instead of (3—(2—(2— amrnoethoxy)ethoxyhnrepanoy})—L~aianirte The purified Conjugate 8m}ad a, STING agenist to Paiivizumab ratio of 4‘ 5.

Exampie 2: Synthesis of KNIT-1519 Conjugate 16,,EEAR 5.3 o PartA Part B 9 Part C 9 kg , 9: h wv. ovum BvdiN-aAOA. RAJEH »»»»»»»»»»»»»»»»» accruu\,/\o/\/‘Lg,\,os aoL>4u\/\O,«\,b\N'/\/0\T(o s 10 «.1 12 “(It ,9 9‘4” mm Par”) °‘ Part1: 4 \1/ PartE ---------------- 5M“CAD.r\,/’kr‘/\/0Y.o/\-«Ao \ H :3 #th fix “ J m 13 14 1 / N' 1’ a x , 1 $355,?“ XMT—1519tfl5r‘o o [554} Part A: To a solution of Compound 9 (04100 g, 0.429 nimoi) in THE? (5 mL) were added HATU (0.196 g, 0.514 mrno1), and 11031: (0.079 g, 0.514 mmo1), the reaction mixture stirred at 0 °C ”or 10 min, then 2*(henzy1oxy)ethanamine (0,0648 mg, 0.429 ) and DIPEA (0.112 rnL, 0.643 rnrnoi) were added. The reaction mixture was stirred at roorn ature overnight, the solution was concentrated, and the residue was purified on siiica gei (040% MeOH in DCM) to afford nd 10 (0,2 g, 100% yield). ESLMS lit/1: Ca1cd for: {1191130N2{)5Na [(MtNaj"): caic. 389.2; found 3892.

Part B: A solution of Compound 10 (150 mg, 0.409 mmol} in EtOH ('10 mL) was degassed with N2 before PdnC (43.6 mg, 0.409 mmoi} was added. The mixture was then degassed with Hz.

The reaction mixture stirred at room temperature under Hz, (1 atm) overnight. The soiid was fiitered off through a pad of , and the fiEtrate was concentrated to afford Compound 11. Crude Compound 11 was used in the next step without further purification. EST—MS mzfz Caicd for : CnstNstNa ‘Naf'l caic. 299.2; found 2992.

Part C: A 100 mL flask containing the residue of Compound 11 (128 mg, 0.463 mmol) and methyipyridinn4namine (11.32 mg, 0.093 mmoi) was flushed with argon, then triethyiamine (129 iii, 0.926 mnioi), acetonitrde (1.544 hi) and EMF (0.772 rnL) were added.

The reaction mixture was corded to 0 CC and stirred for 5 min followed by the addition of 4" henyl carbonochloridate (140 mg, 0.695 mmol), and the resulting mixture was stirred at 20 0C for 2 h, then. concentrated to an oil. The residue was purified over silica gel (040092; ethyl acetate in hexane) to afford nd 12 (50 mg, 24%; yield) as a white solid. E81—MS m/Z Calcd for: Ci4Hi9N307 {(M—Boc-l-HT): calc. 3422; found 342.1. [557} Part D: To a solution of Compound 12 (50 mg, 0.113 mmol} and Compound 1 (36.9 mg, 0.047 mmol} was added DMAP (1.153 mg, 9.44 umol) in DMF (500 uL). ’17he reaction e was heated at 80 0C for 3 h, then Compound 12 t 50 mg, 0.113 rnrnol) was added. The reaction mixture was stirred for r 3 h and then cooled to room temperature. The mixture was concentrated, and the residue was purified over silica gel (030% MeOH in DCM) to afford Compound 13 (20 mg, 39% yield) as a white solid. ESLMS m/e Calcd for: CSJHMNMOB [ennui eale. 1083.4; found 1083.5.

Part E: To a solution of nd 13 (20 mg, 0.047 mmol) in DCM (0.5 1111;) was added TEA (0.1 mL). The reaction mixture d at room temperature for 4 h then concentrated to afford Compound 14 (10 mg, 55% yield) as a solid. ESLMS m/e Calcd for C46H59Ni40u [M+H]"": calc. 9834; found 983.5, [559} Part P: To a. solution of Scaffold 6 (12-115 mg, 10. .17 umol) and Compound 14 (10 mg, l0. ,1? umol) in BRIEF (1 mL) was added HATU (464 mg, 0.012 mmol), HOAt (1881 mg, 0012 mmol) and DIPEA (0.018 mL, 0 .102 mmol). The reaction mixture stirred at room temperature 2 11 then concentrated and the residue was purified on preparative RP HPIC (030% ACN in water) to afford Scaffold 15 (20 mg, 83% yield). ESLMS ”1/1: Calcd for: CioiHiszNzaiOu [wit/211121): calc. 1178.7; found 1178.590. [560} Part G: A solution of XIV/{$1519 {10 mg, 0.069 urnol) was conjugated with Scaffold 15 ((0.823 mg, 0.347 umol in 200 uL Elk/1A) as described in Example 1. The purified Conjugate 16 had a STING agonist to XMT—1519 ratio 0175.3.

Exampie 3: Synthesis stuzumab ate 26 I o l '3 n \ , ‘9 ’Hi 0 9f F\)—/<O NV’K'JLNH‘. \T:\: \ 5 \i wk, N ;’¥‘§“z;“*rf\ ’LNH 9‘5 H‘Nur '\' “”2 N‘N HN—é’ E! HN \: Ya PartA j /\ ,NN/ Parts (x ! “5%” >>>>>>>>>>>>>>g»: o O\ »»»»»»»»»»»»»»»xx» P HHN ~/~~. ‘ 3% ,4 m; E ’ HCL Hz’N \:[RIMA VV"\I 4 . : i > : x‘O’kp’Lr/NVJLV/\v’\o i: >—\:}4 m/ (.- . ” E H : i~ I H N , \ >- {>6NN7: a 7 {N N . ,\,_NH an f‘x, i Hg»: 1:1;‘SWH N" </ V V ’>_“</‘\:'l\iX '/ ,J I: \1/“\\/ 2 1,5 N : i O N" o o N" 133 c.

’J "j 17 18 (kN’fiyiNYkfi/WH Q H 9 \2L 1-- C.‘ H Part C ' : NV '_ E’Nn’ \\_/J: L.“VA.O/\\/O\/\O I Part D \ ' _<<<<< * : ‘ 0 0 \‘0 ""\/U\/ 9 "3H 9H H _______________”b NH OveonvaCKJ/xwrfl NwANAK/ \/\~’01-!: H F“ 0 0645‘ or- 0-4 O:< NH . 0H ”H A)” 9h F0 ’ HO EIXOH , HO w" MN> ,amg;1 3:1 ( \’ WK .»-« > “#0 <’ 3w"' 1 \N e“ 5L \NV/‘K/ \ _/ \ ...

H co»3 kg i we (”Ivar HZN Q H G" Trastuzumab..sx(kv~\I/NVJxN/YNVMKN,H P H C.‘ ,NV/LNwO/‘x/R/wH i = : s H H \ U 0 \O :AGN’UVJ O OH OH NH Ox/ronVfix/‘jra-I‘kfx‘vi'zLN/xX/K/VOH F“ 0 (JAN OH OH °=< W 0H NH 3’ r ?H ”mg-=0 HO }’\,0H HN NA JNHZ 2 \/N\’\‘/) " \flfl > 1194):?) NN/‘KJ/\ N‘- ‘\.xI"! . Yxi I-=\ . Mao» OWNH , Rf E >=o tf‘hlmx HZN' [561} Part A: A solutinn 0f Bec—ala—aiauOH (67 mg, 256 tti‘fioi), CD}. (:70 mg, 435 enrol) and DMF (2 mL) was stirred at ream temperature for 23 h. Then Compound la (prepared as described in 175147A1, 100 mg, 128 timni) and DEPEA (6'7 aL, 384 pmol) were added and the reaction stirred at mom temperature for 23 h. The reaction mixture was concentrated, and the residue was chroinatographed by silica gei (0—2092; CM eiuent). The product, Compound 17 was isolated as a yeliow foam, (109 mg, 83% yield). ESE-MS In/fzi Caicd for (haHmNisOio {Mt-1:1] 1922.5; found 1022. '.

Part B: A mixture ofCoinpound 17 (108 mg, 106 umoi) and 2M, HCi—dioxane (6 mL) was stirred at room temperature for 2 h. The reaction mixture was trated, and the residue was dried under high vacuum to afford Compound 18 is an off-white foam (103 mg, quant.) ESE—MS m/Z Calcd for CuflaiNisOs 1]: 922.4, found 922.4.

Part C: A mixture of nd 18 (80 mg, 84 urnoi). Scaffoid 6 (117' mg, 84 urnol).

HOAt (12mg, 84 umoi), Dil’EA (5:9 uL, 336 umoi) and DEAF (3 mL) was stirred at room temperature for 5 mins, then HA'I‘U (42 mg, 109 umoi) was added and the reaction mixture was stirred at room temperature for 20 h. The on mixture was then concentrated and chromatographed by reverse phase (10'1009/{3 ACN~water w/ 0.1% HCOOH eiuent). Scaffoid 19 was isolated as a white, fluffy solid, (35 mg, 18% yield). ESLMS m/Z Caled for C99H149N25038 [M+2H12+: calc, 1148.0; found 1148.4.

Part B: Trastuzumab (10 mg. 0.067 umo1) was conjugated with Scat‘fo1d 19 (1.237 mg, 0.539 unto} in 200 pl, DMiA) as described in Exampie 1 fo1iowed by purification using CHT type 11 chromatography to give Conjugate 20 The detai1s of the antibody~drug conjugates 20~1 and 20— 2 are given beiow Exampie 311: Synthesis vizumab (Iqmjugate 292:, BAR 5 5 " H I Q. WE\xkKEN”\JL—N\A0,V0\/\ls \ Paiivizumab—‘SK*:/\E/N_[R {A0/\/0V 0 OH OH \ H : g 1‘ 0% H\/\Q»/‘\/V’\ er REV/\f/LE/«x/OB \\ \ NH {1 H 4M 0 OH OH \ 0" \ 0,“ I OH 1 NH 9H H «:0 "R I 119 5/"me ’ HO MN) Neg./ [N112 \JN"' <3 _ I; ~o_ I / -‘\\\.Q’ Hi / " ..\/‘N”“N / 141x V“ H / H3130 ’/ (gym-1 V /’ ’FO / d fw-”\ £1 \ . r"'N 20a {565} Conjugate 2931 was prepared and characterized as described 111 Example 1 except that Pahvizumab was used 1nstead cf XMIE 1 519 Ihe pu11f1ed Conjugate 293 had aWENG aoc111st to Pai1v1zumab 13110 of 5.5. ie 4: Synthesis 01"l‘mstuzumab Conjugate 25, DAR 6.6 N"{ O ,/ I Q j O ':I":~ ”4% ,0 LL N \ «20 a "\ NHz [Lii Nxfi' / I1 \\_ Mi;. i N A». ’11 A0] HN__<, w HNfl /\ Infirm-éjfij IK.¢N\,F(J ‘N '1’” Pattie», x/ a Part B ................... , p_ g e I I ”DI/"v"? ~ xofl/N‘AO K CIA/x0 a 2‘ N" Wi 5 (‘7 ‘1 A» .71 ‘1 2' HyNT/L‘N’jtkfipg'fif‘il a 2HN\£{&JI§[{>"NH 255’“ ZHN‘ Rim/FM 5M é o ‘O’K ,5 o o’l\ I}: ,3 ,‘\ 21 22 23 Part C <2 0 6‘11" /N\-/E\EA.TNx/E-kEAENI{ix/Clifipi\/\-f’VCV\0 Part D __________________a» Q "\L - N\_;\ r/NOO/‘vc’xxj >>>>>>>>>>>>>>>>>>it» OH CH 04 M1 (62‘va ”VA12’xv’I\/\n” OH/R/EYN’OHN O OH O ,0 Mk4 0" 0/ ‘\ hi: / a“ a OH ' " “~\‘ ./ 3&3“?~ \ '_v 2 ; 29w J Hdr '§’\r—OH '2‘ 2:“! f/ o‘ NH2 HQ 0 \\,{)\Nr" ”'4ka o=”'\F,J\'k 24 / 2, ,i \ XMT—1519T°\25\W/x/'I\i\/H\OL‘-’\1(Ev:qg/‘TI‘VJ“«“N\/\.Oto\,—UVANOi. H 3: H .oN‘WCK‘ (”‘O’V0“) v3 3H CH \\ \ :éx‘Ni—l ‘2?ng CW/\/fl\/fi/k%vlex/kOH 1 2’ H : a"\ OH OH i \401 ”N 5' u NH 3 j 4/». 1:)‘\ m - PH ~ » e ,i \ 2, I\ (”i wa h \\ (33‘fib‘z 1S x I ~~f® he x OHr ZEN In. I Aft \ , . 3‘. ’ , NAN, / \ I, halt» ) 3 ,I (FAN : (:15 ‘1”: 25 [566} Part A: To a mixture of Compeuud 21 (prepared as described in US 62/982,935, 38 mg, 0.047 mined) and tertnbutyi (S)n(2~hydroxypropyhcarbamate (9.85 mg, 0.056 mmoi) in EMF (2. mL) were added 3n((ethyIiinino)niethyIene)amino}N,N~dimethyipropan—1"amine hloride (13.48 mg, 0.071) nirnoi) HOBt (10/"’"7mg O 070 mmo1) DIPEA (0 016 mI mm01) and DMAP (5173 mg, 0.047 inmoI). The suspension was then stirred for 2 days at room temperature, The mixture was concentrated to afford a residue which was their purified on si1ioa gei 2) VieOHin DCM) to give Compound 22 as EightVeIIow so1id (20 mg, 4141/6 . ESI—MS m/z Ca1cd for C47H58N. .012 {NHH} oaiei 968,4; found 968.3.

Part B: To a suspension of Compound 22 (‘20 mg, 0.021 minoi) in dioxane (4 mL) was added 4- N HC1 (O, 52 mL, 8219 minoI). The reaction mixture was stirred at room temperature for 5 h, concentrated and used Without purification in the next step. The product Compound 23 (17 mg, 95% yield) was a white solid. ESi—MS m/z Caied for C421-isuNnOm [Mt-H}? 868.3; found 868.4. [568} Part C: To a solution of Compound 23 (17 mg. 0020 rnmoi) and Scaffold 6 (32.1 mg, 0.023 mrnol) in DIVE (2 mL) were added PyBOP (15.3 mg, 0.03 mrnol), and DiPEA (0.034 rnL, 0.196 mrnol). The reaetion mixture stirred at room temperature 2 h, concentrated to afford a residue which was then purified on preparative RE? HPLC (0-8094) ACN in water) to afford Scaffold 24 (26 mg, 59% . ESE—MS mzfz Calcd for C97i-ii43N2iO4ri {IVE-FZEIW: 1120.98; found 1121.06.

Part D: EQ‘VITnl 519 antibody (5 mg, 0.0347 umoi) was ated with Scaffold '24 (0.622 mg, 0.278 urnoi) As bed in Example 1. The crude reaction e was purified by CHT column chromatography to give Conjugate 25 (319 mg, 64% yield). Purified Conjugate 2-5 had a STING agonist to XM'1‘~1519 ratio of 6,6. ’7 ’7 O Exampie 5: sis 0f KNEE-1519 (ianjugate 28, BAR 6.0 c3 ,_ TE: [H(O 0 O ' H ‘s’ paflA 9 H N\ r’\\¢r’)LNH5 A ”NV. 9 «(rm-«II, —»~\,~\ranfi’’” N’\.fi, [KN/mi,“M\/”_N\/\\JWOH " J N \f 0 ‘\ \O’\“O\/ 0 0'4 0'4 ,~ ’ 0 K H ; v. \fif O\ 0/ O\/\0/\VOVv/\T/N L\N,I~\E/~\E,J~\,OH A: HN I H _/\/\ \ 0 iv Or!. CIH. “" ‘i E ° r“ or .¢ l ‘0 I I _oH t? . oxfi/ 2'4)»; \/\ :\ 3,4’ \’ N OKJ‘NH ”:40 “0'10;\A3H CI! 0 ‘/I \k‘fio h/PKFO 3N H4 23 ‘\ r-"i H231 I I’NY’W A ° J- x WY" 0 \T‘xY if” / 41,: I w? [—4.3 \ngéx.

/ S? H, i O H i H \ I“\.I”\5/\\/0\,/‘o \ P Fig KNIT-1513'st \M/\T,»N\/ E*’\~n/"J\ §"\FIN\/ a ' 1 a .

KO g 0'‘ 0 (.2 r’\O«’\/0V 0: Sm 9.1 \ \\ 0/ Ox/‘0/\/0“If”: V'L\. v\J’\x°” \ HN E. \ \ OH CIH \ LNG OH 1 I OH \/\ a 5.x oxiw IN“; \ g H, \,0d O 1»ng 3 “wk-Y" _/ 1,;0 j I3 H’\| \\\ \IISN; /l m 2; (“j/j ’ 0 “(Y I? f o / gr. ,I‘ \ NH? / a” ' / “1:4 / [NH / (I 5 may/”0 \r’k 4I\ [57G] Part A: €011inqu 26 was prepared as described in Example. 1 except that 26 red as descnbed1r: US 0,198”;93 5) was used instead «if (_.<31r1pound I (ornpmmd 27 was obtained as a colariess su-lid (7130 mg,40% vreiId) ESIMS m/z (2110i for 1ra]\220 [IX/IIIZIIIZI: 1 193.52; 1°0qu 1193448 Part B: C(mjugate 28 was prepared as described in Exan'rple I to afford Cmfiuga‘te: 28. The purified Cdnjugate 28 had a STING against to Xh/I’Ill 519 ratio of 6.0.

Example 6: Synthesis of KNEE-1519 (ionjngate 29, BAR 5.5 1’ -'\ o e o 3? °C KNIT-15197L$Vfl\N/\YiNY*¥/\VVNNVA?{A\WIN'VJ 5555N\/’\Q/\~/O\z/\QP H : h; \ :5 H 1 H Conjugate 28 .............au- \ g F. ‘. r . : \\ {‘0‘};t H e \. 0 (.3 I/xo, \,0\/ 0 OH 9H H \\ \ i‘x-t o’rai 0\/’\¢r"\/G\i ”\T/N» INANATjH/KV’OR\ H‘s? b a1 H on on \ , l K 'M-l I OH’ \ D )\ 3/ OH | x_/\ ~ ’ {3 0 \J‘r‘lH “xi? hr.) 8 “two” 3V0 / <0 'r ‘ )\\ \rshl [I ‘\. hi I he a f ~/ \\ r, / ofifi‘ j? )fi/TH /,I \{AN '3 “ szk / 09's 1 “'5 \/”\N4>\ [572} Conjugate 2.8 (6.5 mg) was formulated into PBS, pH 8 with 3 cycles of concentration and on using a 3G {cilia M‘WCO ultrafiltration unit. The reformulated conjugate was then incubated at 37 0C for 24 h and then reformulated to trehalose buffer pH 5.5. Ring—opening was confirmed by LCMS analysis of heavy and light chain following antibody reduction. Good resolution was observed hetween various light chain species: unconjugated, ated with intact succininiide and conjugated with ring—opened imide. pening could also be observed in the corresponding heavy chain species but resolution between the various species was poor. The degree of ring—opening was therefore estimated by focusing on the light chain species. Using this approach, the percentage of ring—opened t in Conjugate 29 ve to intact snccinimide was estimated as 94%. Conjugate 29 had a STING agonist to XMT—l St 9 ratio of 5.5.

Example ’7: Synthesis of KNEE-1519 (ionjngate 32—1, BAR 6.5 :1 9 ii at“ \i /HHN"<.1=,N (N ‘ ””2 ’N‘V "'N\’\o/‘V’G‘~’\o "t |v" N/YI‘JV , “mew \: ‘fl’ : H HN”‘F’N‘~/ ) IN "N W o / O ll 0 / r_ ‘3- rr r” XM'l'—1519~s , y Parts { Q jf g -----------as» \ o \ 04 [5’73] Part A: Scaffold 31 was prepared as described in Example l except that Compound 3% (prepared as described in US 335) was used instead of Compound 1. Scaffold 31 was obtained as a colorless solid (9 mg, 8% yield). ESLMS m/c Calcd for C101H151N23042 [NHZHE‘Y 2; found 117942l.

Part B: Conjugates 32—39 32-2, 32—39 and 32—4 were prepared as bed in Example l to afford the title conjugate The purified Conjugate 32—1, 32—2, 32—3, 32—4, and 32-5 had a STING agonist to )0»le 519 ratio as described in the table below. Conjugate 32-5 had a inAb concentration of >10 mg/rriL, contained <1% of urtconjugated rat/lilo, and <Zl‘?/’r's high molecular weight species.

XMTu1535T-s. AWN/NY 2:: :rN\/ HAW/N" NVA>o-/\/0\/\? \ I c3 ‘ ~ 9 0 o \. /\ ""V \v/ \ “o 0 ,= .i a \ 3 \ 04,4: a“ 0\_/'\O/\v»0\/‘\fl/N\/V NAT/ \ §/'\/0“ ‘\ ”N H \L :3 can CH \ 0,p'\ NH \ I OH r \ e\/\ ‘f l OH oNvakWH- 3‘?.~/‘~ HO3,43 \—OH 1 o HO \‘5 fiji—‘xfiO 0 HM J 1 Hr, I \ /N\2\\ I a j m. o / ’ r; N~§ ‘4’ / G k/\ J N42 I N / \_‘I / N" I [NH 2’ $1 / \/;"’Q ’ C115 \/«NI;‘*\ [575} Conjugates 323,, 3223—1, 32am; 323—3, and 3234 were prepared and characterized as described in Example 1 except that HGT—1:335 was used instead of KEV/{174519. The purified Conjugates 32a, 3234., 323%, 3236, and 3234 had a STING t to XM’T—HSZKS ratio as described in the table below.

Exampie 7b: Synthesis of Palivizumah Conjugate 321), DAR 6.8 / o . a o- o I .' ‘r/Lfi/WNVJKfi/W’N'V/H i H : H 3......“ \ Paiivizumab—fs\( NVAQ/‘VO‘VA‘O \“$3 : 1 2 ' \, Q.' Q \ 0 *0”\’°\/J 9 0H 9H \ \ - . H : : \ \\ 0g \ ‘ MN o\/\0’/\”G\/\IKNY’\’ MAX/Yves“ {3 H I 0H 0H “’”a; W \ 0 \ 0t A S» p on .

V\ .= o \J‘WH—‘ I/ ~ , as ’9 HQ > \,voH 1 ‘L‘f’o “Jyfiy 0 H0 0 MN . w ‘5 r“ ”t .3 , 0% (5/ ”V T / \\ / I” N‘iQ “I, II I: I NL‘K / NH // afiq //d c VA'VJ)“ U 325') [576} Conjugates 32!), SZh—i, and 2 were prepared and characterized as described in Example 1 except that Pahvizumah was used instead ofXMTJ 51 9. The purified Cenjuga‘tes 32b, 32b~1, and SZh—Z had a, STING agonist to Palivizumab ratio as described in the table below ”I"? 5 He 79: S3’nthesis ei’i’afiivizumab mlgGZa Con’u ate 32 BAR 9.1 . E 2?; I' a ,I 9 H C.) H j: H Vi P. \\ Paiivizumab mEgGZa-J-SNJKN fi/NV/ ‘N‘ \TNN --”?‘c\/\U»\ ,Oxv/xo \ g \”‘§\ . : :-E N’ATNH (3 \. 0 O rAe/\/ 0V] 0 OH OH \ c3 . : H g \\ 0’}! OvaQ/N-OWT’,¢ Ne ffixw » ,«x/OH Wk» ' H , 0H 0H , of \\ k INH OH \ “’w E C \V, 1 3/3 1 ‘NH ’4' 9 m \»0H E 1 9 F E ___/ '10 . ‘ \f‘ u \‘ Hh'k I \\ 2.3% ii H N \ r'NxT/ \ 2 I L we j ~. \ o , 04, \(f\\ rfx fem Y I] LL ,/\ ,,J Ming 1” \ ,__., / N—x , [NH / ofik / 770 ’/ d1b \VAN/)\‘ [577} Conjugate 32$. was prepared and Characterized as described above in Example 1 except that Palivimmab migGZa, was used instead of XMTJSEQ. The purified Cenjugate 32$. had a STING agenist to Palivizun'lab ratio of 9,}.

Examgfle 7d: Synthesis- oi' XMT—iSSS mEgGZa Conjugate 32d, DAR 8.8 9 n I: a E :3. 9L H \ XMT..1535m§gQg3T‘\ KEV/‘f \E )N/W. \/H xN,—\q,. ~v/ "N\/\‘O’\V’O\/\C \ , ~\ i") H \ § 0 0 A ,A\,O J e \v 0 cm. \ c r Qh.

H. : \ \\ \ 5L \/ N” "x/OH ”N Qr’\\/‘J\/\q/'N~,. ' \ l an an ,, \ M- \ ‘ 0H 1 V» i A S" OHg: = x ‘NH N 9 ”1.3 x, 0H L‘fp fi'o H0 0 HM r ‘L 3:94 I] ‘“ \\/ \N/ H2 ask / PIN II o— / I; ‘3 / 5115 \AN/ \ Conjugate 326 was prepared and characterized as described above in Example 1 except that XM'I‘—1535 mIgGZa was used instead of XM'I'—1519. The d Conjugate 392:! had a STING agonist to Eff—1535 migGZa rain) of 88 Exampfle 7e: sis ei‘XM,’i‘—1519 AAG Cenj agate 32c, BAR 7.4 / 0 hr a ‘1‘ 5 9 a XMTn‘E 513 AAG—FL‘SVKNAV/NYHfi/WNVJW' \ : gaviu-«NVAOAVOW? O O 1 § (5 —\ (“o/”VCK/ {3 OH \ [579} Cenjuga‘te 322:: was prepared and characterized as described in Example 1 except that XIVHZ 1519 AAG was used instead of KNEE—1519. The purified Conjugate 32c haci a STE NG agenist t0 XM'I'—1519 AAG ratio of 7.4. '?'?7 Example 7—1: Alternate synthesis 0f Cempeund 31 Jr, 0 3'. I j 0 1L:5 ’\j“ PEN- INI\,N /Lo>$:_<"‘IL/:ri1 rm Part A ‘“ \II ‘ Part B H1x I ”j (3’ K."K, 3031 110,,,er I311,'2».on i/ OH (1ij V“H 1‘ HO n’nJ a, ., ., ( o f \I i»\/’\Q’A\/L\'N’;:‘er\u’Si igko“ ‘ 1,4 0 9 ’°\/\ ‘ H ‘1':\--/ a 'H a ’\ N \z'k 0. 1-19 1-10 a? 0/9 1- 1111 1 7 . . o /“0 a; H'\ {/‘wN; AN /N1®.J\N/\n,NVJL MAE/NngA‘r’ Wc”\/ \\ 11 11 ( 0 “artCh ,‘fl .1 a. 1 ,, 0 j j H211~/‘o’\0V v\0 z —9~ G/ANA“ V"o’\a ‘Ne “A “1:. ,1 “ah! x 1 ~1° YU‘HH11 I l 11 .

N312 j F“ 30b “v" 1"}- 31 Oak E f fix: ,0 N", x, x?» L~ 5:112 ”tr-c MIN/1L [580} Part A: Compound 3% (prepared as described in US ,931 5) (500 mg, C1663 mmol, leq), Boc~PEGZ—Ala-OH (0.693 g 199 mmol, 3 eq), EDGHCl (38} mg, 1.99 mmeL 3 eq), and DIViAP (243 mg, 1.99 mmol) were mixed in a vial under air in DMF (26.5 mL). Reaction was complete in 3 hears. Mixture was querielied witli AGGH (0.76 mL, 10 eq), trated, and purified over silica gel (DCh/i:l‘vte0l:i) to afford Compound 36a as a white solid. ESf—MS m/Cz Calcd for CSJHSSNHOM [M-r-Hf: 1684.5; fmind l0844. [581} Part B: Compound 39a (0.663 mmol} was suspended in dioxaiie (10 mL) in a flask under air. HCl (4M in dioxaiie, 6 mL} was added, and the mixture was stirred at room temperature fer l hour. The mixture was concentrated to give a white solid. The solid was ved in pure water and purified by reverse phase chromatography (0259£1 ACN 111 wate1) to afford Compound 3M) (444 mg, 77%) as a white solid. ESl—MS m/z Caled for C46H53N13012 [M+H]+: 984.4; found 984.2.

Part C: To a solution of Scaffold 6 (451’) mg 191.32 nimoL prepared as described in $0181/067193) and. Compound 30!) (351’) mg, 0.072 mmol) in DIVE? ((3.5 niL) were added PyBOP (185 mg, 0.36 mmol) and triethylamine (0.23 mL, 1.62. mmol). The mixture was stirred at RT for 15 minutes and their ed with ACOH (023 mL.’.99 inniol) and purified bv reverse ’7 ’7 8 phase purification (040% ACN in water w," 0. l% acetic acid) to give Compound 31 (408 mg, 55% yieEd). ESI—MS m/CZ Ca1cd for Cmf—iiSiN23O42 [hi-+2113]? 1179.52; found ll79.27. e 8: Synthesis of Trastnzuniah Conjugate 349 DAR 6.9 ‘7 ‘1’- N,N 53 N fire/”km: f" .11 lay-’41:: \ \ N O Q )w E N,J\¢’ /L| / ‘1 iN /‘\”l"NH 1 ‘_ N HN’X’ ! 7 Ox N / K7] (3 Part A 9 ‘3 ’ \ 1 / N J\/\ NAV’ \. / xOA\/0\/\0/\\/o\/’\0/ \//L I,‘e/‘v‘ I \/ H2-" 0 »»»»»»»»»»»»»»»»»a» Rh a «N -4 1 N~i: " / ,/ N\. _ D \" b’wm‘k P affix NV”; ”1/ but. A“: "‘ /’ ’\ 0 0 ,3r" i 33 0 \I a O \\ N"N>»J< Net/“Vi”N““2. \ )‘I/ N4 \\ / ‘\ H g4” \ / KW 0“ 1 ,II a 0 E Part B ‘\ : ’ i RI/V’Ow ”\,/0\¢’\ /\ ,fL xx H \/\C,z 3 if; .4. “(v N o V v G N - I —%Trastuzumab .5 G \wg (N \ / \‘1 )u-N \\ t) \-""N %M /: u N I \ \‘ o / (‘3 I / dtt‘.

Part A: To a solution of Compound 1a (prepared as described in WOZOI? 75147Al, 0.030 g. 0.038 ninioi) in DMF (1.5 mL). was added N—ethyl—N—isopropylpropan—2—ainine (0.067 niL, 0.385 mniol). The so1ution was stirred for 5 minutes at room temperature prior to the addition of 2,5~dioxopyrrolidin~Lyl l~(2,S—dioxo—2,5~dihydro~iH—pyrrol—1—y1)—3,6.9,1 2.1 5, i 8~ hexaoxahenicosanfi1“oate (0,027 g, 0.050 inrno1) in DMF (0.5 mL) and the reaction n'iixture was stirred at room ature for 15 minutes. Then acetic acid (0.1 ml.) was added t‘ol1owed by purification on a preparative HPLC column (Cl 8, 21.2 mm x 100 min), 101 0013/5 MeCN (0.11343 HOAc’) in 1120 (011343 1:10Ac,20 inin. gradient to give Scaffold 33 (0.006 g. 13.05% yield). ESL MS m/Z Calcd for CS7H75N14015 [MH’HY calc. l 195.55; found 1 .

Part B: Trastuzuinab (10 mg, 0.069 iiinol) was conjugated to Scaffold 33 (0,658 mg, 0.550 urnol in DMA) as described in Example 1. The crude Conjugate 34 was purified by CHT type 11 chromatography The ed Conjugate 34 had a S'l‘ING agonist to 'l'rastuzumab ratio of 6.9.

Exampie 83: Synthesis ei‘ihiivizumab Cenjugate 3942a, BAR 7.0 , O \ » «MN MW fl“ H NA/ \ ,/ Q“ 0 a K, I ., 1! . . /“‘\ “Ax/3% New ""\/c"\v/’\‘()/\~/fi\N/‘\/’\C-3 \\i / i Paiwazumabf8~x : 0 0 H ;’ ’ rg_ ‘Nw \ I ’ \ O I“ I? I, ‘ N )rx/k I \ HEN 2%” ‘ ‘11 o / 34a “115 [585} Conjugate 34a was prepared and characterized as described in Example 8 except that Paiivizumab was used instead of Trastuzumab The purified Conjugate 34a had a STING t to Palivizumab ratio of 7.0.

Exainpie 9: Synthesis of KNEE-1519 (ionjngate 45, BAR 6.5 (>37/\0Gigd'\/JE‘cii <;T‘\) 2:33 .

HNYO (F11 0VE E" o “ 9 903)..“ ~ 0 00% H‘N JEx / \I A .. Ti) ‘Q’ELN’\r’EL\N \ PEIEC \ ZEL A EL\ -' \ ‘0 i V G \7 PadA HIAK A0,.r\ /»\\‘.I F‘al‘EB i _/ \l/ H i it _( {i 0, 2/ a I 2 \Ky ”””””””3“ a: / \f} —;9. “MT/x" Fa HM .o = “v; , t) ’I . /E<~o’§'ci’ \, 0 HO’E‘\0 3'5 — 36 V ‘ \ J1 I 3 “W, N‘\ /, t l N /~\ i / c H!\'»{ T \T N" N". x/ / ‘ c» 0 4L .NE \/ EEE\ E0 Pix/‘wkw ”if‘ifl \ )N‘. /‘0 HN<: : I Hm \ifile/ \ 1".0* (7‘1 .3’\ N”\T«4 \ F,( c, I EE¥ K ’ IT M‘ 0 \/E~ L 40 ”(\O 0 H \I o\ /'\0 i . ”N'/I (\ AVOTistr/H JEN/V‘s. N’ E] q“ #42 Part!) ’ H :\ a o C‘x —b~ L NE’. H \--:\. L MN HE: 44 ,1 5/ HZEEm/%”‘N/i i / \v-‘4E/"KN V- V A a ii v E 0 c? o \) / : '3 ‘ n c’,’ r ,//’\-e"‘ H E\ /‘E\ ' 41 L "N kj‘ /. rm or ' G v ‘N E\lr< /\L\ Mix 0 0 . 42 I o > c VE\\€ N; “"‘\’T"v‘NH. /E\oi\>“‘< N ER. «x : / E HNTAKEN!T . I I Hit/I‘M "‘ 0\ 9\ Part6 ~ PanF o ,1 Q E H \Ili Part H \I, ’3‘! ' —9~ LEW» rm, M" i' WW4” —— —;v> \5’ a \/‘~.0 r . '1" EE‘uAF’ \/ 1] :i.'\”‘oi \ \V'EV H 0 ,:vii ( k—ko (E 0 NT K H 9* NE T o H2!\'\n,/[‘*\\/L-N’ )Mi 1 - i N < . : /--N x” /~M v 0%? \r4/ k 0’ HM (m If)» N ,1 {Uik /€\ '5 d o 0 13$ 44 \ ‘\ i449 \ fl“; :ifi'u—J/Nfii/Vf.\ it“”7 \\ \M’E' //' \ [/0 TFA. I i \r (R II J wrists—£5{NW 1‘” {WV\/ {N ’\ ' L \, I i ‘\ / ,. “ z N I g. !!;N. f rm:. _/ 0 0H 1r \V’E‘ N 5,1“ 0,“.\ /' u /.l [586} Part A: To a mixture of Compound ‘35 (400mg, 1688 ttl’fiOi) and rebutyt acetate (8 mL) was added 70"A) Ht104 (302 mg, Mii mmol) The resulting solution was then d at room temperature for 21 n then neutralized with satd. NaHCOi solution. The aqueous phase was washed with EtOAo (2x} and the combined organics then washed with brine, dried (NazSO4), t‘iitered, and concentrated to give Compound 36 is an opaque oil (530 mg, ). ESE-MS m/iz Caled for {fist-{2451104 [Nit—Hf: 294.2; found 2942. [587} Part B: A mixture of Compound 36 (279mg, 782 uniot), Compound 37 (370mg, 938 uniol), HOAt (128 mg, 938 ttmDE), DIPEA (409 uL, 2.35 mmot) and DEVI? (4 mL} was stirred at room temperature for 5 mins, then I-{A'I‘U (416 mg, 1095 ttmDE) was added and the reaction Stirred WO 02984 2021/025556 at room temperature for 2.5 h. The reaction mixture was then concentrated, and the residue chrornatographed by silica gel (20-10094) EtOAc/hexanes ). Compound 38 was isoEated as a white fluffy solid (254 mg, 362 umol, 46% yield). EST-MS m/Z Calcd for Csfi-hsNzOs {Mt-HF: 7023; found 702.4. [588} Part C: A mixture of Compound 38 (254 mg, 362 ttmol), MeOl—l (:6 mL} and 10% T’duC st (50 mg) was stirred under a H2 (l atm) for 2 h at room temperature. The reaction mixture was then fiEtered, and the filtrate concentrated to give nd 39 is a clear oil, (214 mg, 97% yield). ESLMS m/CZ Calcd for CszH42N309 [M+H}+: 6123; found 6123. [589} Part B: A mixture of Compound 39 (58 mg, 64 umol), Compound 41} (prepared as described in US 62/982,993 5, 47 mg, 77 ttmol), HOAt (10 mg, 77 umol), DiPEA (56 uL, 320 uL) and EMF (2 mL) was stirred at room temperature for 5 mins. Then HATU (3 6mg, 96 ) was added and the reaction mixture stirred at room temperature for 16 h, then concentrated to give Compound 41 is a yellow oil, (90 mg, quaint). ESLMS m/fz Calcd for C'70H33Nr4016 {M+Hl+: 1375.6; found 1375.4. [590} Part E: A solution of Compound 41 (90 mg, 64- pmol) in 20% piperidine in DMF (2 ml) was stirred at room temperature for l h. The reaction mixture was then concentrated and clirornatograplied by silica gel (0—4094: MeOH~DCM eluent) to give Compound 42 is a yellow oil, (30 mg, 4l % yield). EST—MS m/fz Calcd for C55H73N14014 [M+H}: l l 53.5; found ll531—‘l. [591} Part F: A solution of Compound 42 (30mg, 37 timel), 2,5~dioxopyrrolidin~l~yl — dioxo—2,5~dihydro~l H—pyrrol-l—yl)acetate (8mg, 44 umol), TEA ('7 all, 74 umol) and EMF (1 rnL) was stirred at room temperature for l7 hr The reaction mixture was then concentrated to give Scaffold 43 as a yellow oil (40 mg, quaint). ESLMS m/fz Calcd for C61H76N15017 {Midi}? 12906; found 1290.3, [592} Part G: A solution of ld 43 {30 mg, 26 urnol) in ice/:3 TFA~DCM (2 mL) was stirred at room temperature for l h, The reaction mixture was then trated, and the residue chromatographed by HPLC (10406.54: ter w/ Gilt/Es HCOOH eluent). Scaffold 44 was isolated as an off—white fluffy solid (6 mg, 20% yield). EST—MS m/Z Calcd for CsfileoNrsOis [hi-twill“? 1134.4, found l l342.

Part B: Conjugate 45 was prepared as described in Example 1 except that 4 equiv TCEP was used. The purified Conjugate 45 had a STING agonist to XMT—l 519 ratio of 6.5.

Example 1i}: Synthesis ot‘XM’l‘4519 Conjugate 511, BAR 8.2 o i G 5" ’ 0 o \ l L “N/K $’\ .1. 5x 1 o / 4 ,] w.' N ‘ ' .1 N N, ,. ‘ ,"‘ 1 MN {,1. /~u His—(l 1V M“ . ,NV/x. fl-wu / s. \I/ 2: “H l" ”r” ‘0 HN—\’ 2i 1’ 0\ Part A if‘l‘i, P3“ 5 0 \ 0\ I i“ f Part C ............i. k4 -----------+ C“ Avg. ( I/ a \ “0 a A: >>>>>>>>>>>>> 7 l\ ’H‘I’ Y i"\"\9 k“, HQN K’ ’N ii/\ 0 “ ( ko"\/\ ‘ < w 0 ‘/ H T j H *4 '~ 1. ’> 1L/ )1: J: ll )- >~~ / r/\ l“ H ( z Y/xx/ u / \,.h 0/ \ / 3‘“ - Hz'kn, VNN) \fl’j\/ 7/"! H N R f D O A I) o f}: E V N /’ /'“\ ‘3 :2” «Uk\ 4:" 47 x \ z \ o \; o o N 4 - o ’\ ’I I},, .. ,. u I] H N /~\ JcL ».\/fl\ 2" ”V’NHI yi/g—é’ i. his ”\x*~ /"‘0 HNJ’ \ll \T m"_ \ , o HNv—xf 1/] xi (*0, “N4 Jr \: MHZ \N AT”! \ 2‘ :g: Part D I" \f/‘ Part E / 'l‘i \ o.\ o \ """"""h H 9 \I 3 x 0\ ............. ll OK i A If; 1 {:91Y 3/. o H , / ”nywiv GAVNQ , N i; I. A {I E: III \, \ CI (I) i) 8| N Ao/\r”“0 N \r, \ I .l . i ‘1) J as c ( ‘l\ j >’g_ o // ‘5' K a\ A: 3—K g, 1 : .I (' XMT-1519'»c l\\/!L5'“f>__H I ’ B: null )N‘ ./ X” r-N “ N . o u // -“ ,’ quy \ .N/I 3 A )l‘flé ()7 on “2' ~ an }'\0,\l\ II ”191 L1 9—N\ I: > <0/" o \ o» DH ‘2’ Y V N o 0 / Zrd‘o-k D C) o ' / (115 48 39 [594} Part A: A mixture of Compound 26 red as described in US ,935, 50 mg 64 urnol), Fi'noe-Deglutamie~0~tBu {54 mg, 128 , DCC (26 mg, 128 urnol). DMAP (1 mg, 6 umol) arid DMF (2 mL) was stirred at roorn temperature for 17 1i. The reaction mixture was trated and used in the next step without purification. Compound 46 was obtained as a yellow oil (135 mg). ESl-MS m/z Calcd for (36315313111014 [Ml-HT: 1190.5; found 1 190.3.

Part B: A e of Compound 46 (135 mg, 64 umol) and 33% TEA in DMF (24 ml.) was stirred at roorn temperature for 4. 5 1i. The reaetion mixture was concentrated, and the residue purified by chromatography by reverse—phase HPLC (10—10096 ACN-wa’ter w/ {1.1% HCOQl—l eluent). Compound 47 was isolated as a yellow powder (27 mg, 44% yield). ESl—MS m/ir Calcd for CMHSSNHOQ Elixir-1:11“? 968.4; found 968.2. [596} Part C: A solution of Compound 47 (25mg, 26 iimol), 2,5—dioxopyrrolidin-l-yl 2- 2,5" dioxo—Zfiudiliydroulll—pyrrol—l—y1)aeetate ('8 mg, 31 umol), TEA r 11 uL, 78 iimol) and DMF (l niL) was stirred at room temperature for 1 li. The reaction mixture was concentrated and used in the next step without purification. Scaffold 48 is a yellow oil (:40 mg, quant.) ESl—MS lit/:2 Caled for C53H61N12015 [NEH]? 1105.4; found 1105.2.

Part 1): A on of Scaffold 48 (40mg, 24 uniol) and 15% TEA in DCM (1 mL) was stirred at room temperature for 2 h. The reaction mixture was then concentrated and chroniatographed by HPLC (10910096 ACNr—water with 0. % HCOOH eluent). Scaffold 49 was isolated as a white. fluffy solid (5 mg; 20% . ESLMS m/iz Calod for C49H53N12015 {ls/HE]? ; found 1049.2. {598} Part E: XM'1‘-1519 (10 mg, 0.069 arm!) was conjugated with Scaffold 49 as described in Example 1. Conjugate St} was purified by CHT type II chmmatography. The purified Conjugate 59 had a STENG agonist to KNIT—1 519 ratio of 8.2.

Exampie 11: Synthesis ai‘XM’i‘4519 Cenjugate 52, BAR 7.7 \, O ., (a \\ I Q O N} \. /‘ , x’L‘O N “I I , ~/\ ” NHZ .- /\,J\ HN-—- \. ‘E / ‘K i \ I i NH; 0 HN—q’ I /i N w r \\ “ KNIT-4519 / 0 Ox O O». —-!9- I, (t) H A ’7 0 H /\//\O i N A / i N/YNx ‘3’ , : (\‘Eflfi/ . / ‘5“ DAV/\Q I‘ ”go 0 N / f': N H <\ I] x40 0 1 4’:fo H < XMT~15193\5' f ‘\ | ,I E I} N H N //“N 0‘ “0H 2 ‘71” N2%er m’4: ,‘L " or, 0H ramp, \v N \ffl ‘ g, o" 0 \/ : 0 I 0 0 / 015- [599} ate 52 was prepared from ld 51 as described in Exanipie 10 except that Fmoc— L~G1u(0ntBu) was used instead of Fines—DwGiutTO—tBu). The purified Ceniugate 52 had a STING against t0 int/1121519 ratio of 7.7.

Example 12: Synthesis oi‘XM’i‘4519 Conjugate 53, BAR 6.5 \ .o 0 5? i) o x / . \ .a\ 9 ,JL \Yl'3“ .9 1L Efi,“\rf\ Am? 1 '-' Nv’fi 'l N H” ( ‘ “5/ “H 3 NH ”Q “K \ l! i 2 j "”1 T NH' = .» N"J\\9{ ‘N‘ ' r’ I' ‘rY- / , PartA ‘x S I Part8 ox Part6 x \ ? ----------------. /' 9 I; “MANN/*0 "\ WE’VA‘D , BocuN‘a/kD/\/\9 S (/"\/-NI . : / VA A,. E.: ,>u-i'H o LL?/»\,.N HM «\ :: "NH :1‘1 k NH 2 1: ./‘N : l1 , 0 ;,,N MagE g d .N >Y~ I 0 I .3 I ‘2 ,2 2b 53 54 N“ ¢ (if Q I) n o \/ N./\ N- x E I . N , f 0/ N“ hm (\y w { | 0 /k; 1-1;» x“ Jim/m " NH _ ‘5" f «\j/L‘NHZ ' “ N; nova “Hui-J / \Ii 0\‘ Part D f. 9 /I gait E 0 '\ “\ ~~e”') o f H .Jic .’ 3’ "x K or\ = ' ' »»»»»»»»»»»»»»»»b b)c”NAfl,N‘ OWN!) I H / i, , , (‘7 7HN’AVN‘2/kfii/‘VAO " "\ : i: = : \f’N\/"‘N/;V’N\/fl‘fi/\/\O ./ .

Y new H a: :- i a LN o a = x, ,Y / ' ’ ‘ ii.“ " ‘N nun . k . Y\\/ M 0/ 2k Hm \VL N’> ix2M\/L\/l'~;f—"l' / 0 T }‘fli\0. == u o’ 0 O’\ 55 55 57 NA \\ xiv) i‘Y—I/‘T‘j\ r/ i .__ N 5 /~\ \ PartF “‘2 \ no G \ YYYYYYYYYYYYYYYYY» 1/ arm—151mg c T t 5 \ i k“? 5/ 0 \ / '5 \rNVi‘u’j‘YN¥J\O/\v/\oH # \é’ t . M X ,l . f i. \—MH KN I HZNW \\/-N / I I: n 0’ / :l (2' Part A: To a mixture of Compound 26 (prepared as described in US 62/982,913 5, 0.105 g, 0.134 mmol), Boc—L—A1anine (50.8 mg, 0.268 mmol), DMAP (50.8 mg, 0.067 mmol) and DCC (0.111 g, 0.537 mmol) was added EMF (2 mL). The suspension was then stirred 2 days at room temperature. The mixture was concentrated and ed on si1ica gel (040% MeOH in DCM) to afford Compound ‘33 as a 1ight—ye11ow solid (0.102 g, 80% yie1d). ESI—MS m/iz Caled for C46HS€SN11312 {Mt-HY: 954.4; found 954.4.

Part B: To a suspension of Compound 5.3 (0.102 g, L. 107 mmoi) in dioxane (10 ntL) was added 4 N HCl (0508 mL, 2.031 mmol). The reaction mixture was stirred at room temperature for 3 h. The sion was then trated and used in the next step without purification.

Compound 54 was obtained as a yeflow solid. ESLMS m/Lz Ca1cd for C41H43Ni 10m {It/{+11} "L: 854.3; found 8543. [602} Part C: To a solution of Compound 54 (0.015 g, 0.017 n1mo1) in EMF (1 mL) was added Boe~D—Glu (Otu)—OH (7.67 mg, 0.025 mmol). foilowed by DIPEA (0.02.6 mL. 0.152 mmol). The reaction mixture was d at room temperature for 5 min. '1‘henPyBGP (13.15 mg, 0.025 mmol) was added, and the mixture was stirred at room temperature for 1 h, concentrated and the residue was purified over silica gel {060% MeOl-i in DCM) to afford Compound 55 (11 mg, 57.3% yield) as a white solid. ESl-MS m/iz Caled for CsanNizGis {Mimi-1T? 1139.5; found 1139.5. [603} Part B: To a suspension of Compound 55 r 11 mg, 0.00966 mnrol) in dioxane (3 mL) was added 4 N HCl (0.241 niL, 0.966 rnnrol). The reaction mixture was stirred at room temperature for 2 h. The suspension was concentrated and used in the next step without purification. Compound 56 was a white so1id, ESLMS m/e Caled for C46H55N120i3 {NEH} 1’: 9834; found 983.4. [604} Part 33: Te a solution of nd 56 (9.5 mg 00096.6. mmol) in EMF (3 mL) were added DIEA (8.44 uL, 0.048 mmol) and Ztfindioxopyirolidin—1—yl 2n(2,5—dioxoflfi—dihydro—1H“ n1nyl)acetate (317 mg, 0.013 inniol). The reaction mixture was stirred at room ature for 1 11, neutralized to pH 6."? with HOAC, then purified over preparative RP HPLC (045% ACN in water) to afford Scaffo1d 57 (3.3 mg, 31% yield) as a white solid. ESI—MS miz Calod for CSZHSSNBOIG [M+H]"": 1120.4; found 1120.4.

Part 1“: thll 1519 (10 mg, 0.069 uniol) was conjugated with Seaffold 57 (0.700 mg, 0.625 unto} in 200 til, DMA) as described in Example 1. Conjugate 58 was purified by CHT type 11 chromatography, The purified Conjugate 58 had a. STING agonist to XMT—i 5,19 ratio of 615, Example 13: sis of XNETdSlE? Conjugate 60 I \ ”N Hg H0 /0 xur—isis ””2 / A} £453“ 1§T[S\ Ho 0 \ /M09 1/ O“ I \ H 9 \1' C» T (N: "it/d“ N\/h‘o’\/“c~ ! /“‘1’; 9 f 0 \ it \’/ /'\\/'T~r«\i/>"Nl:iF” i, H l I . g L o o / “N \ 1‘!“ f/xTN/ w \LN / o 0"\ I‘N / q K ,I 5 0 or // I 011‘: [606} Conjugate 60 was prepared from 59 as deserihed in Example 12 except that Boe—L~Glu (Otu)~OH was used instead of Boe—D~Glqu—tBu. The details of the antibody—drug conjugates 60— 1 and 602 are given bei ow.

Caniu ate 13.41% Exampie 14: Synthesis et‘XM’i‘4519 Cenjugate 62, BAR 6.5 \/ NH“ N {I ‘l \ 6.) HN»" :1 ‘ H H N J‘ ‘ HOTD SWNT/J XMT-u’i 51 9 NH; HO HNM‘? 3 ,0 N . \\ flog. ‘ ------------.. r o at ,- "rVN/LN/xiTANTH ‘3’, 0 : = XMT»15137-;j . . \ o L) E x l H ,I H j o 0 4L N ( “\Sr' V.30 i N” ' ‘ P r” T ‘D’VNO I E‘ \ 7 \ C.) 0 XV“ I HN A TN“ ’I‘h \ 1r “" n />,,.(; 1K HN ’5 'I 6 Q 0 2 W \ L‘N N; .{f‘NE: ’ // lo 0‘ 0* / d:5 Cenjugate 6?; was ed frem Scaffeid 63 as described Example 12 except that - 311; (GHQ—(3H was used instead of BoewD-Giu-O—tBu and Bee-D—Ala was used instead et‘BoeeL— Aia. The purified Conjugate 62 had a S'HNG agenist te XMT—l 519 ratio of 6.5.

Exampfle 15: Synthesis MIKE/{$1519 Conjugate 64, DAR 6.4 54% ’\ \ // \il \ '1 N‘ {\x * Q \ ,/‘~o’ m'\ :.\;: “"2i ‘f,\..4\-’ HN--<’ H,» 0 .NV/s :LNH av w e XMT—‘ES‘EE? r”0 ' ”N " ‘\ Q : H0 0 x k c? XMT—tfi‘igesI NAVY/j 1 4,. o A j W K R N\ \N/\YN\£/L\o/\/’\OH K.) [) J: 5 i E I [III' 0' H J \‘YNV NA]! ‘ 3 3W‘O i ¢;‘\,N \ ‘ H E g: B—NH \G I _/ fu- H“! ,,..N I xw/‘m » </ I:. :' : l /'\\ a / M.

P N N I 0 0A». 1. \F e/ N . of)'\0.,g:\ /' c 1 I (.115 Cenjugate 64 was prepared from Scaffold 63 as described in Example 12 except that Bee“ DwAla was used instead of Boan—Ala. The purified Conjugate 64 had a STING agonist t0 XM’I'n 1519 ratio at 6.4.

Example 16: Synthesis of X‘MTdSW Conjugate 66 7 o o s Nr\> j, I i‘ .3 I < -“ ”R‘ NHu N" (G / o “Haj/I’f : i \ ’ \ . i : ,1 . \ mat—151s 2‘5 Steffi ““2 9 HOT-.0 m ’0 \ ,~‘I o 0x : \ ,_ Q I XMT-tstsis j" NevlL = § JL " \ o / 0 K D\ I NIAW' x Q/NAO {I / ,ll 0 b } _//J-\VNI> \/ \ 'uTil/«TIHX&o/\,/\?' : ' K; “NH ).- \ O x;\ N i HZM \/'L"N/ > (y ’ o.’ "El \ :I . | />---NH g e ”NE/kl” oflgJi /" WO 02984 {6091 Conjugate 66 was prepared from Scaffold 65 as described in Example 12 except that Boon Z-arnino—Z—nrethyipropanoic acid was used instead ofBoo—L—Aia. The details of the antibody—drug conjugates 66—1 and 66-23 are given below.

N A 31 I], 71/ \1/ NH, , ” Pane u ,9 PartE g h g \l, —* “h‘i‘)\O/\*/\ >>>>>>>>>>.. Baum ’Ah/‘Vi'flj’; 0/ \f‘o ’\ u . 5, [42' 71‘ l: 71 72 [610} Part A: A mixture of Compound 26 (prepared as described in US 62/982,935, '75 mg, 0.096 mnioi), j\l"—Boo~(D)nAla—OH (91 mg, 0.48 mnioi), DCC (99 mg, 0.48 n'iino1), and DNLAP (1.2. mg, 9.58 enrol) in BM? (3 n'rL) was stirred at room temperature for 1 h, and then concentrated in vacuo. Purification over siiica gei (DCMfl‘AeOH 60:40 v/V) afforded Compound 67 (82 mg, 90% yield) as a lightnyeliow solid. ESLMS m/s Calcd for N11012 [BAH-11+: 954.40, found: 95443 Part B: To a suspension of Compound 67 (80 mg, 0.084 mmol) in dioxane (5 mL) was added HCI (41% in dioxane, 0.42 niL, 1.68 mnioi), and the mixture was stirred at room temperature for 4 h. The mixture was concentrated in 141615010 afford Compound 68 (72 mg, 16.10% yieid) as a 1ight~yeiiow soiid. ESLMS m/z Calod for C41H48N11010 [TX/HEY: 854.35, found: 854.38 [6121 Part C: To a stirred solution of Compound 68 (48 mg, 0.056 mntoi), N—Bot:-g1yoine (15 mg, 0.084 mmol) and 3 (44 mg, 008411117001} in EMF (3 ml.) was added DIPEA (0.088 ntL, 0.51 mmol}, and the mixture was stirred at room temperature for 2, 1'1. The mixture was concentrated, and the residue was ed over silica gel (DCi’VltMe01-1 60:40 v/v) to afford Compound 69 (53 mg, 939/13 yield} as a white solid. ESI-MS m/z' Caled for C43HsaNr2Ors {Mt-111+: 1011.42, found: 1011.45 [6131 Part 13: To a suspension of Compound 69 i 50 mg, 0.049 mrnol) in dioxane (5 mL) was added 1-1Ci (4M in dioxane, 1 mL, 209/1) WW, and the e was stirred at room temperature for 2 11 then concentrated to afford nd 70 (45 mg, 100% yield) as a white solid. ESLMS m/iz Caled for C43H51N120H {ls/HEY: 911.37, found: 91139 [614} Part E: To a stirred solution of Compound 70 (20 mg, 0.022 mmol), NnBoe—(D)— G1ut0fBu)nOH (10 mg, 0.033 mmol) and PyBOP (17 mg, 0.033 rninoi) in DEAF (2 mL) was added DIPEA (0.03 mi, 022 mmo1), and the mixture was stirred at room temperature for 2 h. The mixture was concentrated, and the residue was purified over silica gel (DCMA‘AeOH 60:40 V/V) to afford Compound '71 (24 mg, 90% yield) as a white solid. ESLMS m/a Caled for N 13016 [ft/H111]? 1196.53; found: 1196.55 [61 5] Part F: To a sion of Compound 71 (24 mg, 0.02 mmol} in DCM (5 mL} was added TFA ( 1 mL, 20% V/V), and the mixture was stirred at room temperature for 12 11. The mixture was concentrated to afford Compound 72 (21 mg,100°/E> yield) as a light—yel1ow so1id. RSI—MS m/z Caled for C48H58N13014 {ls/1411] 1* 1040.41; found: 10402:“: Part G: To a stirred solution of Compound 72 (21 mg, 002 mmol), 2,5—dioxopyrrohdin— 1“};1 2—(2,5-dioxo~2,5“dihydro—1H~pyrro1~l~yl)aeetate (7.6 mg, 0.03 mmoi) in DMF (2 mL) was added DIPEA (0035 , 0.20 mmol), and the mixture was stirred at room temperature for 1 h, The mixture was concentrated, and the residue was purified over RP 1'1PLC to afford Sea1'1b1d 73 (45 mg, 19% yield) as a white so1id. ESE—MS m/z Calcd for (54116115114017 j'h 1177.43; found: 117740 Part H: Conjugate 74 was prepared from Scaffo1d 73 as described in example 12. The purified Coniugate 74 had a ST1NG agonist to X13414 519 ratio of 6.9. e 18: Synthesis othi'i'—1519 Conjugate 76, DAR 7.5 . 0 o \r" "O l N\/\/3: Ere/7""? {NF/\YJLNH; H ”dz \\ ”N "1 A; \, \ “AWN"? \ N “Aw «e f mmsw \. 0 e A 5 \ x a. H H H I .N v/‘L, AVA.

NVi i —» XMT‘IS19/- ~N~VJL AH !/ A I O O \ ”(A\h/ .. [AW/NV \0/"\/'\U \ {5 N W -_ _ fl W vh 0 f = A N i \J a H z 0 ‘ I 3 a“ 0 ° " / w .i's “A ° v ? / , i ' L E / NH -. rA H2N~ Elm k. \ .’ 04km: HENm/A§/‘Nr, / in “ 0 OH T v I“ « - 0/] 0"1\ / O"\\ \ O I] 0 /d15 75 76 [618} ate ’76 was ed from Scaffold ’75 as described in e 17 except that N“ Boe—(L)—A1a,~OH was used instead ofN—Boc—(D)~A}a~OH and N~BOC~(L)—G1U(OIBli)~0H was used instead ofN~Bec~(D)leu(OrBu)—OH The purified Conjugate 76 had a STING agnnist to Dad? 1 519 ratio of 7.5.

Exampte 19: Synthesis of FER/{T4 519 Conjugate 78, DAR 7.4 \f0\. 5/0 ~ Edi. N\/\ /L\NH \\ '-~(!".i....<z“~r\f 2 34/39”/ \d/jq ‘\ A XMT1519 K7 O \I O D 5 we fA \ 1 e \ : H <3 H <3 —5., KNIT—151 31., A A./\- I, “KIN ”WK \ka ”Cw/’7‘ J-konvrxo S ’54 5" V OAVA.) N : TR:‘F i “34% G! j H d ‘ 4. ,N i 7&0 o /: {LTN)"Nl-i\-\ ’I O J. ,L i />"“.“ 3N.- '\ Gale,” Hm A)N’ \g A421“; / 0” “0H 2 7T \/ N k \\ (3 o - o"\/ / G15. 77 73 Conjugate 78 was prepared from Scaffold ’70 as described in Exan'ipie 17 except that N- Bee—(‘tJ-GEi,i(OlBu)—0H was used instead ofN—Bee-(D)*Giu(0rBu)-OH. The purified Conjugate 78 had a STING st to XMT—1519 ratio of 7.4.

Exampte 2i}: Synthesfis of FER/{T4 519 Conjugate 80, DAR 7.5 ,0 0 \r2‘l” o 9 s I/ ” ”S N A )L \rii I,“ NH \ we? HN—fi l;. i M12 H’s—x . \ \ \J/ A, r" N N" e- s mar-1519 ' 14‘ \ T ' O\ xmrutsuy o x q o we H. e 1 than/NI“ "r“‘rl‘eAf‘e‘ -A A . - I {LA] Lg.

‘\ I/'”x/ ‘4/t‘rx'"‘V’ A r /\ [K : . <3 , 1 \\ ' ‘ \’§o 7,1 H i (\Er,N \x I 0 _ N , I” :’N§_NH >\ \ HN >43“ Ann "' ‘T/\/\N 2"“ Has \ k5 i / N \ 4/ "‘H - ,h j .: ‘. \«f N H,0"J\5 \ : ’ .: if: Q, \ o r 0 \l/dpI [620} Conjugate 89 was prepared from ld 79 as deseribeci in Exampie 17 except that N— Boe—(L)—A}a—OH was used insteaci ofN-Boe-(D)—Ala—0H. The purified Conjugate 80 had a S'i‘ING agenist t0 KEV/[111519 ratio of 7. 5.

Exampie 21: Synthesis ei‘XM’i‘4519 Cenjugate 82, BAR 5.7 I O ”.43 {/0 W. \ r HAP-<2“ i ’Ni/LNH? 0 N L \\ Ho \ T,0 N z; XMTH1519 (A0. HN__<, l N52.

, \ H0 40 o / N. I// \ i e g o \ , H ,/ XMTAS‘iQrs‘ o 1 5 1 \r’NNAN’2YN\/J\Q/\//\O \ \ .l ”1/ 0 g H .0 Ii = r’)’ 5 '\ «l/"NVJ N/YNx/i OAV‘ \Q ’ H hi f i I )t-"N'H H 0 0 4x (”I I ”Mk/‘x N’ , N ‘ / z“ \ \ f 1 2 NH ‘L / 6’ Wk» o HZNW/\ v \/' ff '1’ e c"\ o / / d1»: 3‘! " [621} ate 82 was prepared from Scaffold 8]. as described in Example 12 except that Bee Glycine was used instead of Boe~(L)~Aia—OH. The purified ate 8]. ha d a STING agenist t0 Xk/iT-l 5] 9 ratio of S. 7.

Exampie 22: Synthesis ei‘XM’i‘4519 Cenjugate 85, BAR 6.5 u- 0. / O o . o o , - H - i . I I H H2?!" (\"S\__. V :\\'[N )\ ./ «N\_/L“N' Par-EA K| xNx/LKN/\Iq Nx/JH /’\ox’\/0\/\Q : f—N / r ........._;.. a fir a H H = \N N ”J t C; g’ "\K 0 \ ‘0 f”*O‘AVQV’ 0 OH OH H k ; (\r . \ o\/\e/\,o\/\H/N N H J\a/\/Q" xo / HNI e e” OH OH / .. W cm ” H N \ 5.: 7 /dc“\ / . N )7 \ L4 QMNH “‘ZN‘K HG ”\N’UH o 0 I” .L o c: ,J V ‘/X“\k : ‘S .. a~\ N?{ A 5; $3 \_‘.\ JN ‘VN/k‘f’q 0 r i! 5' Hz"~ “J I \\/ “N 0/” L617:N” 0”Jjw—G \a “N / e 0 o a ed/‘KNAT/NYJ‘NWNVJLN ”\vat, H H : : H \ I g ; “v“crN/C’vm \ Part B E \_4 é :- H H O b /\ /\V.0~v/‘l \ \\ o 0 OH a: \ on \ O ‘v H \ \ l : \ 01/ OVNC’AVOV" ,,-N. \N/V /\/.D.i \ \ HN \if Ed I A \ L 0 /, OH OH a \ . 0H 1 ok ”x on ’ ’ ’ \ o \WL ,q NH 0 H0 ’8 ‘vou F N I V0 HO / 15 ! ofi N“. I L? g N__,l\\ )3I II H N o __/' N’Kw / ~\‘\/ II 9“ I O \ / 1.47— \V’L‘N N / t ,v / NL'K / Gav. 2 {J15 \\/J\N~.~ [622} Part A: Scaffeid 84 was prepared as described in Example 1 except that nd ‘83 (prepared as described in US 335) was. used instead of Compound 1. Scaffold 84 was obtained as a white fluffy seiid (3.3 mg, 0.5% yieid over 5 steps). RSI—MS m/z Caled for C101H143N22042$ [NHZHPZ 1 187.49; found 1L 7.78. [623} Part B: Conjugate 85 was prepared as described in Exanrpie 1 to afford the title conjugate.

The purified Conjugate 85 had a STING agenist to XIR/IT—l 519 ratio of 6.5.

Exampie 223.: sis afi’aiivizumab Cenjugate 852a, BAR 7.4 / Q r: 9 H E: H_ "T H \ ?aiivizumamsV“AN_nw(Nx/LKNE an' ‘E/fir'NV’l"uN‘V’\o‘”\x’O\/\o \ : ’ ' E " I O O \ Lfia 0 N: (“a/WEN“ 0 cm H 9H \ \ 0%“: Ox/\rr"v‘0\/’\ ,,»N\,¢\\,jL.N./~ ,«L /‘\\/0H \ \ mi; ‘ T: I \ \F 0 9L H 0H 0H \ L 0 NH \ ' 0“ o\ ’\ o 3H 1 - , c/NvleNiH\ no x cm c: HzNNfO I “ QC {f.~\\ \ [i N% S I ‘\ x I 0 HN a e&& / 2. do O)\Q\N,"\»( e ‘ _F i \v/J‘ N" // N':j\ / EH / V care I d1- \ JL/ N ij'ugate 85:: was prepared and characterized as described in Example 1 except that Paiivizxmtab was used instead of XMT—i 519. The ed Conjugate 85a had a STING ageriist to Paiivizumab ratio at“ 7‘4.

Exampfle 23: Synthesis of FER/{T4 519 ate 88, DAR 6.6 2 \ ‘3‘ 'I O o. 0 1.3"“? “L /\Y/LNH2 “Ck-"f N ./\\“r’iLP-H; / G H I O) ‘ : “1/ HO 0 ”MK! «,4 7r FEVER ‘1: N, N Part3 k}; *****************1" [Pg/<11 (NV V,E “1%,«i? I." iii/Mt» h H~Kxeeet, , ,4 .N\ wK\LJ§«t; :L 0” o' 0/ o" 87 Pfi"\ '{lc N N}: \\\ I /'\0’ “H3 WV“ ram—(II; \ XMT—‘ifs‘iQ-{zs} 0 j Q ‘N’ \ I 9 H. 0: {SK/ILMia/N\/L\o/\’/\9 ,/ \\ \IT, j \‘5 H xix A1 ( I Hfigrfg¢y/if 0/ 0"\ ll \. 1/61: Part A: Scaffold 87 was prepared as described in Example 12 except that Compound 86 (prepared as described in US 62/982,935) was used instead of Compound 26‘ ESLMS m/z Caled for SNMOH [IVHHTY 10914; found 1091.2. [626} Part B: Conjugate 88 was prepared as described in Example 12 except that Scaffold 37 was used instead of id 57. The purified conjugate had a S'I‘ING t to XM’I—I 519 ratio 0176.6.

ExampIe 233.: Synthesis eI‘I’aiivizumaI} Cenjugate 89, BAR 5.9 93%120 O ’[MO ”II—“3:19 NH? \\ H0 0 Paiévizumab/;<>\ Y ‘\ 1"?” 1 ‘9 O / H / {NE/”‘xO/‘xv/xo i I \° 0 " HZI‘LH/O ——N {N I N>uNH>,....{I// EN / o 0 0 // / 915 [627} Conjugate 89 was prepared and characterized as described in Exainpie 12 except that Paiivizumab was used instead 0f XIVII 1519 Ihe ed Conjugate 89 had aSTING agenist t0 I’aIiVizumah ratio 0175.9.

Exampie 23b: Synthesis of C'I‘Ln481;§2____mIgG2a Conjugate 89a, BAR 8.8 CTLméfi-S‘I 32mmIgGZa" x 893 / 1- E628j ate 893. was prepared and characterized as described in Exanipie 12 except that CIL—48132 mIgGZa was used instead ct XIVIIl1519 ”Ihe purified Corijugate 89a had a‘I'I‘ING agenist t0 C'I'L—48132pjjjangGZa ratio of 8. 8.

ExampIe 239: Synthesis cf RII‘I’5___1nIgG2a ate 89h, III/Isis 9.0 WO 02984 2021/025556 MFPfimmigGZa{5 1+sz \ Aug—N; </ N 1/ o 0“\ [629} Conjugate 89b was prepared and characterized as described in Examp1e 12 except that CTLASBZJn‘EgGZa was used instead ot‘XMTd 519. The purified Coniugate 89b had a. STING agonist to MFPS_mIgGZa ratio of 9.04 Exampie 24: Synthesis i‘4519 Conjugate 92, BAR 7.6 9 o , HEN/L‘Y/‘TS _ 3‘? gigfr~ o HEN/11‘ fl \\{I 7]" fl/fi N>nN JN HO ,0 ETIE}:N ‘ /‘;“”N ‘f/ Part B { > PartA /’5 \f K I; \ » ------------------x- » ‘1)»A?!) 0 He”\-/‘“o g" ,N\/L\N/ ViQ~W5N <\\. x/kwi‘! 0' H 1\ E! ,b—NH / i?“ xix/x“ “2N1%: I ”qu 0 “j My» 37“! o N 99 Q1 o \ 0 .. ; gmfig \ HQ to ”L I \ mar-151 J ,s\ N. \ a \r T |/\‘?” Q t“ O ,/O 5’) \\a 1 x 1 a i .'If N/ T V1LWAN/"o " a H i 2 i f I \ O - ”TX-’ NH 0" / H N2 \ . \n, N a \ L: / o / {630} Part A: ld 91 was prepared as described in Exantpie 12 except that Compound 99 1:prepared as described in US 62/982,931 5) was used instead of Compound 26. ESI—MS m/Cz Calcd for Cpl-{53154130158 {Md-11f: 1136.4; found 1136.2. [631} Part B: Conjugate 88 was prepared as described in Exantpie 12 except that Scaffold 87 was used instead of Scaffold 57. The purified conjugate had a STENG agonist to XM’I‘d 519 ratio of 7,6.

Exampie 243.: Synthesis afi’aiivizumab (Iqmjugate 93, BAR 6.7 m/\ HZNJ T15 —N\$\M<NJ‘§’ Paiwszumab. .. / , ‘\ To N ,J "(I f‘v/ D 5 13‘ o N ’\ E JL \ f’ V , I N \E/ Q \ o I o :2 \ >fi‘2” 5. [632} Conjugate 593 was prepared and characterized as described in Example 12 except that Paiivizumab was used instead of A 519: The purified Conjugate 93 had a STING agmist to Pa} ivimmab ratio of 6. 74 Examgfle 25: Synthesis anMT-ESW Cunjugate 1999 BAR 7.8 HO‘FA//\ H0 "‘0 -,s 0 30d ‘N/ “\F/N/\O } 2N‘AI/OYK/fix 4%ka \ o y» \N t;I.-(.,D:V:Ap o (a .4/ ,N ,;\-—NH '0\/ E Ii ,)-N.Lv: ‘ Q“ i“ ii / N )—NH O ”N \v/ ‘ N I! Par‘lA “ZN /\\~/\N E PaatB “ZN ’ T \ ’\\/“~.\s \[i/ ,g/ \,.N T ,(I \,-N Y N \ v o’ o o , o ’ “/J 94 95 96 4‘ P o r o , V U Om/ ; O . 0-- l I \ 1 \ V , _) : no rtNJ‘ (kap?‘""\ K 3: H2N/l\€’\~/5\>_\W\: "'Ie N ,f ‘ ’¢\N’ ”.4" A k " o ./o 2 CL 3 ,0 O O HO Ca 8ccHM ,k \‘ \\ 0 V \: ’3 .

\E l/\ /~\, 12: \/1k f .\ \ /\ A F’yBOF‘, D:PEA i T \l/\‘ O a E T? V (:3 ( \~. 0 \ C‘ . {fix . T" Paric = i ' i . f i : Part0 % ,_/L H2!». h-N, .

- I 1/N»I; "NH o\./ , -\ A , \ Q \Q ’<, 2 \fl/ \u §.

. . Q/ ./ N . 0.4.- W N. o 0 \ 98 -J o 0 ’ , /‘~ ,3 w E HZN HZ” E! “F 3=N’ \I’N \ /2‘~ .’ I Q Q ,2. / W A. ,2 \ f\T// Q/' ’\ 0% X0 \ ,0 \ N 1L”N N o I o V ., .i Q -\ o ‘ , \ L :3 ,u\ I E: x. \ ___________a, fl 0 O O ,Iav 2.. \VAO ' Fr .qu \NN, ' '” 5 : H l n ( _ HN/‘TOW/\vfl W?”s XMT 151 ,L j. g O \2 \. Q ‘ O ,x 1 g; ”N D:F’EA ‘ 0 : \ - wH .. , ,2C / i ,HZN, l x/"w 0/ \OH '10, O Hark. §/ \i />~v<\\li l PartE \ A O (N / \ ” ,J / d 100 ‘5 Part A: To a stirred solution of Compound 94 (prepared as described in US 62/982, 35 45 mg, 0.054 mi'nol) in DMF (3 mL) was added (3)4—(Boc-arnino)propan-2—ol (l9 mg, 0.ll mi'nol), EDC (l 7 mg, 0. l 09 minol) and DMAP (33 mg, 0.027 ), and the mixture was stirred at room temperature for 12 hours: The reaction was concentrated in mom), and the residue was purified over silica gel (DCh/l:lvle0l:l 60:40 V/V) to afford 95 (49 mg, 92% yield) as a white solid.

EST—MS: CMHSVNioOizS (Nit-H): calc. 985.38, found: 98521. [(334] Part B: To a stirred sion of Compound 95 (49 mg, 0.05 nunol) in DCM (5 mL) was added TFA (l mL, 20% v/V DCM), and the e was stirred at room temperature for l2 hours.

The resulting mixture was concentrated to afford Compound 96 (44 mg, 3100943 yield) as a light yellow solid. EST—MS: CazlhsNioOioS (NH-H): calc. , found: 885} 8. [635} Part C: To a stirred solution of Compound 96 (50 mg, 0.05 mmol) in EMF ('2 mL} was added Boc~Glu~OtBu (86 mg, 0.28 mmol), PyBOP (118 mg, 0.23 mmol), and DTT’EA (0.12 mL, 0.68 mmol), and the e was stirred at room temperature for 30 minutes. The mixture was trated in town, and the residue was purified over silica gel (DCMh/kOH 60:40 v/V) to afford Compound 97 (45 mg, 77% yield) as a white solid. EST—MS: C56H72Ni iOisS (ls/RH): calc. 1170,49, found: 1170.29.

Part E): To a stirred suspension of Compound 97 (45 mg, 0.038 rmnol) in DCM (5 mL) was added 'l‘FA (1 mL, 20% v/V DCM), and the mixture was stirred at room temperature for 12 hours. The mixture was concentrated in, vacuo to afford Compound 98 (38 mg, 100% yield) as a light yellow solid. ESl—MS: C47H56N110BS (MJrH): calc, 1014.37, found: 1014.20 [63 7] Part E: To a stirred solution of Compound 98 (20 mg, 0.02 minol) in Dix/[F (2 ml.) was added oxopyrrolidin~l~yl —dioxo—2,5~dihydro—lH—pyrrohl—yl)acetate (5 mg, 002 mmol) and DTPEA (0.034 mL, 020 mmol), and the mixture was stirred at room temperature for l5 minutes, The reaction was quenched with acetic acid (0.034 mL, lzl V/v DTPEA) and directly purified via HPLC using a C18 stationary phase (waterACN) to afford Scaffold 99 (4.7 mg, 20% yield) as a white solid ESl—MS: CsalrlssNizOreS (Mt-H): calc: 1151438, found: llSl,l 8. [63 8} Part 3?: Conjugate 100 was prepared as described in Exai'nple 12 except that Scaffold 99 was used instead of Scaffold 574 The purified ate had a. STING agonist to XMT-1519 ratio 01°78.

Example 2:335 sis nt‘lhlivizumah Conjugate llll, BAR 6.5 9 o o / HEN/IL %—r3_, «I I E >rN / ”N” AWN N VI NW6») WAG . . . 1.

H ‘ Palivizuma l i O \ O / N O r !- Q—NH \r/O~ / \\ m Y w. , b O //045 [63 9} ate 191 was prepared as described in Example 25 except that Palivizuniah was used instead ofXMT~l 5l9. The purified conjugate had a STING agonist to Palivizurnab ratio of 6.5, Example 26A: Cancer cellvtargeted wild type or 17c mutant STENG~ADC activity in cancer celli’THPl Luciferase er cell cowcultures [6540} Generation of NaPizh Fe silent dies: NaPiZb inAb with its Fc region engineered to h Po effector function (airti—NaPi2h~(AAG)) was designed with three mutations in the heavy chain constant region 1.4234A, 1235A and P3 296 (AAG; Kabat Eu nunihering) and generated tl'irougli rd molecular hiology procedure, The antibody was expressed and purified. Briefly, DNA encoding variahle region of the heavy chain of NaPiZh dy with constant region ot‘liurnan lgGl carrying1..234/3i 1..41235‘i and P3298 mutations and variable region of light chain of an tieNaPiZb antibody Wltl’l hunian lrappa light chain were cloned into mammalian expression vectors: The heavy chain and light chain of NaPiZthiAG) were coexpressed in llEK293 cells, and the antihody was ed from cell supernatants hy standard Protein A affinity chromatography. {641} Induction of STENG pathway in immune cells: The induction of STING pathway in immune cells by NaPi2h—targeted S’l‘1NG ADC was evaluated by a cancer I'lll’lulRFB- Luciferase reporter cell co—culture assay. OVCAR3 human ovarian carcinoma cells were seeded in 96uwell nd surface tissue culture plates (l 5,000/well} and allowed to attach for 6 hours in RENE—1640 medium with 10% FBS and. % penicillin/streptomycin. A range of dilutions (0.4 nM to lOO nl‘v’l based on payload; Sufold serial dilutions in growth medium) of the test articles Conjugate Sit—1,, Conjugate 8f, Conjugate Sci—1,, and Compound 1 were added to each well and the plate was incubated for 20 rnin at 37 0C. 'l'lil’l—dual reporter cells (30,000) were then added to each well and the incubation continued for 20 hours at 37 “C in a humidified atmosphere of 59/5 CO; Cell culture supernatants (‘2‘:In. ul} from each incubated sample was added to resuspended QUAN'l‘l—Luc (50 ul) and the lurninescent signal was measured immediately using a Spectrah/laa' MS plate reader (Molecular Devices). The ECso value was ined from the dose response curve. Table 1A provides the liCso values in Tl-ll’l —Dual cells co—cultured with OVCARB cancer cells.

Table 1A Test Article Cnnnaate Sbl nnWrnate 8f 3.2 3: lfld l [642} As shown in Table 1A, Conjugate Sir—i with wild type Fc effector function exhibits greater than lOO—fold increased activity compared to the free agonist, Compound 1, and ~1000u fold increased activity compared to Conjugate Si and Conjugate Sit-:1, confirming the role of the Fc receptor for activity. Results shown are ECso values of a representative ment.

Example 2682 Cancer cell—targeted wild type or Fe mutant S’l‘lNGnADC activity in cancer l‘lll’l erase reporter cell comenltnres [643} Generation of HEan Fe silent antibodies: Trastuzumab mAb with its Fc region engineered to abolish Fe effector function (antinHerZ—(AAGD was designed with three mutations in the heavy, chain constant region L234Ar L23 5A, and P3298 (AAG; Rabat Eu numbering) was ted as described in Example 26A. {6-44} induction of STlNG pathway in immune cells: The induction of STING pathway in immune cells by HerZ—targetcd STING ADC was evaluated by a cancer oell/THPl —lRF3~ Luciferase reporter cell ture assay using SKBR3 human breast carcinoma cells as described in Example 26A with test articles ate Sail? Conjugate Sj, ate 802, and Compound 1) Table lA provides the ECso values in THPl~Dual cells co~cultured with SKBR'} cancer cells Table iii est e ,oniu:ate Set-2 :3 , 31onnate file—2 Zionnrll_nn [645} As shown in Table 13, Conjugate Eta—2 with wild type Fc effector function exhibits ~50" fold increased activity compared to the free agonist, Compound 1, and ~lOOO-fold increased activity compared to Conjugate 8j and Conjugate Sic—2, confirming the role of the Fc receptor for activity. Results shown are ECso values of a entative experiment.

Example 2'7A: Cancer cell—targeted wild type or 17c mutant STING ABC activity in T8121 rase reporter cells cultured on tumor cell antigen—coated plates Human NaPiwaderived e (QlNV'I‘VPS'l‘ANCTSPSLCW'l'BGlQNW'l‘l‘leN) was coated onto the surface of each well of a 96—well plate by incubation with the peptide (1 rig/nil; in PBS), overnight at 4 OC. The wells were then washed 1); with PBS—T and blocked by incubation with BSA (3%in PBS1) for l hour at room temperature After washing with PBS"1 (2x) PBS (lX), and growth medium (RPMI l640, l0% lE'BS, 1% penicillin/streptomycin, lX), a range of dilutions (0.4 nM to l00 nM based on payload; 3—fold serial dilutions in growth medium) of the test articles (Conjugate 813—1 Conjugate Sf Conjugate 3c—1 and Compound 1) were added to each well and the plate was incubated for 20 min at '37 0C THPl—dual reporter cells (50,000) were added into each well and incubated for 20 hours att3? Cin a fied atmosphere of 5% C02 Cell culture supernatants (20 pl) from each incubated sample was added to resuspended QUANTI— Luc (50 pl) and the luminescent signal was measured immediately using a Spectralsrlax MS plate reader (Molecular Devices) The ECso value was ined from the dose response curve Table 2A provides the ECso values in THPl “Dual cells cultured on NaPiZb recombinant peptide coated plates Table 2A ‘CnuiuateSt"BCsr nM for)uate 8c— 1 :01}:mound1 <0022 [647} As shown in Table 2, Conjugate Shel with wild type Fc exhibits ~l00~fold increased activity ed to Compound 1.. ate Sf has no activity and Conjugate So]. has ~1000— fold lower activity compared to ate Sb*1, confirming the role of the Fc receptor for activity.

Results shown are ECsa values of a entative experiment Example 273: Cancer argeted wild type or Fe mutant S'l’lNG ABC activity in 'l‘lrll’l rase reporter cells cultured on tumor cell antigen—coated plates [648} Human HERE/ErbBZ protein (l-lis—tagged, ECD, don'iain l‘V, 17.1 kDa) derived e was coated onto the surface of each well of a 96—well plate by incubation witli the peptide ('1 ug/niL in PBS} overnight at 4 OC and the assay was performed as described in Example 27A except that 3-fold serial dilutions (0.09 nl‘vl to 206 hM based on payload) of the test articles Conjugate 8a- 3, Conjugate Sj, Conjugate 8&2, and Compound 1 were used. Table 213 provides the ECso values in '1‘H1’l~l‘3ual cells cultured on Her'l‘. recombinant protein coated .

Table EB :est e Conugate Eta—3 ’ ‘3 "asonuate Sit-2 ornround 1 T-faw [649} As shown in Table ZB, Conjugate 821—3 with wild type Fe exhibits ~lUD—t‘old increased activity cornpared to Compound 1. Conjugates 83' and Conjugate 843—2 have no activity? eonfirrning the role of the Fe receptor for activity. Results shown are ECso values of a representative experiment.

Example 28A: Activity ol‘t'nnior cell ed Nal’iill) ADC in cancer cell/PBMC tures {650} OVCAR3 human ovarian carcinoma cells stably sing nuclear restricted niKate fluorescent red protein were generated by transduction with lncuCyteCC‘) NuoLight Red Lentivirus reagent. Stably transduced cells (designated as OVCAR3—NucRed cells) selected in puroniycinn containing media (2 ug/niL) for two days, were seeded in 96~well tissue culture plates /well) and d to attach overnight in ln164i) medium with 10% PBS and % penicilliii/streptomycin. Culture medium was replaced with fresh media (59 uL). Test aitic-les (3x concentrated, Conjugate 8b~l (100, 10, and 1 11M), Conjugate 8f (100, 10, and l rn‘vl} Conjugate Sol (100 and 1011M) and Compound 1 t 1i‘i‘i and 10 nM); conjugate concentrations were based on the payload) were then added to each well in media (50 uL) and the plate was incubated for 20 min at 37 °C Frozen human peripheral blood mononuclear cells (PBMCS) were thawed according to the supplier’s instructions and were added to each well (40,099 PBMCs in 50 pl, media.) and the plate was placed in an lncuCyte© live eell imaging instrument in an incubator (3’7 °C, 5% 02) and scanned every r-‘l hours over 2 days. The number of red objects (cancer cells) were quantified using the lncuCyte© Zoom software. Red object contluency in each well was normalized to its own “:10 time point red object confluency. {651} Flt}. lA plots the red object confluency as a function of time and shows the robust induction of cancer cell g by PBlVle in response to Conjugate Elli—l at a 10th lower payload concentration compared to Compound 1. Conjugate 8? also exhibited activity although at reduced levels ed to Conjugate 8b. Conjugate 8ch had significantly lower activity compared to Conjugate Sbml.

Example 2882 Activity of tumor cell targeted Nal’iZh ABC in cancer ’llMC consultures OVCARB—NucRed cells were seeded in 96mwell tissue culture plates (20,000/well) and allowed to attach 6 hours in RPh/llnlo4t) medium with 10% FBS and 1% penicillin/streptomycin. e medium was replaced with fresh media (50 uL). Test articles (3): concentrated, Conjugate Sl and Compound 1 (each at 100, 10, and 1 11M) and Conjugate Rm (100 old); conjugate concentrations were based on the payload) were then added to each well in media (50 uL). The assay was performed as described in Example 28A except that 30,000 PBMCs were used. The number of red objects (cancer cells) were quantified using the lnouCyte© Zoom re Red object numbers in each well was normalized to its own T:O time point red obj ect numbers.

EEG. ill plots the red object numbers as a function of time and shows the robust induction of cancer cell killing by PBh/le in response to Conjugate Sl at a lOOX lower payload concentration compared to Compound ll Conjugate 3m had no significant activity and the se in red object nurnber (cell growth) over time was similar to the untreated control. Inset shows that none of the test articles (l 00 nM) inhibited growth of OVCARES —NucRed cells in rnonocultures. e 29: Flow cytometry analysis of €314, Fey Receptors, and C133 expression in PBMCs and isolated te subpopnla’tions [6543 Frozen human PBMCs (lxl 03) were thawed and aliquoted into three tubes: one aliquot was subjected to human yte enrichment [StemCell Technologies} ("cote depleted tes”), one aliquot was subjected to human monocyte enrichment without {iDlo depletion ell Technologies] (”enriched monocytes”), and one aliquot was not subjected to enrichment {”PBh/le“). For flow cytonietry, cells 0) from each group were transferred to a U—bottoin 96—well plate 1114replicates washed with PBS and d with live/dead fixahle Aiqua dead cell staining dye 1:Molecular ) ed by staining with fluorophore conjugated target specific (triplicates) or isotype control dies (Pacific Blue anti—human CD14, Fl'l'C anti" hu1nanCD3 APC/Cy7 CDlo PE anti-human CD32 PE/Cy.1 anti—human (31364). Cells were fixed and surface expression of the proteins of interest was determined by flow cytonietry analysis on a MACSQuant flow cyton'ieter. Data analysis was performed by Flowlo software. Table 3 provides the ncy (% of single/live cells)) of CDlél-l’CDlé-t, (:D14-/’CD16, CD14" zoos/'31,. CD141/Ct3321. eminence, CDl4+,/CD64+, and {1314—1033: cells in PBMCs, enriched tes, and CDlt’jndepleted yte populations Table 3 C1316+ CDl6+ C1332+ C‘1)32+ (3964+ $364+ CD3+ 1.11.1131 211131.118 l5.5:1:.08 311 .1c.11»-133475.1(17 Cllléllepleled 58:13.5 242:1 1 4e.s__1_1.5 .,:01 Monocvtes *Gated on single/live cells Table 3 shows efficient depletion ofCD3+ cells and enrichment of cytes, and diminished levels of CD16 positive cells in the CD16~depleted monocytes postisola‘tion, (11364 staining results indicate that all CD14 positive cells express (11364 (Fcle) in PBMCs as well as in the enriched monocyte suhpopulations.

MG. 2 demonstrates efficient depletion ofCD3+ cells and enrichment of n'ionocytes, and diminished l eyels ol' (IN 4 positive n the CD16depleted inonocytes post isolation Example 3%: Cancer celluliilling ty of the Fc-mntant tumor cell targeted Her}! ADC in in: vitm co—cnltnres ol'" PBMCs and S'l‘th} wild type or lsnocli out SEER} cells {657} Generation of STING knock out single cell clones expressing nuclear cted niliiate fluorescent protein: SKBR3 cells were seeded in 24 well plates (SQUOO/well) and transfected with a non—targeting ngNA and three different ngNAs targeting the human S'l'lNG gene using the TrueGuideTM Synthetic gRNA, TrueCutTM Ca39 Protein VZ, and LipofectamineTM CRISPRMAXTM Transfection Reagent from Thermo Fisher according to the manufacturer’s protocol. ngNA sequences were: sgNT (non—targeting): AAAUGUGAGAUCAGAGUAAU; sg#3: 'l‘AC'KXIC'l‘CCCAAA'l‘GCGG'lT; sg# : C'I'CGil)AGGCAC’I'GAACATCC; and sg# : G'lII‘AAACEKIGGTC"I‘EKIAGCC Seven days post transfection, single cells were sorted in 96—well plates containing 100 [L of DMEM with 20% FBS and 1% penicillin!" streptomycin and clones were fonned in 2-3 weeks (media was refreshed once—twice a week). Multiple clones were tiypsinized and ed to analyze STING sion by Western blots using rabbit monoclonal anti-STING (Cell Signaling logies) and anti—B—Actin (Licor) antibodies. The clones with no STING protein expression (as determined by n blots) were selected to stably express the nuclear restricted mKate fluorescent protein as described in Example 28‘ Killing assay: STING wild type (sgNT-Z: Non-targeting ngNA, clone 2) and knock out (sg#3-2: ngNA#3 clone 2) SKBR3 cells expressing NucRed were seeded in 96 well plates (liOOO/well) in RPMI with 19% FBS and 1% penicillin/ streptomycin and the PBMC killing assay was performed as described in Example 29 using Conjugate Sa—S, Conjugate Sj, Conjugate tit—2, and Compound ’1 each at. ”EGO? 25, 5 and l nM (conjugate concentrations were based on the payload). {659} and FIG. SB plot the red object confluency as a function of time and shows the killing of STING wild type (sgNT—Z) SKBR3 cells / PBMC co—cultures and knock out (:sgiifiB-Z) SKBIB cells I’BMC co—cultures respectively. Conjugate 8a—3 induced robust killing of both STING wild type and ltnocl: out SKBlB cells/I’BMC cocultures at all doses . Conjugate 8j had high activity at 100, 25, and 5 nM in STING wild type (sgN'I'—2) SKBR3 cells / I’BMC con cultures and low activity in the STING knock out {sg#3—2} SKBR3 cells f I’BMC co—cultures. nd 1 showed activity in S'I'ING wild type (sg'N'll—Z) SKBR3 cells / PBMC co—cultures at 100 nlvl only and no activity in the STING knoclt out (sgtitB-Z) SKBR3 cells .1" PBMC co—cultures at all doses. Conjugate 801 did not have any activity in either STING wild type (sgNT—Z) SKBR3 cells I’BMC tures or S'I'lNG knock out (sgthZ-f) SKBR3 cells / PBMC tures.

Example Lilli/t: Activity of tumor cell ed Her: ADC and HerZ antibody in S’i‘LNG wild type or SIlNG knock out SKBR3 cancer cell/PBMC co—cnltnres STING wild type 'nZ: non—targeting; ngNA, clone 2) and knock out (sgt’t3n2: ngNAtfi clone 2) SKBR3 cells expressing NiicRed were co~cultured with PBMCS and killing assay was performed as described in Example 30 using a dose range of Conjugate Sit—3, Conjugate 8nj (200 nM based on payload? 4x dilution) Unconjugated wild. type Fe Trastuzumab and AAG Fe mutant Trastuzurnab were closed based on the antibody concentration corresponding to Conjugate 8a—3 antibody concentration. As shown in li’IGo 4A and l?1G. sill Conjugate 821-3 exhibited robust killing of both S'l'lNG wild type and lrnoclc out SKBRB cancer cells, whereas the Fc mutant l-lerZ—targeted ADC, Conjugate S—j showed killing ty only in the STING wild type SKBlU co—cultures, which was nearly diminished in the S'l'lNG knock out SKBR3 co—cultures. Both Fc wild type and AAG mutant unconiugated 'l‘rastuzuinah antihodies showed low activity in both STING wildtype and knoclt out cancer cell coucultures. These data demonstrates that the cancer cell killing activity of the Fc niutant cancer argeted S'l‘lN G—f‘iDC ty in immune cell co—cultures is contributed front the tumor cell—intrinsic S'HNG activation. e 31: Activity of tumor cell targeted NaPEZh ABC in cancer celllhuman munocyte co— cnltures in the absence el‘ '1‘ cells [661} G‘s/"CAR3—NucRed cells (generated as described in Example 28) were seeded (l 5,0r’li’l/well) in 96~well CellBind e tissue culture plates (Corning) and d to attach for 6 hours in RPMlnloélO medium with 10% FBS and 1% penicilhit/streptomycin. Culture medium was replaced with fresh media (50 uL). Test articles (Conjugate Sbnl and Compound 1, each at 20 and 4 nlx/L conjugate concentration based on payload concentration) were then added to each well in media (50 uL), and the plate was ted for 20 min at '37 “Cr PBMCS, enriched monocytes, and CDlovdepleted nionocytes were prepared as described in Example ‘29, Live PBMCS (3 0,000/well), enriched inonocytes (20,000/well), and (it‘ll o—rlepleted nionocytes 0/well) were then added to the wells in culture medium (50 Rule) and the plate was placed in an lncuCyte© live cell irnaging instrument in an incubator (37 ”C, 5% O2) and scanned every 4 hours over 2 days. The number ofr'ed objects (cancer cells) were quantified using the lncuCyte© Zcorn software. Red object conthiency in each well was normalized to its own TIC} tirne point red object confluency The red object confluency is plotted in FIGS. 5A to SC as a function of tinre and Sl’lO‘WS that (1293+ T cell-depleted rnonccyte populations have comparable cancer cell g activity in response to turnor cellstar'geted ADC activity. ate Shwl, at 4 nM and 20 anl payload concentrations induced robust killing of OV’Cr‘iRSwNucRed cancer cells by PBMCS (FIG, 5A), enriched rnonocytes (FIG. SB), and CDlé ed rnonocytes (). Compound 1 at 20 nM induced cancer cell killing only in enriched nionocyte and CD1 «fa—depleted rnonocyte co~cultures Example 32: in vitrn measurements of human {:XCLM and cell binding nf HERE ed Antibody Drug (Ennjugntes to HCClQSét human breast cancer cell line [662} 1954 breast cancer ceils were grnwn tn ~811—95‘Vu cnnflueney in RPME 1640 medium supplemented with PBS (1 %) and pemc1ll1n/st1epton1v1n (1%). Cells were harvested and added to wells of a l flat bottom plate (40,000/well) and incubated overnight at 37 0C, % (:02. Cells were then treated with test HERZ antibody~drug conjugates (20 11L} as indicated in Table 4, at concentrations ranging n 1 pM and l0 1.1M, and incubated for 24 hours at 37 0C, % C02. Plates were centrifuged (300 g 5 minutes), the supernatants were collected (100 mL) and subjected to ELISA analysis for human CXCL l 0 (Human CXCLI O/lll l O). Developed plates were read at 011450 on a Spectrah/lax MS plate reader. The values for each treatment were plotted, and 11135values calculated with GraphPad Prism software using a fourparameter curve fitting.

For the determination of cell hinding ofHERZ antibody—drug conjugates to HCC1954 cells were grewn t0 ~80—950/o cenfluency in RPMI 1640 medium supplemented with PBS (1119o) and penicillin/streptomycin (1%). Cells were harvested and added to wells of a 96—well V bottom plate (SOfiOOO/well} The cells were pel1eted (300 \ g 5 minutes) and resuspended in ons of test HERZ antibody—drug conjugates as indicated in Table 4 at concentrations ranging n 00 l 11M and 100 nh/l' and incubated on ice for 3 hours. Cells were then washed in ice cold PBS (3);), ed (300 g 5 minutes), and incubated with detection antibody (_Goat Anti Human lgG —Alexa~ 6-47 (H +1.1 chain) for 1 hour at 4 0C. Cell suspensions were ed (300 g, 5 minutes) washed 3 times in ice cold PBS and tixed by resuspending 111 solution of paraformaldel1yde .) Resuspended cells were then subjected to tlow cytornetry analysis on a, MACS Qttant Flow Cytometry. Single events (10,000) were collected for analysis tion gating and Mean Fluorescent Intensity (11/1171) analysis was performed with FlowJo Software. [664} Table 4 summarizes the mean 13650 values for cell binding and CXCMO induction in 11011954. . lCXCLlO ELISA Cell BindingE0511 1 7 1est Articlesfl DAR. l1:Lot (11M) (nM) 1 (301111 ate 25 6.6 0.l3 iCon unate asI 6.5 0.17 6.8 10.51 = Con uuate 28 f“3 0.16 . .5 10.18 1‘ .CXCLIO ELISA Cell Binding, We 65 020 \D Con agate 64 CL)ii negate ’74 >l0 l6 -_‘f‘5 igate 662 I I I I . s I .....................................................................+................................................. ...........................................g I I Con u I ate 32- 1 I ND Not Determined [665} As shown in Table 4, treatment of HCCIQS4 cells with HerZ targeted dy—drug conjugates resulted in suh— to low nai'iomolar ECso values for ii'iduction of CXCLIO. Where determined, ECso values for binding of llerZ targeted ADCs to HCCl954 cells were also in the low nanornolar range.

Example 33): Activity nl‘tunier cell targeted ltlERZ Antibody Bring Conjugates in cancer cell/’PBMC cemeultures [6663 Cancer cell killing activity: SliBRB human breast cancer cells stably expressing the nuclearurestricted niKate fluorescent protein were generated as described in Example 28A and designated as SliBlB NucRed cells. 20,000 cells l:per well) SKBlU NucRed cells were seeded in 96—well tissue culture plates and allowed to attach 6 hours in 50 uL of lU?hxll—164Cl medium with % H38 and 1°/0 penicdlin/streptonivcni A range ot dilutions (0 01 nM to 200 nM based on payload; 4wfold serial dilutions in growth medium) of the 50 uL test articles gate Sam; Conjugate 8—3, Conjugate Sell and Compound 1) were added to each well and the plate was ted for 20 min at 37 0C. PBMCs er primary human monocytes (isolated from PBMCs as described in e 29) (50,000) were then added to each well and the assay was performed as described in Example 28. Table 5 shows the leo values (killing activity) of the test articles in cancer cell / PBMC and isolated primary human monocyte ltures (cancer cell killing activity). _rticle'Eest ‘ , 1on1 ugate 8c—2 R1111:1111111d l Carine $111111? (11:1 “\IA EC50 'nh’l)- Monecvte__5 '31 _11—5l [667} As shown in Table 5, Conjugate 8:112 exhibited ~810th and ~lSOx greater potency compared to Compound l in PBMC and yte co~~eultures respectivelV. The potency of Conjugate 8“} was ~30); and SGX lower relative to Conjugate 3:142 in PBMC and monocyte co~ cultures respectively: Conjugate 811—2 had minimal activity in both PBMC and rnonocyte co- cultures.

CXCLH} induction: Table 6 shows the ECsn values of the test articles in cancer cell PBh/lC and isolated primary human monocyte coecultures for CXC‘LlG induction.

Table 6 Test A1title lNA :61 l 3 As shown in Table 6‘, Conjugate 83.le exhibited ~400x a11d ~700x greater potency for CXCLlO induction compared to the Compound 1 in PBMC and nionocyte co—cultures, respectively, The potency of Conjugate 3~j was ~50); lower relative to Conjugate 8a~2 in both PBMC and te tures. Conjugate 81:12 had minimal activity in both PBMC and n1onocyte co~eultures. [670} Type III TEN induction: Co—cultures of SKBR3 cells and PBMCS were set up as d eseribed above to analyze the ion of Type III lnterferons in supernatants 24 hours post ent using a human lLZQ/ILZS‘U ELISA kit Table 7 shows the ECso values of the test articles in cancer cell/”PBh/lC coecultures for LZSh induction (IFNM ,Ipfi)’.

Table ‘7 i i lest Article ‘ ' 1 ‘ , , , , 1on1Augate 8c—2 A11111111111d l “(:50 ‘ {ERA — light/EC [671} As shown in Table 7, ate 8:1.“2 exhibited 14001: greater potency for ILZEP/ILZSb induction compared to the Compound 1 in PBth concultures. The potency of Conjugate 8~j was ~65; lower relative to Cnnjugate 83-1 in PBMC cnucultures. Conjugate Sic—2 had mininral activity in PBMC cn—cultures {672} The data in Tables 7 -9, demonstrates that mutation in the Fc region of ADC that abrogates FcyR interactions reduces but does not eliminate the cancer cell killing activity of the targeted ADC y suggesting an li'cuindependent contribution of the ADC.

Example 34: tlen {if STlNG pathway in immune cells The induction of STING pathway in immune cells by Her2~targeted S’l‘lNG ABC was evaluated by a cancer cell/”l‘HlPl~lRF3nLucifei'ase reporter cell cor-culture assay. SKOV3 human evarian adenocarcinonra cells were seeded in 96wwell CellBind e tissue culture plates (20,000/well) and allowed to attach for 6 hours in McCoy’s 5a medium with l0% PBS and l% penicillin/streptomycin. A range of ons (0.01 nM to 300 nM based on payload; 4—feld serial dilutions in growth medium) of the test articles Conjugate 32—5, Conjugate 32c and Compound 30 were added to each well and the plate was incubated for 20 nrin at 37 °C 50,000 THPlvdual er cells were then added to each well and the incubation continued for 20 hours at 3’7 °C in a humidified atmosphere of 5% CO; Cell e supernatants (20 uL) from each incubated sample was added to resuspended ~I.,uc (50 lull) and the luminescent signal for 1RF3 was measured immediately using a SpectraMax M5 plate reader (Molecular Devices). The ECsc- value was determined from the dose response curve. Table 8 provides the ECsc values in THE-W ~Dual cells co~cultured with SKOVB cancer cells: Table 8 Test Article ° ‘uga’te 32c Cum mind 30E As shown in Table 8, treatment of SKOV3 and nl concultures with Hei'Zntargeted ADCs resulted in suhnanomolar to low nanomolar ECso values for induction of STlNG pathway in the THP~1 immune cells.

Example 35: Activity of turner cell ed HERE Antihnily Bring Conjugates in cancer cell/PEMC cevcultures [675} CXCLM inductinn: Calu3 human lung adeiiccarcinoma cells were seeded in 96—well tissue culture plates and allewed to attach overnight at 37 0C, 5% C02 in l00 ul, of ENlEM medium with 10% FBS and 1% penicillin/streptoniycin. Culture medium was replaced with fresh media (100 uL). A range of dilutions (0.0.1 nivl to 200 nM based on payload; 4—fold serial dilutions in growth medium) of the 50 uL test articles (Conjugate 32—4, Compound 30 and Conjugate Slit—l were added to each well and the assay was performed as bed in Example 33—CXCL1 0 ion. Table 9 shows the ECso values of the test articles in cancer cell ./' PBMC and cancer cell monocultures for CXCLlQ induction.

Table 9 'l‘est Article ‘4 lon‘uuate 32h~l Elialttllill: E As shown in Table 9, Conjugate 3'24 exhibited subnanornolar potency ed to Compound 30 in cancer cell/PBMC co~cultures and monocultures respectively. Conjugate 32bnl had minimal activity in both cancer BMC co—cultures and ltures Example 36: Activation of the STING pathway in cancer cell monocultures in the presence of conditioned media from untreated immune cell cultures [677} Conditioned media was prepared by ing 1.5 \ 106/inl, PBMCs (two different donors), l \ 106/mL primary monocytes (isolated from PBMCs from two ditt‘erent donors, as described in Example 29), or l x l06/mL THPl cells in REPNlLlor-‘ll'l medium with 10% PBS and 1% penicillin/ streptomycin for 24 hours at 37 0C, 5% ()2, in an incubator. The atants were collected and spun down at 2000 rpm for 10 min to remove any cells SKOV3 cells were seeded in 96 well plates (30,000/well) and allowed to attach overnight in RPh/ll—1640 medium with 10% FBS and ill/is penicidin/streptomycin. Culture medium was replaced with either 100 uL/well of fresh media ol) or conditioned media from untreated immune cells followed by the addition of Compound 1 (50 uL/well, 100 nM final concentration) or control media (no treatment) to each well, After tion for 24 hours at 37 “C, 59%: O2 supernatants from the 96 well plates were analyzed for CXCLlO production using a human CXCLlO ELlSA lcit. Table 10 shows the OD. 450 values for (IXCLlO produced by the SKO‘V3 cancer cell monocultures.

Table 10 Q1)” 4'56 Control 9-13. 450 Conditioned MediaA (No S’l‘lNG aeonist treatment + 100 nM Commmg 1 0.02 -+—/— 0.023 0.16 -+—/— 0.038 0.47 -+—/— 005.3 2.22 -+—/— 0.034 0.13 -+—/— 0.019 0.83 -+—/— 0.030 0.04 -+—/— 0.010 1.62 -+—/— 0.05.0 h/lonocyte {DonorZ} 0.21 +/— 0.018 0.97 +/— 0.142 THPl 0.00 »+-/— 0.007 0.18 »+-/— 0.043 [6’78] As shown in Table 10, SKOV3 cells responded to STING agonist treatment in monocultures only in the presence of conditioned media from PBMCS and primary human monocytes but not from THP1 cells, thereby suggesting that PER/{Cs or y human monocytes secrete factors that can sensitize cancer cells to STING agonist treatment.

Examgle 37: Tumor Growth Response to Administration of HERZ Antihridywflrug Conjugate in SKQVS [679} Female €8.17 SCID mice were inoculated subcutaneously with SKOVE human ovarian cancer cel1s (10 x 10*" cells per mouse}. Animals were randomized into ent groups when tumor volumes were between mm3 (mean 65.4rnni3/group) 10/group). Vehicle, Trastuzumah (3/0 rng/ltg), {liABZl STTNG agonist (0/5 mg/kg), Conjugate Seal (1/004 rng/ltg or 3/012 mgr/kg), or Conjugate Saul (1/003 mg/kg or 3/009 rug/leg) were dosed intravenously as a single dose on day 1 (all doses are given by antibody/payload). Transient body weight loss, within able limits, was observed 2~3 {lays following treatment, with no additional clinical observations, for diABZl STING agonist (0/5 mg/kg) and Conjugate 80ml (3/012 trig/kg). Body weight loss at later timenpoints correlated with tumor progression, indicating tumor modelninduced caeliexia. [680} PEG, 6 provides the s for the tumor volumes of SKOV3 bearing mice treated with 'I‘rastuzumab, diABZl STING agonist, Conjugate 80—1, Conjugate Sad or vehicle. Treatment with Trastuzumah (3/0 , {liABZI STTNG t (0/5 rng/kg) or Conjugate 80ml (1/004 mg/kg or 3/0.le mg/kg) resulted in 47.5, 36,9, 13.5 or 25.5 % TGL respectively. ent with Conjugate Saul (1/003 mg/kg or 3/009 nag/kg) resulted in 86.5 and 100% tumor regression, respectively Example 38: Serum {3ytokines Following Administration of 8123312 Antibody—Bring Conjugate in SEQV3 Female (78.1? SCID mice were inoculated subcutaneously with SKOV3 human ovarian cancer cells (10 Xli)6 cells per mouse). Animals were randomized into treatment groups when tumor volumes were between l08-l72 mrn3 (mean 128ul3lo mmg). Vehicle, diABZl S'HNG agohist (0/5 rng/kg), Conjugate Sit—1 i 3/012 rug/kg) or ate Sa—l (3/009 nig/kg) were administered intravenously on day 1 (all doses are given as antibody/payload, 11:10 for each group). Serum was collected at 6, 12, 24, and 72 hours post—dose (n=5 per group) and snapmfrozen on dry ice for serum cytokine analysis. 'l‘umors were collected at 12 and 72. hours postwdose (r1 = for each time point) and processed into fornialinnfixed paraffin embedded (FFPE) blocks.

Serum cytoliines (Eotaxin, G~CSF, , IFNV, anltx, lbw, lL—IZ, 11—3, an4, an5, ice, 1L7, lL—Q, lL—lO, iL—iz (p40), lL~lZ (p70), an13, an15, $17, CXCL'lO (rein), CXCLl (KC), LlF, LIX, l‘vlCP—l, l‘v‘lnCSF, l‘vflG, l‘xflP—ltt, l‘vflPwlB, l‘v’flPnZ, RANTES, TNFd, and VEGF) were analyzed using mouse cytokine/chemokine magnetic bead panel on a lapfl) inex instrument. Belysa linniunoassay Curve Fitting Software was used for data analysis.

FEGS. 7A~7H provide measured cytolzines as a function of time. Only cytokines with a measurable increase relative to e control are shown (CXCL—l 0 (lP~lO), lL~6, TNFn, ll'i‘Ny, CXCLl (KC), MIG, MlP—l u and RANTES), inserts in each plot show ne levels induced by Conjugate Sa—i and Conjugate Sic-l relative to vehicle. The intravenously stered diABZl STING agonist induced significantly higher levels of serum ines than Conjugate 321—1 or Conjugate Sc—i, with fold differences as high as l 00 fold for lLo and as low as 6 fold for CXCLl 0.

For most cytolrines, there was no significant difference between control and targeted ADCs, and few cytokines showed difference between the vehicle control and the ADCs. Y-axis scales in the main plot and insert ize the difference between diABZl STING agonist and the rest of the treatments in this study.

Example 39A: Pl) Responses Following Administration of HERE dy—Bring ate in SEQ)V3 {684} Six-week—old t‘emale {78.17 SUD were inoculated subcutaneously with SKOVB human ovarian cancer cells (10 X 106' cells per . Animals were randomized into treatment groups when tumor volumes were between 75-1226 nnn3 (inrean 94.5-96.7 oup). Vehicle, Conjugate Sit—1 (3/012 mg/hg) or Conjugate Saul (3/009 trig/kg), Conjugate 8cm} (3/012 nrg/kg) or Conjugate Saul (3/009 mg/kg) were administered intravenously on clay 1 (all doses are given as dy/payload, n===12 for each group). Serum was collected at 6, 12. 24, and '72, hours post- dose (n===6 for each group) and snap—frozen on dry ice for serum cytolrine analysis (data not shown).

Tumors were collected at 12 and '72 hours (n====6 for each group) and fixed in FFPE bloclrs for further processing.

For Real Time qPCR Analysis, RNA was extracted from the 14141315 hlocks using the Qiagen Rneasy FFPE lrit. Samples were equalized hased on rianodrop g and cDNA produced using the '1‘herniofisher SuperScript 1V V1L0 Master Mix with exDNase Enzyme Gene expression assays for mouse CXCLl O, interferon" beta. and the were set up with the TaqMan Fast Advanced Master Mix. A131 assays for mouse Interferonnfi (IFNh), 1136. and CXCLlO were used with GAPDH, and R1’L3i’) as housekeepers. Levels ofinRNA relative to GAPDH were calculated using Z'At‘CT .

FEGS. SAVSC provides levels of mouse CXCLlO, interferon—l3, and 11..»6 mRNA in SKOV3 tumors measured at 12 and 72 hours Treatment with Conjugate Saw]. resulted in the highest levels of CXCMOI, Interferon—h, and 1L6 mRNA, at 12 hours relative to the Vehicle or Conjugate 8434. For Conjugate Sal, CXCLlO, interferon—l3, and Hui—o mRNA ayes all decreased at 72 hours. [687} FIG. E9pr'ovides CD45 irnniunohistocheniistry (111(1) staining of FFPE tumor tissue sections with rabbit anti—CD45 onal antibody at 12 and 72 hours for ate 821—1, Conjugate Sit—1 or vehicle. As shown, there was increased CDzlS—positiye mouse immune cell ation into the 8K0 73 tumors in SCID mice at 72 hours post treatment with Conjugate Sa—l.

Exarnole 39B: Empress-ion of STENG pathway genes in 8K0 '73 human tumor xenogral‘ts in SCH) mice in response to HERZ Antihody-fir‘ug Conjugate. [688} Female (313.17 SCID rnice were inoculated subcutaneously with SKOV3 human ovarian cancer cells and treated with Conjugate Eta—i (3/009 ing/kg) as described in Example 38. Tumors were harvested and processed into PEPE. as described in Example 31. RNA was extracted using the Qiagen Rneasy FFPE kit according to kit ctions. 150 ng RNA per sample was analyzed on NanoString ei' Max system using the nCountei' ncer human or mouse inimune profiling panels and nCounter Standard Master Kit. 1‘ables ll shows the logZ fold changes of selected S'I'ING pathway genes at l2 houi's.

Table ll.

Human Mouse Gene Name 3LogZ fold,.

Std liner {‘1ng fold- Std EE‘FOR" Lhange {name CXCII0 8m 5.0482 4.82 ' ' 3 . ithi 0 334 6.68 0.542 WI III (III I72 0.403 New IIcaIIIe NoIIIetecIecI As shown in Table ll a single administration at Conjugate 8a 1 resulted in inarkId induIti on of both mouse and human STING pathway genes, suggesting that the tumor—targeted SHNG agonist ADCs may induce intrinsic STING pathway activation in tumors in viva. e 48: l3harmacokinetic analysis of Hers}? targeted ADC in €33.17 SCH) mice [690} Ten-weekold female CB l7 SCH) inice weie dosed intravenously as a single dose on day l with vehicle or Conjugate 8a-l (3/01 mg/kg} (doses are given as antibody/payload, n23 for each group). Blood was seiially collected tiom all animals at l, 24, 48, 72, 96, ”E68, 246, and336 hours following treatment (n23 for each group) Whole blood was inunediately diluted l:lO with acidic buffer (06% BSA (VII/,SV) mM EDTA in 100 ml”, PBS + 15.34ml 10 L citric acid), for a, total volume of Ol niL. Diluted wl'lole blood was snap-li‘ozen on dry ice and stored at ~80 0C until analysis for total antibody and conjugated drug. [691} MG. 19 provides the results for circulating plasma concentrations of total dy and conjugated drug. Plasma concentrations of~25.l ug/n'iL and ~046 ug/mL were achieved for total antibody and ated drug, respectively, and a corresponding clearance rate of ~617l rnL/day/kg for total antibody and ~20}: rnL/"day/ltg for conjugated drug. {692} Similarly, Conjugate 3253 (1.14/004 nigx’ltg) was administered, and total antibody and conjugated drug was assessed at 0.25, 24, 72, l68, 240, and 336 hours. Data is reported in Table Table 12 Cniax (ng/mL) ife (day) AUG» Cl_obs (Dayl‘ng/nilu) (rnL/Day/Kg) Conjugated 562:16 , _ 2880:468 14.2:251 Total Antibody l2200 :l: H60 869:l: l (20 96300 :l: 7l 90 l l.9 :1». 0.88 Example 41: Expression of S'l‘ll‘tG pathway genes in SKBR3 cancer cell / PBMC com cultures in response to Hei'zstargeted STING agonist ADC treatment. [693} riag Analysis: Cancer cells were seeded (250,000 cells/well) in ates in l2 well plates in 075 mL culture medium (RPMl—1640 medium with l0% PBS and l% llim’streptomycin) and allowed to attach at 37 0C in a 5% C02 incubator overnight Culture medium was removed and Conjugate 8a~3 or vehicle was added into the wells in 0.5 mL culture medium at a final concentration of 50 nM (based on payload). 500,000 PBMCS per well were added to each well in 0.5 mL medium. After 5 hours of incubation at 37 °C, the suspended cells were collected in an Eppendorftube and spun down . Supernatant was removed and the tube was placed on ice. The attached cells were lysed with the RNA lysis butler and transferred onto the suspension cell pellet. RNA was extracted using the Qiagen Rneasy rnini kit according to kit instructions 150 ng RNA per sample was analyzed on NanoString er h/lax system using the nCounter pan—cancer human immune profiling panel and nCounter Standard Master Kit, Table l3 shows the logZ fold changes of the selected STING pathway genes by Conjugate Ella—3 vs vehicle treatment (5 hours) in SKBR3 cancer cell and PBMC co—cultures in Vitro 50 nM Conjugate Eta—3 ent led to marked induction of S'l‘lNG y genes and the type lll lnterferons (IFNM and lFNQlUZ), confirming that the tumor-targeted STING agonist ADCs treatment induces STING pathway genes as well as Type III lnterferons also in in with) cancer immune cell eeueultures.

Table 13 Logz feld P—Vaiue 7 Gene Name Std liner» Change ' {PIT} ...............................................................................................................................................t i : , . SA-tE~t)8 IFIHI .. .; 64913—08 my? ' .I , . 2.92E—06 [695} thCR is- M’ type Hi interfere“ genes: SKB‘EB/PBMC (JO—cultures were set up as described above and treated with 5G HM and 1 HM (based en d) 0f Conjugate 33—3 or Conjugate Sc—Z for 5 hours. Cells were ted and RNA was extracted as described above. qPCR analysis was med as described in Example 39A. ABE assays for human {FNM (11929), {FNAZ (114283), and EFNM (lLZSb) were used with GAPDH, and ACTB as housekeepers. Levels of mRNA relative to GAJ,’D}-{ were calculated using Z‘AACT methed as ShOWIl irr Table 14.

Table 14 Relative mRNA Expi'essien Treatment, ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ IFNM (ILZQ) EFNIsZ (1142821) IFN33 (ILZSb) Cenjugate 83-3 50 nM 190.5 £189 297 i 2.0 Cenjugaie Ste-2 ——1 nM —-—.— [696} Table 14 shows the inRNA expression of type III interferons relative to the eeping genes in response to targeted ADC and control ADC treatments of the cancer cell PBMC eo- eultures. Conjugate Sit—=3 treatment at both 50 nM and l nl‘vl payload dose induced marked upregulation of type 111 interferon genes. Conjugate Set-:2 treatment did not induce type 111 erons icantly at the doses used in this ment.

Example 42: luduetien of ’I‘ype HI Interferons in STING wild type or knock out SKBEB cancer cell and PBMC ice—cultures [697} STING wild type or knock out cancer cell (as described in Example 30) and I’BMC con cultures were treated with Conjugate Sa~3t Conjugate flint, Conjugate 80-2 and Compound 1 (0.0.1 nM to 200 nM based on payload; 4—fold serial dilutions) and Type III lnterferons lFNM/ IFNAS (IL29/1L28b) cytoltines were measured as described in Example_3 — Type III Inteiferon Induction.

Table 15 shows tlie Type III lnterferons IFNM/ IFN‘M (lLZ‘Q/ILZSb) cytokine production in STING wild type or STING knock out SKBR3 and PBMC ltures in response to Conjugate 83,43, Conjugate 8334., Conjugate 8&2 and Compound 1.

Table 15 Conjugate Elia—3 Conjugate 313—1 Conjugate 8t~2 nd 1 Cancer Cell Line ECse Bantu: Bmax Ellso ECsa , Binax l’, 3 3 =, {ER/i3 _ (313459) ({3});er “EVE STING wild type STlNG ltnoel: out STING knock out ’s RNAtiau {699] Both Conjugate 321—3 and Conjugate Sj—l induced robust Type III Interferons lFNltl/ 117 NA?) IL28b) eytokine production in STING wild type SKBRB eell tures. Compound 1 also induced 'I'ype Ill lnterferons IFN’Al/ IFNM lLZSh) production in this eo-cultui‘e setting but with a ~4OOX lower ECso concentration whereas Conjugate 80-2 had no significant activity. The amount of lli'Nll/ llE'NB (IL29/28l3) production in the STING knock out SKBR3 cell co—cultures was niarltedly reduced in response to all treatments as evident from the very low Bniax (OD450) levels.

Example 43A: Effect el‘lh‘NM (1129) er IFNAZ (ILZSA) neutralizing antibodies on the killing activity of the Her2—'l’argeted S’ETENG ABC in cancer cell and PBMC eomcultures [7001 Cancer cell killing activity: SKBR3 and PBMC tutes were conducted as described in Example 33 'I'ype Ill eron Induction to assess the killing activity of Conjugate Sta—3: (l nl‘vl or 0.1 nM based on payload) in the presence of IFNM (IL29) or IFNM (le813) neutralizing antibodies (16, 2, 0.4, 0.08 ug/rnL). As shown in FR}, 11 the IFNM neutralizing antibody at l0, :2, and 0.4 ug/inL inhibited the cancer cell killing activity Conjugate 8an3 at 0.1 nM (based on payload) in PBMC cowcultures. No tion was observed with the IFN?»1 antibodies when Conjugate 834:} was used at the l anI (based on payload) IFNhZ neutralizing antibodies did not inhibit cancer cellnltilling activity even when Conjugate 83—3 was dosed at 0.1 nM Example 433: EFNM/ IFNAS (’L29/28b) eytokine induction Sister plates of the assay described in Example 43A were used to analyze IFNS‘il/ IFNB (II..29/28b) in the supernatants 24 hours post treatment using a human ILZQ/‘Zfib ELISA kit as bed in Example 33 — Type III Interferon Induction Table 16 shows the DIM-50 values obtained from each ion, Table 16 Conjugate lama," 1min = IFN M niAb ‘ (lL29/"28b) = 0134503 :. 0,034 .................................................................................. : 0.008 Example 43C: CXCIJIL lFNfi, Eliot and TNFu Cytekine Induction atants from Example 438 were used to analyze for CKCLl 0, IFNEB, lLé and TNth cytokine production using a human ne/cheinokine magnetic bead panel on a Flexk/Iap3l) Luminex instrument. Belysa Immunoassay Curve Fitting re was used for data analysis.

Table l7 shows the CXCLl 0, IFNB, 1L6, and 'l'NFu cytokihes ed in the supernatants.

Table 17 [703} Together these data demonstrate that 'l‘ype Ill Interferon production is important for the tumor cell~targetetl S'l‘lNG~ADC activity, especially at the low concentrations.

Example 44: Tumor Growth Response to Administration of Nafilb Antibody—Bring Conjugate in Q'V'CAkmfi Five weeknold female CB. l7 SCH) mice were inoculated subcutaneously with OVCARUB cells (5 x 106 cells per mouse) Animals were randomized into treatment groups when tumor volumes were between S2—l 80mm3 (mean: @394 roup). e, diABZl STING agonist 0/5 mg/kg), Conjugate Sew} (3/l'll2 rng/kg), ate Sly]. (3/009 mg/kg), or Conjugate 8f Z rng/kg) were dosed intravenously as a single dose on day l (all doses are given by antibody/payload n=8 for each group group). Transient body weight loss was observed 3~l3 days following treatment with no additional clinical ations for diABZI STING agonist (0/5 mg/kg) ate Sol, Conjugate 8b]. and Conjugate St: [705} MG. 1?; provides the results for OVCAR~3 tumor-hearing mice treated with vehicle, diABZl STING agonist, Conjugate Sit-l, Conjugate Sit—l or Conjugate 8f. When compared with vehicle control, treatment with Conjugate Sc—l (3/012 nig/kg) resulted in a mean increase in tumor giowth ot 706°/o, Treatment with diABZl 81”th} agonist (0/5 nig/kg) iesultedin 427% TGE Treatment with Conjugate Silt-T (3/009 mg/kg) resulted in 98.4% TGl. Treatment with Conjugate 8f resulted in 83.1% TGT.

Example 45: Tumor Growth Response to Administration of Chimeric {thGli-mlgflaa) NaPiZh dy—Brug Conjugate in GVCAR-S [706} Female CB. 17 SCTD mice were inoculated subcutaneously with OVCAR—3 human ovarian cancer cells (5 X 10" cells per mouse). Animals were ized into treatment groups when tumor volumes were between 53~223 mm3 (mean = 112n122mm3/group). Vehicle, Conjugate SdmZ (3/’O.l mg/kg), or ate 8e (3/01 rug/kg) were administered intravenously on day 1 (all doses are given as antibody/payload, n=8 for each group). Transient body weight loss within acceptable limits was observed 3n15 days following treatment with no additional clinical observations for Conjugate 8d~2 (3/91 mg/kg) and Conjugate 8e (3/91 mg/kg). [707} FTC“, 13 provides the results for the tumor volumes of OVCARm3 tumor—bearing mice treated with vehicle Conjugate 8d~2 or ate 8e. Treatment with Conjugate 3dr: (3/01 rug/kg) resulted in 37.3% TGI. Treatment with Conjugate 8e (3/01 rug/kg) resulted in 958% TGT, Example 46: Tumor Growth Response to Administration of Target C Antihodydflng ate in a Triple Negative Breast Cancer Xenograft Model [708} Eight~week~old female NCr nu/nu mice (Charles River laboratories) were implanted with human triple negative breast cancer (TNBC) tumor tragments (l mml) expressing Target C.

Animals were randomized into treatment groups when tumor volumes were between 63—l08mm3 (mean 807-82 mmh’group) Vehicle, diABZT STING agonist (0/5 trig/kg), ate 815—1 (1/004 rug/kg), or Conjugate 8h (1/004 mg/kg) were dosed intravenously as a single dose on day l (all doses are given as an tibody/payload, r129 for each group). Transient body weight loss Witl’lil’l able limits was observed 2 days following treatment with no onal clinical observations for diABZl STlNG agonist {0/5 mgr/kg).

FTC; 14 provides the results for the tumor s of human TNBC tumor—bearing mice d with vehicle, diABZl STING t, Conjugate 80-1, or Conjugate 811. Treatment with diABZl STING agonist (0/5 rug/kg) resulted in 52.4% TGT. Treatment with Conjugate Soul ed in 35.1% TGT. Treatment with Conjugate 8h (1, 1/004 nig/ltg) resulted in 51.8% TGT.

Example 47: Tumor Growth Response to Administration of Target D AntibotlymDi/‘ug Conjugate in Colon Cancer {7710} Seven to eight-week-old feniale CS'7Bl/6 mice were inoculated aneously on the right flank with rn'urine colon cancer cells (5x105 cells per mouse). Animals were randomized into treatment groups 10 days following inoculation, when tumor volumes were between 63-l 08mm3 {mean 78 nirnl/group). Vehicle, Conjugate Sirius (1/004 mg/lrg), or Conjugate 83:; {009/004 nig/kg) were dosed intravenously as a single dose on day 1 (all doses ented as antibody/payload, n=lO for each group). Transient body weight loss within able liniits was observed 2 days following treatment with no additional clinical observations for Conjugate 8g (0. 9/0. 04 ing/kg). [71 1} Fit?“ 15A provides the results for the tumor volumes of niui‘ine colon cancer tuinornhearing mice treated with vehicle, Conjugate 3dn3 or Conjugate 8g. Treatment with Conjugate 8d~3 resulted in 42% T61. whereas treatment with Conjugate 8g resulted in 92.5% 'l'Gl. [7l2l EEG. 158 shows the Kaplan Meier survival curves for murine colon cancer tumor~bearing mice treated with e, Conjugate 8&3, or Conjugate 8g. Treatment with Conjugate 3&3. Mice treated with Conjugate 3g had the longest survival.

Example 48: Rewhallenge Study for Tumor Growth Response to Administration of Target i) Antihodydkug Conjugate [713} free mice (6 out of 9,) previously treated with Conjugate 3g (OB/"0,04 rng/ltg) from Example 36 shown in A and age-matched untreated animals from Example 38 were inoculated aneously on the left flank with rnurine colon cancer cells (5x105 cells per mouse) and on the right flank with murine lung cancer cells (lxlO6 cells per mouse) to 6 and 12, respectively). The tumor growth of mice previously treated with ate 8g 04 mg/kg) when re—challenged with m urine colon cancer cells or m urine lung cancer cells are shown in Flt}. 163 and Fit}. 16C, respectively, lr’revious treatment with Conjugate 8g resulted in 25.5% T61 of murine lung cancer model and 99.8% 'l‘Gl of murine colon cancer model when compared with the respective untreated controls. e 49: 'I‘umoi" Growth Response to Administration of Target E Antibodymfirug Conjugate in Embryonic Cancer [7l4l Female Balb/C mice were inoculated subcutaneously with niurine embryonic cancer cells (5X105 cells per . Animals were randomized into treatment groups when tumor volumes were between 56—108 mm3 (niean 75.3-76.5 nuns/group). Vehicle, diABZl STING agonist (0/5 mg/kg) Conjugate 8d—3 (55/018 mg/kg}, or Conjugate 8i (32/018 mg/ltg) were dosed enously as a single dose on day 1 (all doses are given as antibody/payload, n===l0 for each . Transient body weight loss within acceptable limits was observed 2 days following treatment with no additional clinical observations for diABZI STING agonist (0/5 rug/kg) and Conjugate 8i (32/018 mg/kg). [715} FIG, 17 provides the results for the tumor volumes of murine embryonic cancer tumor— bearing rnice treated with e, diABZI STING agonist, Conjugate 8d—3, or ate 8i.

Treatment with Conjugate 8d—3 (55/018 nig/kg) resulted in 27.5 and 66.5% 'I‘GI, respectively.

Treatment with diABZI STING agonist (0/5 mg/kg) resulted in 93.8% 'I‘GI. Treatment with ate Si (32/018 nag/kg) resulted in 205 and 100% TGI, respectively Example 50: Tumor Growth Response to Administration of HERE Aiiitibodyfirug ate in SKQVS Ovarian Cancer [7l 6} Female C817 SCID mice were inoculated subcutaneously with SKOV3 human ovarian cancer cells (l0 xl06 cells per mouse). Animals were randomized into treatment groups when tumor volumes were between 63-75 mm3 (mean 6544 mud/group). Vehicle, Conjugate 34a *3/04l l mg/ltg), Conjugate 34 (3/0ll rug/kg), Conjugate 20:: (3/009 rug/kg), or Conjugate 20—1 (3/010 mg/kg) were dosed intravenously as a single dose on day I (all doses are given as antibody/payload, nIIlO for each group). Transient body weight loss within acceptable limits was observed 2—3 days following treatment with no additional clinical observations for ate 34a, ate 20a, Conjugate 34, and Conjugate 204. Body weightless at later time-points correlated with tumor progression, indicating tumor model—induced cachexia. {717} provides the results for the tumor volumes of SKOV3 tumor-bearing mice treated with vehicle, Conjugate 34a, Conjugate 34, Conjugate 20a, or Conjugate 20—1 (3/009 rug/kg).

Treatment with ate 20a (3/009 ) or Conjugate 394a (3/011 rug/kg) resulted in 21 and 65.9% 'l'Gl, tively. Treatment with Conjugate 34 (3/011 mg/kg) or Conjugate 20—1 (3/010 mg/kg) eacli resulted in 100% mean tumor regression.

Example ‘31: Tumor Growth Response to stration of HERE Antibody—13mg Conjugate in SKOVS Ovarian Cancer Female CB17 SCID mice were inoculated subcutaneously with SKOV3 human ovarian cancer cells 1 10 X106 cells per mouse). Animals were randomized into treatment groups when tumor volumes were between 88—126 mm3 (mean = 112.8n1 13.2 ninth/group). Vehicle, Conjugate 28 (03/001 mg/kg or 1/003 mg/ltg'), ate 29 (02/001 mg/ltg or 0.8,I’i’1.021ng/kg), Conjugate 8451.50.3/0.0l mg/kg or 1/004 mg/lig), Conjugate 25 (03/001 mg/lig or U004 , or Conjugate 45 (03/001 mg/kg or 1/004 mg/ltg) were dosed intravenously as a single dose on day l (_all doses are given as antibody/payload, n=10 for each group). Transient body weightless within acceptable limits was observed 3 days following treatment with no additional clinical observations for Conjugate 25 (1/004 ). Body weight loss at later timepoints correlated with tumor ssion, indicating tumor niodel~indueed oachexia. [719} provides tumor volumes of SKOVS tumor~bearing mice treated with vehicle, Conjugate 28, Conjugate 29, Conjugate 8w2, Conjugate 25 or Conjugate 45 Treatment with Conjugate 28 (03/001 nig/kg or 1/003 mg/kg), Conjugate 29 (0. 2/001 mg/kg or 08/002 org/kg), Conjugate 34’; (03/0101 mg/kg or 1/004 ing/kg} Conjugate 25 (03/001 rng/kg), or Conjugate 45 (03/001 rng/ltg or 1/004 m/g'kg) resulted in 17,67 97.6,, 121, 86.97 10.9, 703, 81.9, 15.8 and 272% TGI, respectively. Treatment with ate 25' (1/004 trig/kg) resulted in 745% mean 11110101 regression. ie 52: Tumor Growth Response to Administration of HERZ dywflrug Conjugate in SKQVS Qvarian Cancer [720} Female €8.17 SCID mice were inoculated subcutaneously with SKOVE hui'nan ovarian cancer cells (10 X 106' cells/mouse 50% Matrigel). Animals were randomized into treatment groups when tumor volumes were between 63—75 mm3 (mean 67.8 mmS/group}. Vehicle (saline, q3dx3 or quXS), diABZl G agonist (1.5 mg/kg q3dx3 or 0.128 mg/kg tjdxl ), Compound 30 (1.5 mg/lrg q3dx3 or 0.128 mg/kg tjdxl), Conjugate 32134; (342/0128 mg/kg qul). KEV/1111519 (3.00 mg/lrg quxl). Conjugate 32—5 (0100/0004, 0300/0013, 42 or 300/0128 mg/kg qul}, Conjugate 32c (l lid/0.039 or 300/0117 ntg/kg qul) were all dosed intravenously ng on day l (all doses are given as antibody payload, n====lU for each group).

EEG. 29 provides the tumor volumes of SKG‘V 3 tumor-bearing mice treated with vehicle, diABZl SilNG agonist, Compound 30, Conjugate ”fab—=2, mil—1519, Conjugate 32—5, or Conjugate 32c Treatment with ate 32-5 at 042 or 3.00/0.l l7 mg/kg resulted in lU/l 0 complete responses.

Example 53: "former Growth Response to Administration of HER? Antibodynflrng Conjugate in «i’i‘i ered to Express Human HERZ Female Balh/C mice were inoculated subcutaneously with 4T1 murine breast cancer cells engineered to express human HERZ (4'l‘l—liHER2) (2 all)6 cells per mouse). Animals were randomized into treatment groups when tumor volumes were between 75n88 mm3 (mean = ”71.5“ 80. 3 inin3,"group). Vehicle, Conjugate Sit—3 (1/004 rug/kg), or Conjugate 8k 4 ing/lig) were dosed intravenously as a single dose on day 1 (all doses are given as antibody/payload, n=10 for each group). Transient body weight loss within acceptable limits was observed 2 days following treatment with no additional clinical observations for Conjugate 3k (0,9,I’Oi'lr-‘l ing/kg) EEG. 21A provides tumor volumes of 4Tl—hHER2 tumor~bearing mice treated with vehicle, ate 3&3 (_ 1/004 nag/kg), or Conjugate 8k (OB/0,04 trig/kg). ent with Conjugate 8d~3 (_ U004 trig/kg) resulted in 854% TGl (3) Treatment with Conjugate 8k 4 rug/leg) resulted in 93.2% mean tumor regression, with 8 out of l0 animals tumor-free at the end of study (EEG. 21C).

Example 54: Tumor Growth Response to Administration of Fe mutant HERE Antibody,“ Bring Conjugates in SKQ‘B fiverian Cancer Female C1317 SCID mice were inoculated subcutaneously with SKGV3 human ovarian cancer cells (l 0 x 1G6 triouse), Animals were randomized into treatment groups when tumor volumes were n 63-75 min3 (mean 72.6 'gi'oup). Vehicle, Conjugate 892—2 (3.17/0. l O nig/kg), Conjugate Sta—2 (27/010 mg/lrg or 081/003 trig/leg), Conjugate Elli (ZN/0.16 rug/kg or 081/003 trig/leg), or diABZl lV STl NG agonist (0/5 mg/lrg) were dosed intravenously on day l (all doses are given as antibody/payload, n===ll3 for each group). [7241 R{Go 22 provides the turnor volumes of SROV3 tumor—bearing mice treated with Conjugate 862-2, Conjugate Sax-2, Conjugate Sj, or diABZl 1V S'l’lNG agonist 'l'reatrnent with Conjugate 811-2 (276/010 rng/kg} resulted in 113 CR and l0 1711‘; 'l'reatrnent with Conjugate 8111—2 0030 trig/kg resulted in 2 RR, SCR and 3 IFS. '1‘reatrnent with Conjugate Sj 1’271/010 nig/kO) resultedin 2 PR and 6 CR lreatrnent with ate 81 (0 81/0 03 trig/leg) resultedin 3 PR and 1 CR. 'l'reatrnent with diABZl 1V S’l‘1N6 agonist (0/5 rug/leg) resultedin '7 PR.

Example 55: 111 Vitrn g of 111211”th Antihedynflrug Conjugates to Human Ovarian Cancer Cells OVCAR3 cells were crown to81395% conflueneyin RPMI 1640 medium supplemented with PBS (20%) and penicillin/streptomycin (1°/o1 Cells weie harvested and added to wells of a 91’)well U hettoni pla-‘-e (50 001’?l/Vv’ell) 1he cells were pelleted (31313 X g 5 minutes) and resuspended in solutions of test es as indicatedin Table l8 at concentrations ranging between 0.01 nM and 300 nM and incubated on ice for 1 hour. Cells were then washed in ice cold PBS (3):}, pelleted (300 g, S m111utes), and incubated with detection ant1110dy (Goat Anti—Human lgG ~Alexa—647 (11 +1. 1:11ain) foi l hour at-lle. Cell suspe11s1ons weie ed (300 g 5 1nin11tes1 washed 3 times in ice cold PBS, and fixed by ending in solution ot‘paraformaldehyde ( 1 9/11). Resuspended cells were then subjected to flow cyton1etry analys1s on a MACS Quant Flow Cytometry. Single events (10000) weie collected for analysis tion gating and Median stent lntensity (V1131) analysis was performed with M1\(‘53‘SQuant Software Table 18 summarizes the mean ECsn valures for cell binding.

Table 18 Binding ECso 'nM , 1Q, .. 1 KNIT—1535 — c129 XMT153S ”N.-x1111 15351111 on ND Not Determined; NA: Not Applicable As shown in Table l8, ECsu values for binding of NaPiZb ADCs to OVCLARZA cells were in the low nanomolar range whereas the isotype controls did not bind to ()‘VCARB cells.

Example 56: Functional Activity al’iZb Biocnnjngates in a Ctr—Culture Assay of {EVCARS Human Gvarian Cancer and Tl-TRl Reporter Cells [727} The induction of STING pathway in immune cells by Nal’iZb—targeted STlNG ADC was evaluated in a cancer cell/'l‘HPl—lRF3nLuciferase reporter cell co—culture assay. OVCAR3 human n carcinoma cells were seeded in 96mwell CellBind surface tissue culture plates (205)00/well) and allowed to attach for 6 hours in RPl‘v‘fl medium with 29% FBS and 1% penicillid/streptomycin. A range of dilutions (0.0l ni‘vl to 300 nl‘vl based on payload; 3~fold serial dilutions in growth medium) of the test articles were added to eaclr well and the plate was incubated for 20 min at 37 0C. 50,990 'l'HPl —dual repoiter cells were then added to each well and the incubation continued for 20 hours at 37 0C, 5 % C02. Cell culture supernatants (2% ul) from each incubated sample were added to resuspended QUANTl—l..uc (50 rd) and the luminescent signal was measured immediately using a Spectrahrlax MS plate reader (Molecular Devices) The ECsc- value was determined from the dose se curve. Tahle l9 provides the ECso values in THPl ~Dual cells tured with OVCAR3 cancer cells Table 19 iliéé’iiliiiié’ié” ifiiiii;iiiiEiié3iililting;iii’i’il’siii iiiiiiiiiiiiiiéiiii?lifiiii’fiéiiiééiii-l’i iiiiiEiiii-éiiiéisiils Enggnl‘l/l) 029 j .47 at :NA all As shown in Table 19, treatment of OVCAR3 and 'l‘Hl)~l co—cultures with ’ihn ADCs resulted in subnanomolar ECso values for induction of STING pathway in the "l‘l—ll’d immune cells whereas the isotype controls had no activity.

Example 57: Tumor Growth Response to stration of NaPEZh Antibodyvfirug Conjugates in Z‘; [729} Female CB. l 7 SCH) nrice were inoculated subcutaneously with UVCAR—3 human ovarian cancer cells (5 x l06 mouse}. Animals were randomized into treatment crou s when tumor . , 0 volumes were hetween 56 — 122 mm} mean = 839 — 84.7 thing/urea ate 32b~l \J } e, Con'u (3.39/Ql0 mgr/kg): Conjugate 32a, 93 or 3.,l2/lltl0 trig/kg), Conjugate 32c (ZlZ/l'llfi , Conjugate 32d (220/010 mg/kg), or diABZl lV STlNG agonist (0/5 mg/ltg) were closed intravenously on day l i:all doses are given as antiblady/payload, n====l 0 for each group). {7730} F18. 23 provides the results for the tumor volumes of ()VCAR—3 tumor—bearing mice treated with Conjugate 312th}.9 Conjugate 32a, Conjugate 32c, ate 32d, or diABZl l‘v’ Example ‘38: Tumor Growth Response to Administration of NaPiZb STING dy—fling Conjugate in combination with a Nal’iZb AF—HPA Antibodwarug Conjugate in G‘VCAR—B [731} Female C317 SCID mice were ated subcutaneously with 0VCAR3 human ovarian cancer cells (5 x l06 cells/mouse). Animals were randomized into treatment groups when tumor volumes were between 68 — 247 mm3 (_mean = 148 — 148.2 mmg’x’group) vehicle; a combination of mab AFUHPA ADC (075/0023 mg/kg) and ate 89—2 (40/0126 mg/kg); XM'I‘~1535 AFnHPA ADC (075/0024 mg/lcg); Conjugate 8b~2 (20/0071 or 40/0142 nig/lcg); a combination of mll’lln1535 AFUHPA ADC (075/0024 mg/kg) and Conjugate 8on2 26 mg/kg ); a combination of Rituxiinab AF~HPA ADC (075/0023 nag/kg) and Conjugate 8b~2 (40/0142 mg/kg); a combination of Rituximab AF~HPA ADC (075/0023 nag/kg) and Conjugate 8b~2 (2.0/0.07l mg/kg); a. combination ofXMT—1535 A17—HPA ADC (0 75/0024 mg/kg) and Conjugate 8bw2 (ll-010.1432 ing/kg); a ation of XMT~1535 AF—HPA ADC (075/0024 mg/kg) and C injugate 8b~2 (2.0,/007l mgr/kg); a combination of 535 AF~HPA ADC (075/0024 mg/kg) and LYNN-1535 (4.0/0 ; or XMT~l 535 (4.75/0 mg/kg) were dosed intravenously on day 1 (all doses are given as antibody/payload, new for each group). [732} Fifi. 24 provides the results for the tumor volumes of ()VCAR-3 tumor-bearing mice treated with e; a combination of Rituxii'nab ARHPA ADC and Conjugate git-2; XMT~1535 AF—HPA ADC; Conjugate Sir—2; a combination oi‘XMTw1535 AF-HPA ADC and ate 8c— 2; a combination of Rituximab AF-HPA ADC and Coningate Sir—2; a combination ot‘Rituximab AF—HPA ADC and Conjugate Sir—2*, a combination of XMT~1535 ARHPA ADC and Conjugate Sir—2; a combination ot‘XM'l-l 535 AFuHPA ADC and Conjugate Sir-2; a combination ot‘XMfll l535 AE-HPA ADC and Xh/l”1‘-1535; or KNEE—1535. All calculations were based on values on day 22 Treatment with a combination of Rituximah AF—l-lPA ADC (075/0023 mg/kg) and Conjugate Sic—2 (40/0126 mg/kg) resulted in T61 of 25.1% (nIlO) Treatment with Xl‘x/lT-lftfii Ali—1113A ADC (075/0024 rng/lcg) resulted in a 'l'Gl of 71.6% (In-9) and a mean tumor shrinkage of 71.2% (n--1) Treatment with Conjugate 8&3—2 42 trig/kg) resulted in a T(31 of 72.9% } and a mean tumor rage of 48% (n===1). Treatment with Conjugate Sir-2 (20/0071 mg/ltg) resulted in a T61 of 59. 8% (n====10). Treatment with a combination ole‘x/l'l‘d 535 AF—HPA ADC 024 mg/kg) and Conjugate Sic—2 (40/0126 mg/kg) resulted in a TGl of 85.1% (r1119) and a mean tumor shrinkage of 24.6% (n====.1) Treatment with a combination of Rituxiniab AF-HPA ADC (075/0023 mg/kg) and Conjugate .8me (40/0142 mg/kg) resulted in a T‘Gl of 61.7% (n===:10). Treatment with a combination of Rituximab AF—l-TPA ADC (074/0023 mg/kg) and Conjugate 88—2 (20/0071 mg/kg) resulted in a 'T‘Gl of 55.7% (11:10). Treatment with a combination ofXMTn1535 AEHPA ADC (075/0024 mg/kg) and Conjugate 813% (40/0142 mg/kg) resulted in a T61 of 95.2% (n=4) and a mean tumor shrinkage of 60.5% (n=6), Treatment with a combination of KNIT—1535 AF“ HPA ADC (075/0024 mg/kg) and. Conjugate 8on2 (20/0071 rug/kg) ed in a TGI of 98.2% 01:8) and a mean tumor shrinkage of 17.8% (n=2). Treatment with a combination of XM'1‘~l 535 AFnHPA ADC (075/0024 nig/lrg) and XM'1‘~1535 (4.00/0 nig/lrg) resulted in a T81 of 68.2% 01:9) and a mean tumor shrinkage of 23.7% (n=1). Treatment with XM'1‘~1535 (4.75/0 ntg/'l<rg) resulted in a T61 of 0.6% 01:10) Example 59: Tumor Growth Response to Administration ofHERE Antibodywflmg Conjugates in SKQVfi an Cancer Female C817 SCID mice were inoculated subcutaneously with SKOV3 human ovarian cancer cells (10 x 106 cells/mouse). s were randomized into ent groups when tumor volumes were 1oetween 75 —100 mm3 (mean 84 ~ 85 mrn3/group). Vehicle, Conjugate 32b (085/003 rug/kg), Conjugate 32—2 (090/003 rug/kg), Conjugate 88 (0. 87/003 org/kg}, Conjugate 85 (287/010 rug/leg)? Conjugate 92 (236/010 , Conjugate 100 (223/010 rng/kg), Conjugate 89 (099/0030 rug/kg), Conjugate 85a (259/010 ), Conjugate 93 (285/010 ), or Conjugate 303 (270/010 org/kg) were dosed intravenously on day 1 (all doses are given as antibody/payload, niel 0 for each group). [734} EEG. 25 provides the results for the tumor volumes of SROVS turnor— bearing mice treated with Conjugate 32E), Conjugate ;a Conjugate 88, Conjugate 85, Conjugate 92, Conjugate 100, Conjugate 89, Conjugate 85a, Conjugate 93, or Conjugate 101.. Treatment with Conjugate 32—2 (090/003 mg/kg) resulted in 1 PR and 9 CR and 5 T‘FS. Treatment with ate 88 (087/003 nrg/kg) resulted in 6 CR and 4 TFS. 'l‘reatnient with Conjugate 85 10 nig/kg) resulted in 9 CR and 8 TFS. 'l‘reatment with Conjugate 190 10 nig/kg) ed 1 PR.

Example en: Turner“ Growth Response to stration ell-HERE Antihotly4h‘ug Conjugates in SKQV3 Female CB1? SCID mice were inoculated subcutaneously with SKOVS human ovarian cancer cells (10 X 10‘3 cells/mouse). Animals were ized into treatment groups when tunror volumes were between 75444 mm3 (rneair=l l2~l l4 rnrn3,"groiip) (11:1 O/grou'p). e, Conjugate 28 (0,99/00325 trig/kg), or Conjugate 6'2 (OS/"0.6.3325 rug/kg) were dosed intravenously on day 1 (all doses are given as antibody/payload, n=l0 for eaclr group). [73 6} FEG‘, 26 provides the results for the tumor volumes of SKO\/’3 turnorwbearing mice treated with e Conjugate 28, or Conjugate 62 (HERZntargeted). Treatment with Conjugate 28 (OW/0.0325 mg/ltg) resulted in 1 PR, 8 CR, and 5 'l‘FS. Treatment witlr Conjugate 62 (O.92/’i’l0325 nrg/kg) resulted in 2 PR, 7 CR and 4 TFS.

Example 61: l3harmacokinetics of HERZ Antibodyflrug Conjugates in SKQVS [73 ’7} Female CB. l7 SCID mice were treated with a single, intravenous injection of vehicle, Conjugate 28 (3.0/0 l0 nag/kg), or Conjugate 62 (2.84.1"Olt'l rug/kg) (all doses are given as antibodyx’pafload, n=4 for each group). Plasma was ted at l 5 minutes, l, 6, 24, 48, 72, 96, 168, and 336 hours following treatment. Plasma was d 1:l0 in 1.33 trig/ml citric acid for a total volume of 0.1 ml Diluted plasma was snap~frozen on dry ice and stored at —80 0C until PK analysis EEG. 27 provides the results for circulating plasma concentrations of conjugated drug.

Plasma concentrations of ~25 ug/rnL and ~18 gig/mt were achieved for Conjugate 28 and Conjugate 62, respectively, and a corresponding clearance rate of ~905 /kg and ~l4.6 rnl/day/ltg respectively EQUlVALENTS The details of one or more embodiments of the invention are set forth in the accompanying description above. Although any methods and als similar or equivalent to those described herein can he used in the practice or testing of the present disclosure, the methods and materials are now described. ()ther features, obj ects, and advantages of the disclosure will be apparent from the description and from the claims. ln the ication and the appended claims, the ar forms include plural referents unless the context clearly dictates otherwise. 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. All patents and publications cited in this specification are orated by reference. [740} The foregoing description has been presented only for the purposes of ration and is not intended to limit the invention to the precise forin disclosed, but by the claims appended hereto.

Claims (2)

What is claimed is:

1. An antibody-drug conjugate (ADC) being or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises the XMT-1519 antibody comprising a variable heavy chain complementarity determining region 1 ) sing the amino acid sequence SMN (SEQ ID NO: 20); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 21); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence DL (SEQ ID NO: 22); and a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSYLA (SEQ ID NO: 27); a variable light chain complementarity determining region 2 (CDRL2) sing the amino acid sequence GASSRAT (SEQ ID NO: 28); and a variable light chain complementarity ining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO: 29), and d15 represents an integer ranging from about 1 to about 20.

2. The conjugate of claim 1, wherein the XMT-1519 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24.