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WO2024118785A2 - Tlr7 agonists and antibody-drug-conjugates thereof - Google Patents

Tlr7 agonists and antibody-drug-conjugates thereof Download PDF

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Publication number
WO2024118785A2
WO2024118785A2 PCT/US2023/081614 US2023081614W WO2024118785A2 WO 2024118785 A2 WO2024118785 A2 WO 2024118785A2 US 2023081614 W US2023081614 W US 2023081614W WO 2024118785 A2 WO2024118785 A2 WO 2024118785A2
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WIPO (PCT)
Prior art keywords
och
compound
heteroalkylene
adc
abd
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Ceased
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PCT/US2023/081614
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French (fr)
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WO2024118785A3 (en
Inventor
Alina Baum
Amy Han
Christos Kyratsous
Thomas Craig Meagher
William Olson
Christopher Petro
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to JP2025531228A priority Critical patent/JP2025540064A/en
Priority to KR1020257021135A priority patent/KR20250128394A/en
Priority to AU2023403422A priority patent/AU2023403422A1/en
Priority to EP23836674.4A priority patent/EP4626553A2/en
Priority to CN202380092608.9A priority patent/CN120659785A/en
Publication of WO2024118785A2 publication Critical patent/WO2024118785A2/en
Publication of WO2024118785A3 publication Critical patent/WO2024118785A3/en
Priority to MX2025006112A priority patent/MX2025006112A/en
Priority to IL321396A priority patent/IL321396A/en
Anticipated expiration legal-status Critical
Priority to CONC2025/0008795A priority patent/CO2025008795A2/en
Ceased legal-status Critical Current

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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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Definitions

  • TLR7 agonists and antibody-drug-conjugates thereof.
  • ADCs antibody-drug-conjugates
  • BACKGROUND Toll-like receptors (TLRs) are a subset of pattern recognition receptors (PRRs) and play a key role in the innate immune response. TLRs are divided into two groups depending on subcellular localization, with endosomal TLRs being of pharmaceutical interest. Of these endosomal TLRs, TLR7 has been extensively studied as a target for small molecule agonists. See, Patinote, et al., Eur. J. Med. Chem., 2020, 193:112238; U.S.
  • TLR7 agonists have been reported to have antiviral and antibacterial activity, as well as activity as vaccine adjuvants and in the treatment of allergic diseases and asthma. Of interest herein, TLR7 agonists have been studied as cancer immunotherapeutics. One TLR7 agonist has been approved by the U.S. FDA, Aldara® (imiquimod) which is indicated for treatment of actinic keratosis, superficial basal cell carcinoma and external genital warts.
  • ADCs combine the power of antibody specificity with the ability to site specifically target a particular type of cell or tissue with a payload.
  • ADCETRIS® currentuximab vedotin
  • KADCYLATM anti-trastuzumab emtansine
  • TLR7 agonist payloads have been reported. See, e.g., U.S. Patent Nos.10,472,420, 10,780,180, 10,548,985, 10,722,591, 10,675,358; PCT Publication No. WO 2020/181050. However, no such ADCs have been approved for human use. [0006] Thus, there is a continuing need for TLR agonists and ADCs thereof for treatment of various diseases, including cancer and chronic hepatitis B.
  • the present disclosure provides TLR7 agonists of Formula I for use in the compositions and methods provided herein: or a pharmaceutically acceptable salt thereof, wherein: R 1 is H, halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 ; R 2 is H, halo, or alkoxy; R 3 is -CO 2 R 23 , -CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly,
  • the present disclosure provides TLR7 agonist-linkers of Formula II for use in preparation of ADCs provided herein: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 and X are as defined for Formula I; R 9 is a divalent group formed by removal of a terminal hydrogen (i.e., a hydrogen distal from the phenyl group to which R 9 is attached) from an R 3 group, as defined in claim 1; and L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD.
  • the ADCs provided herein are useful in methods of treatment, methods of imaging, or methods of diagnosis.
  • ADCs antibody-drug-conjugates
  • ADCs comprising an antigen- binding domain (ABD) specific to an antigen and a Toll-like receptor 7 (TLR7) agonist
  • the antigen is an HBV surface antigen (HBS sAg) and the disease is chronic Hepatitis B.
  • HBS sAg HBV surface antigen
  • the present disclosure provides an antibody-drug-conjugate (ADC), comprising (a) an antigen-binding domain (ABD) having binding specificity to a hepatitis B virus surface antigen (HBV sAg) and (b) a Toll-like receptor 7 (TLR7) agonist.
  • the ADC further comprises a divalent linker that links the ABD to the TLR7 agonist.
  • the ADC is according to Formula IV: or a pharmaceutically acceptable salt thereof, wherein: L 1 is a divalent linker; R 1 is H, halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 ; R 2 is H, halo, or alkoxy; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 9 is a divalent group formed by removal of a hydrogen from R 3 , R 3 being a group attached to the phenyl group at the position of R 9 ; R 3 is -CO 2 H, -CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y
  • the ADC comprises ABD linked to a compound of Formula III: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 and X are as described elsewhere for Formula I; L is any group or moiety that links to ABD; R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl
  • ABD-L 1 is linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39.
  • the ABD is linked to a compound selected from LP1, LP6, LP7, LP8, LP10, and LP11.
  • the ADC is according to Formula V: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 and X are as described elsewhere for Formula I; R 10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene- PEG-NH-;
  • ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 1)
  • the ADC is according to Formula VI: or a pharmaceutically acceptable salt thereof, wherein: L 1 is a divalent linker; R 1 , R 2 , R 16 , R 11 , R 12 , R 13 , R 14 , R 15 , X, and x are as defined for Formula III; and k is an integer from one to thirty.
  • the ADC comprises ABD-L 1 linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39.
  • k is 1, 2, 3, 4, or 5. In some embodiments, k is 2.
  • the ABD comprises a heavy chain and the C-terminus of the heavy chain is conjugated to L 1 . In some embodiments, the ABD comprises two heavy chains and the C-terminus of each of the two heavy chains is conjugated to L 1 . In some embodiments, L 1 is linked to a cysteine residue of the ABD.
  • the ABD is an antibody against a HBV sAg or a fragment thereof. In some embodiments, the ABD is a human antibody or a humanized antibody. In some embodiments, the ABD is IgG1 or IgG2a. In some embodiments, the ABD comprises a scFv having binding specificity to a HBV sAg.
  • the ABD comprises V H chain and V L chain of an antibody against a HBV sAg.
  • the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody against a HBV sAg.
  • the ABD comprises an Fc region, wherein the Fc region comprises a modification for enhanced binding to Fc ⁇ R.
  • the present disclosure provides a pharmaceutical composition comprising the ADC disclosed herein and one or more pharmaceutically acceptable carriers, excipients, or diluents.
  • the present disclosure provides a method of treatment, comprising administering to a subject in need thereof an effective amount of the ADC or the pharmaceutical composition disclosed herein.
  • the subject has chronic Hepatitis B.
  • the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or the pharmaceutical composition.
  • the method further comprises, before the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject.
  • the method further comprises, after the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject to assess therapeutic efficacy of the ADC or the pharmaceutical composition.
  • the step of administrating the ADC or the pharmaceutical composition is repeated. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated twice, three times, or more. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated at least at 1-week intervals, at 2-week intervals, at 3-week intervals, or at 4-week intervals. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated at 1-week intervals, at 2-week intervals, at 3-week intervals, or at 4-week intervals. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated at 1-month intervals, at 2-month intervals, or 3-month intervals.
  • the ADC or pharmaceutical composition is administered by oral, intravenous, intraperitoneal, inhalation, intranasal, intramuscular, or subcutaneous administration.
  • One aspect of the present disclosure provides the ADC or the pharmaceutical composition for use in treatment.
  • the ADC or the pharmaceutical composition is for use in treatment of chronic Hepatitis B in a subject in need thereof.
  • Another aspect of the present disclosure provides the ADC or the pharmaceutical composition for manufacture of a medicament.
  • the medicament is for the treatment of chronic Hepatitis B in a subject in need thereof.
  • FIG.1 shows in vitro plasma stability of anti-HER2 Ab-LP1 ADC (Example 68).
  • FIG.2 shows in vitro plasma stability of anti-HER2 Ab-LP6A ADC (Example 68).
  • FIG.3 shows in vitro plasma stability of anti-HER2 Ab-LP11A ADC (Example 68).
  • FIG.4 shows in vitro plasma stability of anti-HER2 Ab-LP7A ADC (Example 68).
  • FIG.5 shows the conjugation scheme for conjugating antibodies with linker payloads provided herein (Examples 63, 64 and 69).
  • FIG.6 is a preparative SEC chromatogram of a conjugation mixture demonstrating a clean separation of ADC monomer from aggregates (“HMW”) and unconjugated linker payload (“Free drug”).
  • FIG. 7 is an analytical SEC chromatogram of an SEC purified antibody-TLR7 conjugate showing a monomer purity of 99.7%.
  • FIG.8 shows a LC-ESI-MS spectrum of a deglycosylated and reduced ADC sample. The calculated average DAR value was 1.84. The deconvoluted mass spectra exhibited light chain species (LC, LC1) and heavy chain species (HC, HC1, HC2, etc.). The average DAR can be calculated from the LC and HC drug-loading.
  • FIG.9 shows HIC chromatograms of an antibody and its LP11A conjugate, revealing a mixture of three species: DAR2 species (51%), DAR4 species (28%) and unconjugated antibody (21%). The average DAR of this ADC is 2.1.
  • FIG. 10 depicts results following a single treatment of anti-HER2 Ab-LP6A ADC (Table 3) in the N87 xenograft tumor model. Dosing was performed at Day 0.
  • Regression of tumor was observed after treatment with 5 mg/kg (gray circle) of anti-HER2 Ab-LP6A ADC, while treatment with 1 mg/kg (gray square) anti-HER2 Ab-LP6A ADC resulted in tumor stasis, when compared to saline treated animals (open circle).
  • Regression of N87 gastric tumors was not observed in the N87 xenograft mice treated with 5 mg/kg of isotype control Ab-LP6A ADC (Table 3) (black circle) or 0.5 mg/kg (gray triangle) anti-HER2 Ab-LP6A ADC or 0.1 mg/kg (gray diamond) anti-HER2 Ab-LP6A ADC when compared to saline treated animals (open circle).
  • FIG.11 depicts results following treatment of human N87 xenograft tumors with a single dose of anti-HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC or anti-HER2 Ab-LP7A ADC (Table 3). Dosing was performed at Day 0. Regression of tumor was observed after treatment with 5 mg/kg of anti-HER2 Ab-LP6A ADC (gray circle), 5 mg/kg of anti-HER2 Ab- LP11A ADC (gray square) or 5 mg/kg of anti-HER2 Ab-LP7A ADC (gray triangle), when compared to saline treated animals (open circle).
  • FIG. 12 depicts results following treatment of trastuzumab-resistant HER2 medium human JIMT-1 xenograft tumors.
  • Dosing was initiated at Day 0 and subsequently every 7 days for a total of 4 doses of anti-HER2 Ab-LP6A ADC (Table 3) or in combination with pertuzumab. Regression of tumor was observed after treatment with 5 mg/kg of anti-HER2 Ab-LP6A ADC in combination with 5 mg/kg pertuzumab (gray square), while treatment with 5 mg/kg of anti- HER2 Ab-LP6A ADC alone (gray circle) resulted in tumor stasis for 45 days, when compared to 5 mg/kg unconjugated mAb2 alone (open circle) treated animals.
  • FIG.13 depicts results following treatment of MC38 tumors engineered to express human CD20.
  • tumor bearing mice were treated with 3 total doses of anti- CD20 Ab-LP6A ADC (Table 3) with each dose separated by seven days. Tumor regression was observed in four of five mice after treatment with 5 mg/kg of anti-CD20 Ab-LP6A ADC (gray square) when compared to saline treated animals (open circle). Regression of MC38hCD20 tumors was not observed in the MC38hCD20 syngeneic mice treated with 5 mg/kg of anti-mIgG2a Ctrl Ab-LP1 ADC (black circle) (Table 3) or 5 mg/kg unconjugated anti- CD20 Ab (open square), when compared to 5 mg/kg saline treated animals (open circle).
  • FIG.14 depicts HBV sAg levels measured in the chronic hepatitis B (CHB) disease mouse model after treatment with an anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS three times, two weeks apart subcutaneously (SC). (Example 51; Experiment 1).
  • CHB chronic hepatitis B
  • mAb3+LP1 or mAb4+LP1 anti-sAg mAb-TLR7 agonist
  • LP1 TLR7 agonist
  • FIG.15 depicts HBV sAg levels measured in the chronic hepatitis B (CHB) disease mouse model after treatment with an anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS three times, two weeks apart subcutaneously (SC). (Example 51; Experiment 2).
  • FIG.16 depicts results following parental MC38 tumor cell rechallenge in mice having initially cleared MC38.hTAA Pos engrafted tumors (the same as used in Fig.13).
  • mice On day 60 after MC38.hTAA Pos tumor cell inoculation, tumor free mice (black square) were rechallenged with parental MC38 cells without overexpression of human TAA. Compared to control na ⁇ ve mice (open circle), mice previously treated with anti-CD20 -LP6A conjugate are protected against tumor rechallenge. Data represent mean tumor volumes (mean+/-SEM) over time (post-rechallenge). [0047] FIG.
  • FIG. 17 depicts results following treatment of mice having been inoculated with MC38.hTAA Pos tumor cells with 3 doses every seven days of anti-CD20-LP11A conjugate in wild type mice (closed symbols with solid lines) and in humanized IFNAR mice (open symbols with dashed lines) that lack the ability to respond to murine type I IFN. Regression of tumor was observed after treatment with 5 mg/kg of anti-CD20-LP11A conjugate (closed triangle) when compared to saline treated animals (closed circle) and isotype control antibody conjugate (closed square).
  • FIG.18 depicts results following treatment of mice having been inoculated with MC38.hTAA Pos tumor cells with 3 doses every seven days of anti-CD20-LP6A conjugate with or without 5 doses every four days of anti-CD20 x anti-hCD3 bispecific antibody in mice humanized for TAA and human CD3.
  • FIG.19 depicts the ring opening of the imide bond of the antibody-drug conjugates from the conjugation of the cysteine thiol with the maleimide of the linker-payload. Ring- opening of the imide bond under physiological conditions affords two regio-isomers that one is the thiol attached to the alpha carbon and the other is the thiol attached to the beta carbon to the carboxylic acid group, respectively.
  • FIG. 20 is a scheme showing one possible metabolic pathway for the compound referenced herein as Q o -LP11A.
  • FIG.21 is a scheme showing another possible metabolic pathway for the compound referenced herein as Q o -LP11A.
  • FIG. 22 shows hepatitis B virus surface antigen (HBV sAg) levels measured in a chronic hepatitis B (CHB) disease mouse model after treatment with an anti-sAg monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate-buffered saline (PBS) 5 times, 1 week apart subcutaneously.
  • mAb3+LP6A was effective in reducing the HBV sAg levels as compared to the PBS control.
  • FIG.23 shows anti-hepatitis B virus surface antigen (HBsAG) IgG titers measured in a chronic hepatitis B (CHB) disease mouse model at day 120 (D120) post-first treatment with anti-surface antigen (sAg) monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate- buffered saline (PBS) 5 times, 1 week apart subcutaneously.
  • sAg anti-surface antigen
  • mAb3+LP6A monoclonal antibody-TLR7 agonist
  • PBS phosphate- buffered saline
  • Biological activity thus, encompasses therapeutic effects and pharmacokinetic behavior of such compounds, compositions and mixtures. Biological activities can be observed in in vitro systems designed to test for such activities.
  • "antigen-binding domain” or “ABD” means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide- containing construct that is capable of specifically binding a particular antigen of interest.
  • “antigen-binding domain” includes antibodies and antigen-binding fragments of antibodies.
  • proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species.
  • the phrase “specifically binds,” or “binds specifically to,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10 -8 M or less (e.g., a smaller K D denotes tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • Antibodies can, for example, be identified by real-time, label free bio-layer interferometry assay on an Octet® HTX biosensor, which bind specifically to a target antigen. Moreover, multi-specific antibodies that bind to one domain in the target antigen and one or more additional antigens or a bi-specific that binds to two different regions of the target antigen are nonetheless considered antibodies that “specifically bind”, as used herein. In addition to neutralizing antibodies, antibodies that bind specifically to the target antigen, but are non- neutralizing, also can be used within the scope of the present disclosure to generate antibody- drug conjugates. Such antibodies may function, for example, to deliver a payload to the cells expressing a target antigen.
  • high affinity antibody refers to those mAbs having a binding affinity to a target antigen, expressed as K D , of at least 10 -8 M; preferably 10 -9 M; more preferably 10 -10 M, even more preferably 10 -11 M, even more preferably 10 -12 M, as measured by real-time, label free bio-layer interferometry assay, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORETM, or by solution-affinity ELISA.
  • slow off rate refers to an antibody that dissociates from a target antigen, with a rate constant of 1x10 -3 s -1 or less, preferably 1x10 -4 s -1 or less, as determined by real-time, label free bio-layer interferometry assay, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORETM.
  • label free bio-layer interferometry assay e.g., an Octet® HTX biosensor
  • surface plasmon resonance e.g., BIACORETM.
  • unrelated antigens are proteins, peptides or polypeptides that have less than 95% amino acid identity to one another.
  • antibody means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen.
  • CDR complementarity determining region
  • the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, C H 1, C H 2 and C H 3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region.
  • the light chain constant region comprises one domain (C L 1).
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • HCDR1, HCDR2, and HCDR3 Three CDRs of V H are referred to as HCDR1, HCDR2, and HCDR3, and three CDRs of V L are referred to as LCDR1, LCDR2 and LCDR3.
  • antigen-binding fragment of an antibody means any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • human antibody means antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • Human antibodies may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the term “humanized antibody” means chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • a humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
  • Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function.
  • recombinant human antibody means all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed in WO 2016/100807 or US 2016/0176953 A1, each of which are incorporated herein by reference in their entirety.
  • a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
  • the phrase “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98%, or 99% sequence identity.
  • the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcoreTM system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).
  • K D means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., antibody-antigen interaction). Unless indicated otherwise, the K D values disclosed herein refer to K D values determined by surface plasmon resonance assay at 25° C.
  • salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'- ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and inorganic salts, such as but not limited to, sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral
  • the terms “treat,” “treating,” or “treatment” refer to the reduction or amelioration of the severity of at least one symptom or indication of the disease, e.g., cancer or hepatitis B infection, due to the administration of a therapeutic agent such as a disclosed antibody to a subject in need thereof.
  • a therapeutic agent such as a disclosed antibody to a subject in need thereof.
  • the terms include inhibition of progression of disease or of worsening of infection.
  • the terms also include positive prognosis of disease, e.g., the subject may be free of infection, the subject may have reduced or no viral titers, the subject may have tumor shrinkage, upon administration of a therapeutic agent such as a disclosed antibody or antibody-drug conjugate.
  • the therapeutic agent may be administered at a therapeutic dose to the subject.
  • the terms “prevent,” “preventing,” or “prevention” refer to inhibition of manifestation of any symptoms or indications of a disease (e.g., cancer or hepatitis B infection) upon administration of a disclosed antibody or antibody-drug conjugate. The term includes prevention of the spread of infection in a subject exposed to the virus or at risk of having hepatitis B infection.
  • the phrase “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
  • amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the compound or pharmaceutical composition.
  • the IC 50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
  • moieties are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical moieties that would result from writing the structure from right to left, e.g., -CH 2 O- is equivalent to -OCH 2 -.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain saturated hydrocarbon radical.
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkyl.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, including those groups having 10 or fewer carbon atoms.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having six or fewer carbon atoms.
  • alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n- heptyl, n-octyl, and the like.
  • alkenyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain hydrocarbon radical having one or more carbon-carbon double bonds.
  • alkenylene by itself or as part of another substituent means a divalent radical derived from an alkenyl.
  • an alkenyl (or alkenylene) group will have from 1 to 24 carbon atoms, including those groups having 10 or fewer carbon atoms.
  • a “lower alkenyl” or “lower alkenylene” is a shorter chain alkenyl or alkenylene group, generally having six or fewer carbon atoms.
  • alkenyl groups include, but are not limited to, vinyl (i.e., ethenyl), 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and the higher homologs and isomers.
  • alkynyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain hydrocarbon radical having one or more carbon-carbon triple bonds, which can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C 1 -C 10 means one to ten carbons).
  • alkynyl groups include, but are not limited to, ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkoxy alkylamino
  • alkylthio thioalkoxy
  • alkoxy alkylamino
  • alkylthio thioalkoxy
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, containing at least one heteroatom in the chain selected from O, N, P, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may have an alkyl substituent to fulfill valency and/or may optionally be quaternized.
  • the heteroatom(s) O, N, P, Si and S may be placed at any interior position of the heteroalkyl group (i.e., not at the point of attachment to the rest of the molecule).
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH 2 -O- CH 2 -CH 2 -, -CH 2 -CH 2 -O-CH 2 -CH 2 -, -CH 2 -O-CH 2 -CH 2 -NH-CH 2 -, -CH 2 -CH 2 -S-CH 2 -CH 2 - and - CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • cycloalkylene and “heterocycloalkylene” by themselves or as part of another substituent means a divalent radical derived from a cycloalkyl or heterocycloalkyl.
  • cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornanyl, bicyclo(2.2.2)octanyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, 1- or 2-azabicyclo(2.2.2)octanyl, and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (in some embodiments from 1 to 3 rings) which are fused together or linked covalently.
  • heteroaryl refers to aryl groups that contain from one to four heteroatoms selected from N, O, and S in the ring(s), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • arylene and heteroarylene by themselves or as part of another substituent means a divalent radical derived from an aryl or heteroaryl.
  • aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzo
  • heteroarylium refers to a heteroaryl group that is positively charged on one or more of the heteroatoms.
  • substituent moieties for cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups also include substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl.
  • R', R", R"' and R" each in some embodiments are independently are hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring.
  • -NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and –CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • haloalkyl e.g., -CF 3 and –CH 2 CF 3
  • acyl e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • Two of the substituent moieties on adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -Q'-C(O)-(CRR') q -Q''-, wherein Q' and Q'' are independently –NR-, -O-, -CRR'- or a single bond, and q is an integer of from 0 to 3.
  • two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently –CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula – (CRR') s -X'-(CR''R'') d -, where s and d are independently integers of from 0 to 3, and X' is –O-, -NR'-, -S-, -S(O)-, -S(O) 2 -, or –S(O) 2 NR'-.
  • the substituent moieties R, R', R" and R'" are, in some embodiments, independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • halo by itself or as part of another substituent, means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(C 1 - C 4 )alkyl is meant to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.
  • oxo as used herein means an oxygen atom that is double bonded to a carbon atom.
  • heteroatom or “ring heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • TLR7 Agonists [0095] In one aspect, provided herein are TLR7 agonists for use in the compositions and methods provided herein.
  • the TLR7 agonists are compounds of Formula I: or a pharmaceutically acceptable salt thereof, wherein: R 1 is H, halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 ; R 2 is H, halo, or alkoxy; R 3 is -CO 2 R 23 , -CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR 5 R 6 or -COZ; Z is -OH, alk
  • the compound of the formula (I) is not a compound of the formula: [0001]
  • R 5 and R 6 are selected from (i), (ii), and (iii): (i) R 5 and R 6 each H; (ii) R 5 is H and R 6 is alkyl; (iii) R 5 and R 6 , together with the N to which they are attached, form a heterocyclic ring; and R 7 and R 8 , together with the N to which they are attached, form a heterocyclic ring.
  • the TLR7 agonists are selected with the proviso that R 4 is not substituted with hydroxyl.
  • the TLR7 agonists are selected with the proviso that the alkylene and heteroalkylene portions of R 3 are not substituted with oxo. [0098] In some embodiments, the TLR7 agonists are selected with the proviso that the compound is not 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine, which corresponds to P3 in Table 1; or (4-((2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3-methoxyphenyl)methanol, which corresponds to P1 in Table 1.
  • R 1 is H. In some embodiments, R 1 is halo, -NHR 4 , -OR 4 , -NH- OR 4 or -R 4 , and is a straight chain of 6 atoms in length. In some embodiments, R 1 is halo. In some embodiments, R 1 is -NHR 4 . In some embodiments, R 1 is -OR 4 . In some embodiments, R 1 is -NH-OR 4 . In some embodiments, R 1 is -R 4 .
  • R 1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH 2 CH 2 -OEt. In some embodiments, R 1 is -NH- n-pentyl. In some embodiments, R 1 is -NH-O-n-butyl. In some embodiments, R 1 is -O-n- pentyl. In some embodiments, R 1 is -n-hexyl. In some embodiments, R 1 is -NH-CH 2 CH 2 -OEt. [0100] In some embodiments, R 2 is halo. In some embodiments, R 2 is alkoxy.
  • R 2 is methoxy. In some embodiments, R 2 is H. [0101] In some embodiments, R 3 is -CO 2 R 23 , -CONHR 23 , , -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y. In some embodiments, R 3 is -CONHR 23 . In some embodiments, R 3 is - alkylene-Y. In some embodiments, R 3 is -heteroalkylene-Y. In some embodiments, R 3 is - heteroalkylene-arylene-Y.
  • R 3 is -(hydroxy)heteroalkylene-Y. In some embodiments, R 3 is -(amino)heteroalkylene-Y. In some embodiments, R 3 is alkylene-PEG-Y. In some embodiments, R 3 is -CONH 2 , -COOH, -CH 2 -Y, -CH 2 -O-heteroalkylene-Y, or -CH 2 -O-alkylene-Y. In some embodiments, R 3 is -CH 2 -Y, -CH 2 -O-heteroalkylene-Y, or -CH 2 - O-alkylene-Y.
  • R 3 is -C(Me) 2 OH, -CO 2 H -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2
  • R 3 is -C(Me) 2 OH, -CO 2 H, -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OH, - CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH
  • R 4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl. In some embodiments, R 4 is n-butyl. In some embodiments, R 4 is n-pentyl. In some embodiments, R 4 is n-hexyl. In some embodiments, R 4 is ethoxyethyl. [0103] In some embodiments, R 5 and R 6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring. In some embodiments, R 5 and R 6 are each H. In some embodiments, R 5 is H and R 6 is alkyl.
  • R 5 and R 6 together with the N to which they are attached, form 1-piperazinyl.
  • Y is OH.
  • Y is a divalent glycine group of the formula -NHCH 2 C(O)-.
  • Y is -NR 5 R 6 .
  • Y is -COZ.
  • Y is -OH, -NH 2 , 1-piperazinyl, -COOH, -COOEt, -CONPr 2 or -CO-1-piperazinyl.
  • Y is -OH, -NH 2 , 1-piperazinyl, -COOH or -CO-1- piperazinyl.
  • Z is -OH.
  • Z is alkoxy.
  • Z is -NR 7 R 8 .
  • Z is -OH, ethoxy, -N-n-Pr 2 or 1-piperazinyl.
  • Z is -OH or 1-piperazinyl.
  • R 7 and R 8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1-piperazinyl.
  • R 7 and R 8 together with the N to which they are attached, form 1-piperazinyl.
  • the TLR7 agonist is selected from compounds P1-P39 and P41-P48 in Table 1 and pharmaceutically acceptable salts of any of these: Table 1 [0108]
  • a TLR7 agonist can be a known TLR7 agonist, e.g., 852A, imiquimod, resiquimod, gardiquimod loxoribine, bropirimine, 3M-011, 3M-052, DSR-6434, DSR-29133, SC1, SZU-101, SM-360320, and SM-276001.
  • TLR7 agonists are described in, for example, Chi et al., Front. Pharmacol.8:34, 31 May 2017, which is hereby incorporated by reference in its entirety. III. Synthesis of the TLR7 Agonists [0109]
  • the TLR7 agonists of the disclosure can be synthesized in any suitable fashion. Non- limiting examples of synthetic schemes for the synthesis of the TLR7 agonists of the disclosure are presented herein in Schemes 1-7. [0110] Scheme 1. Synthesis of Intermediate Aa starting from Compound 1 [0111] Scheme 2. Synthesis of Intermediates Starting from Compound 5
  • Scheme 3 Synthesis of payloads P1, P2, P20, P23, P27, P29, P32, P33, P37 and P39 [0114] Scheme 5. Synthesis of payloads P22, P25, P31, P35, P21, P24, P30 and P34.
  • the ADC comprises ABD linked to a linker-TLR7 agonist according to Formula II: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 and X are as defined elsewhere for Formula I; R 9 is a divalent group formed by removal of a hydrogen from R 3 , R 3 being a group attached to the phenyl group at the position of R 9 ; and L is any group or moiety that links, connects, or bonds an antigen-binding domain (ABD), as defined elsewhere herein, with a compound of Formula I.
  • R 1 , R 2 and X are as defined elsewhere for Formula I
  • R 9 is a divalent group formed by removal of a hydrogen from R 3 , R 3 being a group attached to the phenyl group at the position of R 9
  • L is any group or moiety that links, connects, or bonds an antigen-binding domain (ABD), as defined elsewhere herein, with a compound of Formula I.
  • the compound of Formula (II) is not a compound of the formula: .
  • R 9 is -alkylene-Y 1 -, -heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 , or - alkylene-PEG-Y 1 .
  • R 9 is -alkylene-Y 1 -.
  • R 9 is - heteroalkylene-Y 1 -.
  • R 9 is -heteroalkylene-arylene-Y 1 -.
  • R 9 is -(hydroxy)heteroalkylene-Y 1 . In some embodiments, R 9 is - (amino)heteroalkylene-Y 1 . In some embodiments, R 9 is -alkylene-PEG-Y 1 . In another embodiment, R 9 is -CH 2 -Y 1 -, -CH 2 -O-heteroalkylene-Y 1 -, or -CH 2 -O-alkylene-Y 1 -.
  • R 9 is -C(Me) 2 O-, -CO-, -CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazin-4-yl-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -((4-NH-)-1-phenyl), -CH 2 OCH 2 COO-, -CH 2 OCH 2 CH 2 OCH 2 CO-, -CH 2 OCH 2 CH 2 OCH 2 OCH 2 CH 2 O
  • Y 1 is -O-. In some embodiments, Y 1 is a divalent glycine group of the formula -NHCH 2 C(O)-. In some embodiments, Y 1 is -NR 5 -. In some embodiments, Y 1 is - COZ 1 , wherein Z 1 is -O-, -NR 7 -, -O-alkylene-, or 1-piperazin-4-yl. In some embodiments, Y 1 is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl. [0120] In some embodiments, Z 1 is -O-.
  • Z 1 is -NR 7 -. In some embodiments, R 7 is H. In some embodiments, R 7 is alkyl. [0121] In some embodiments, Z 1 is 1-piperazin-4-yl. In some embodiments, Y 1 is 1- piperazin-4-yl. In some embodiments, Y 1 is -CO-1-piperazin-4-yl. [0122] In some embodiments, linkers L for use herein may be found, for example, in Antibody-Drug Conjugates and Immunotoxins, Phillips, G.
  • the L group for the ADCs provided herein is sufficiently stable to exploit the circulating half-life of the antigen binding domain and, at the same time, capable of releasing its payload after antigen-mediated internalization of the ADC.
  • Linker L can be cleavable or non-cleavable.
  • Cleavable linkers for use as L herein include linkers that are cleaved by intracellular metabolism following internalization, e.g., cleavage via hydrolysis, reduction, or enzymatic reaction.
  • Non-cleavable linkers for use as L herein include linkers that release an attached payload via lysosomal degradation of the antigen binding domain following internalization.
  • Suitable L linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction labile linkers, self- immolative linkers, and non-cleavable linkers.
  • Suitable L linkers also include, but are not limited to, those that are or comprise peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, and para-aminobenzyl (PAB) units.
  • PEG polyethylene glycol
  • PAB para-aminobenzyl
  • PEG groups include, but are not limited to, the following [insert ChemDraw structures].
  • PEG groups can have any suitable molecular weight, such as from about 60 g/mol to about 6,000 g/mol, about 60 g/mol to about 600 g/mol, about 100 g/mol to about 500 g/mol, about 300 g/mol to about 1,200 g/mol, about 200 g/mol to about 800 g/mol, about 200 g/mol to about 1,000 g/mol, about 500 g/mol to about 1,000 g/mol, about 500 g/mol to about 2,500 g/mol, or about 800 g/mol to about 2,200 g/mol.
  • L linker is a cleavable linker. In other embodiments, the L linker is a non-cleavable linker.
  • L linkers that can be used in the ADCs provided herein include linkers that comprise or consist of e.g., MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine- alanine), dipeptide site in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide site in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof. Additional examples of L linkers that can be used in the ADCs provided herein are disclosed, e.g., in U.S.
  • the L linkers are stable in physiological conditions.
  • the L linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value.
  • an L linker comprises an enzyme-cleavable moiety.
  • enzyme-cleavable L linkers include, but are not limited to, peptide bonds, ester linkages, and hydrazones.
  • the L linker comprises a cathepsin-cleavable linker.
  • the L linker comprises a non-cleavable moiety.
  • the L linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L- or D- ⁇ -amino acids.
  • the L linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or combination thereof.
  • one or more side chains of the amino acids is linked to a side chain group, described below.
  • the linker comprises valine and citrulline.
  • the L linker comprises lysine, valine, and citrulline.
  • the L linker comprises lysine, valine, and alanine. In some embodiments, the L linker comprises valine and alanine. [0128] In some embodiments, the L linker comprises a self-immolative group.
  • the self- immolative group can be any such group known to those of skill in the art.
  • the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof.
  • PAB p-aminobenzyl
  • PABC p-aminobenzyloxycarbonyl
  • the L group can be modified with one or more enhancement groups.
  • the enhancement group can be linked to the side chain of any amino acid in L.
  • amino acids for linking enhancement groups include lysine, asparagine, aspartate, glutamine, glutamate, and citrulline.
  • the link to the enhancement group can be a direct bond to the amino acid side chain, or the link can be indirect via a spacer and/or reactive group.
  • spacers and reactive groups include any described herein.
  • the enhancement group can be any group that imparts a beneficial effect to the payload, linker payload, or ADC including, but not limited to, biological, biochemical, synthetic, solubilizing, imaging, detecting, and reactivity effects, and the like.
  • the enhancement group is a hydrophilic group.
  • the enhancement group is a cyclodextrin.
  • the enhancement group is an alkyl sulfonic acid, heteroalkyl sulfonic acid, alkenyl sulfonic acid, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, heteroalkenyl amine (e.g., quaternary amine), or heteroalkenyl sugar.
  • sugars include, without limitation, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like.
  • sugars include sugar acids such as glucuronic acid, further including conjugated forms such as glucuronides (i.e., via glucuronidation).
  • exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like.
  • Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like.
  • the cyclodextrin can be any cyclodextrin known to those of skill. In some embodiments, the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or mixtures thereof.
  • the cyclodextrin is alpha cyclodextrin. In some embodiments, the cyclodextrin is beta cyclodextrin. In some embodiments, the cyclodextrin is gamma cyclodextrin. In some embodiments, the enhancement group is capable of improving solubility of the remainder of the ADC. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or non-substituted.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 ) 1-5 SO 3 H, –(CH 2 ) n –NH-(CH 2 ) 1-5 SO 3 H, –(CH 2 ) n –C(O)NH-(CH 2 ) 1-5 SO 3 H, –(CH 2 CH 2 O) m –C(O)NH-(CH 2 ) 1-5 SO 3 H, –(CH 2 ) n –N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , –(CH 2 ) n – C(O)N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , or —(CH 2 CH 2 O) m –C(O)N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1- 5 SO 3 H) 2 ,
  • the alkyl or alkenyl sulfonic acid is —(CH 2 ) 1-5 SO 3 H.
  • the heteroalkyl or heteroalkenyl sulfonic acid is —(CH 2 ) n –NH-(CH 2 ) 1-5 SO 3 H, wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH 2 ) n –C(O)NH-(CH 2 ) 1-5 SO 3 H, wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 CH 2 O) m –C(O)NH-(CH 2 ) 1-5 SO 3 H, wherein m is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is — (CH 2 ) n –N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 ) n –C(O)N((CH 2 ) 1- 5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH 2 CH 2 O) m –C(O)N((CH 2 ) 1- 5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , wherein m is 1, 2, 3, 4, or 5.
  • L comprises a maleimido (for conjugation with a thiol, e.g., cysteine, of an antigen binding domain), an N-hydroxysuccinimido ester (for conjugation with an amine, e.g., lysine, of an antigen binding domain) or cyclooctynyl group (for conjugation with an antigen binding domain using click chemistry).
  • a maleimido for conjugation with a thiol, e.g., cysteine, of an antigen binding domain
  • N-hydroxysuccinimido ester for conjugation with an amine, e.g., lysine, of an antigen binding domain
  • cyclooctynyl group for conjugation with an antigen binding domain using click chemistry.
  • the maleimido group in L reacts with a cysteine residue on an antigen binding domain to form a carbon-sulfur bond.
  • L contains an N-hydroxysuccinimido ester group.
  • the N-hydroxysuccinimido ester group reacts with a lysine residue on an antigen binding domain to form an amide bond.
  • L contains an alkyne which can react via click chemistry with an azide, e.g., to form a click chemistry product.
  • the alkyne group reacts with an azide on a modified antigen binding domain.
  • L contains a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc. 1961,357-396; Wang et al. J. Am. Chem. Soc. 2003, 125(11), 3192-3193; and Agard et al. J. Am. Chem. Soc.2004, 126(46), 15046-15047).
  • the reactive group is an alkyne that is capable of undergoing a 1,3- cycloaddition reaction with an azide.
  • Alkynes that may be used in such embodiments include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • strained alkynes e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC)
  • cycloalkynes e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • Alkynes that may be used in such embodiments also include, but are not limited to, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted, e.g., fluorinated alkynes, aza-cycloalkynes and bicyclo[6.1.0]nonyne.
  • alkynes are useful for conjugating antibodies that have been functionalized with azido groups.
  • Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups.
  • such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, e.g., heavy chain Gln295, with a compound bearing an amino group and an azide group, in the presence of the enzyme transglutaminase.
  • L is selected from 2-maleimido-1-ethyl, 2-maleimidoacetyl, and 3-maleimidopropanoyl. In certain embodiments, L is selected from: , .
  • L is a group selected from 2-maleimido-1-ethyl, 2- maleimidoacetyl, 3-maleimidopropanoyl, , [0136]
  • the linker-TLR7 agonist is selected from those in Table 2 and pharmaceutically acceptable salts of any of these:
  • the payload i.e., TLR7 agonist
  • the payload can be converted to a prodrug prior to attachment to a linking group and formation of the ADC. See, e.g., WO 2020/146541.
  • the payloads of Formula I can be converted to linker-TLR7 agonist of Formula III:
  • L is a linker as defined elsewhere herein;
  • R 1 , R 2 and X are as defined elsewhere for Formula I;
  • R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl;
  • R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7- OR 8-membered heterocyclyl;
  • R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R 13 is
  • the linker-TLR7 agonist has Formula III, wherein R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or 6-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 11 or R 14 to form a
  • R 16 is the moiety of R 3 Formula I that links the phenyl ring to the oxygen atom of the alcohol.
  • the TLR7 agonist used in preparing the linker-TLR7 agonist according to Formula III is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, or P39.
  • V. Synthesis of the TLR7 Agonist-Linkers [0140] Scheme 8. Synthesis of Linker-payloads LP1, LP2, LP3 and LP4. [0141] Scheme 9. Synthesis of Linker-payload LP5. [0142] Scheme 10. Synthesis of Linker-payload LP6A.
  • Scheme 11 Synthesis of Linker-payloads LP7A and LP10A.
  • Scheme 12. Synthesis of Linker-payload LP8A. LP8A
  • Scheme 13. Synthesis of Linker-payloads LP9 and LP12.
  • Scheme 14. Synthesis of Linker-payload LP6A, LP6B, LP7A, LP7B, LP7C, LP7D LP10A, LP10B, LP11A, LP11B, LP11C, LP11D, LP12 and LP14
  • the present disclosure provides an antibody-drug-conjugate (ADC) comprising an antigen-binding domain (ABD) (e.g., an ABD having binding specificity for a target antigen such as HBV sAg or a tumor specific antigen) and a TLR7 agonist.
  • ADC antigen-binding domain
  • the ADC further comprises a divalent linker that links the ABD to the TLR7 agonist.
  • the ABD can bind to the TLR7 agonist with or without a linker, at any location along the ABD as long as the ABD is able to bind its target.
  • the ADC is according to Formula IV: or a pharmaceutically acceptable salt thereof, wherein: L 1 is a divalent linker; R 1 is H, halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 ; R 2 is H, halo, or alkoxy; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 9 is a divalent group formed by removal of a hydrogen from R 3 , R 3 being a group attached to the phenyl group at the position of R 9 ; R 3 is -CO 2 H, -CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R 23 is H,
  • the ABC comprises one TLR7 agonist molecule conjugated to an ABD having binding specificity for a target antigen.
  • the ADC comprises more than one TLR7 agonist molecules per an ABD.
  • two, three, four, five or more TLR7 agonist molecules are conjugated to one ABD.
  • k can be 1, 2, 3, 4, or 5.
  • k is 2.
  • k is 1.
  • k is 4.
  • k is 5 or more.
  • the ADC is selected with the proviso that the ADC does not comprise 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine or (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl)methyl)-3-methoxyphenyl)methanol.
  • R 1 is halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 , and has a straight chain of 6 atoms in length.
  • R 1 is halo. In some embodiments, R 1 is - NHR 4 . In some embodiments, R 1 is -OR 4 . In some embodiments, R 1 is -NH-OR 4 . In some embodiments, R 1 is -R 4 . In some embodiments, R 1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n- pentyl, -n-hexyl or -NH-CH 2 CH 2 -OEt. In some embodiments, R 1 is -NH-n-pentyl. In some embodiments, R 1 is -NH-O-n-butyl.
  • R 1 is -O-n-pentyl. In some embodiments, R 1 is -n-hexyl. In some embodiments, R 1 is -NH-CH 2 CH 2 -OEt. [0156] In some embodiments, R 2 is halo. In some embodiments, R 2 is alkoxy. In some embodiments, R 2 is methoxy. In some embodiments, R 2 is H.
  • R 3 is -CO 2 H, -CONHR 23 , -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y.
  • R 3 is CONHR 23 .
  • R 3 is - alkylene-Y.
  • R 3 is -heteroalkylene-Y.
  • R 3 is - heteroalkylene-arylene-Y.
  • R 3 is -(hydroxy)heteroalkylene-Y.
  • R 3 is -(amino)heteroalkylene-Y. In some embodiments, R 3 is alkylene-PEG-Y. In some embodiments, R 3 is -CONH 2 , -CH 2 -Y, -CH 2 -O-heteroalkylene-Y, or -CH 2 -O-alkylene- Y. In some embodiments, R 3 is -CH 2 -Y, -CH 2 -O-heteroalkylene-Y, or -CH 2 -O-alkylene-Y.
  • R 3 is -C(Me) 2 OH, -CO 2 H -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2
  • R 3 is -C(Me) 2 OH, -CO 2 H, -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OH, - CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH
  • R 4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl. In some embodiments, R 4 is n-butyl. In some embodiments, R 4 is n-pentyl. In some embodiments, R 4 is n-hexyl. In some embodiments, R 4 is ethoxyethyl. [0159] In some embodiments, R 5 and R 6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring. In some embodiments, R 5 and R 6 are each H. In some embodiments, R 5 is H and R 6 is alkyl.
  • R 5 and R 6 together with the N to which they are attached, form 1-piperazinyl.
  • Y is OH.
  • Y is a divalent glycine group of the formula -NHCH 2 C(O)-.
  • Y is -NR 5 R 6 .
  • Y is -COZ.
  • Y is -OH, -NH 2 , 1-piperazinyl, -COOH, -COOEt, -CONPr 2 or -CO-1-piperazinyl.
  • Y is -OH, -NH 2 , 1-piperazinyl, -COOH or -CO-1- piperazinyl.
  • Z is -OH.
  • Z is alkoxy.
  • Z is -NR 7 R 8 .
  • Z is -OH, ethoxy, -N-n-Pr 2 or 1-piperazinyl.
  • Z is -OH or 1-piperazinyl.
  • R 7 and R 8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1-piperazinyl.
  • R 7 and R 8 together with the N to which they are attached, form 1-piperazinyl.
  • ABD-L 1 is linked to a compound selected from P1-P43 by removal of a hydrogen from the group at the position corresponding to R 3 of the compound.
  • R 9 is -alkylene-Y 1 -, -heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 , or - alkylene-PEG-Y 1 .
  • R 9 is -alkylene-Y 1 -. In some embodiments, R 9 is - heteroalkylene-Y 1 -. In some embodiments, R 9 is -heteroalkylene-arylene-Y 1 -. In some embodiments, R 9 is -(hydroxy)heteroalkylene-Y 1 . In some embodiments, R 9 is - (amino)heteroalkylene-Y 1 . In some embodiments, R 9 is -alkylene-PEG-Y 1 . In some embodiments, R 9 is -CH 2 -Y 1 -, -CH 2 -O-heteroalkylene-Y 1 -, or -CH 2 -O-alkylene-Y 1 -.
  • R 9 is -C(Me) 2 O-, C(O)-, -CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazin-4-yl-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -((4-NH-)-1-phenyl), -CH 2 OCH 2 COO-, -CH 2 OCH 2 CH 2 OCH 2 CO-, -CH 2 OCH 2 CH 2 CH 2 NH-, -CH 2 OCH 2
  • Y 1 is -O-. In some embodiments, Y 1 is a divalent glycine group of the formula -NHCH 2 C(O)-. In some embodiments, Y 1 is -NR 5 -. In some embodiments, Y 1 is -COZ 1 , wherein Z 1 is -O-, -NR 7 -, -O-alkylene-, or 1-piperazin-4-yl. In some embodiments, Y 1 is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl. [0166] In some embodiments, Z 1 is -O-.
  • Z 1 is -NR 7 -. In some embodiments, R 7 is H. In some embodiments, R 7 is alkyl. [0167] In some embodiments, Z 1 is 1-piperazin-4-yl. In some embodiments, Y 1 is 1- piperazin-4-yl. In some embodiments, Y 1 is -CO-1-piperazin-4-yl. A. L 1 Divalent Groups [0168] In some embodiments, the ADC of the present disclosure comprises a TLR7 linked to an ABD indirectly via a linker. In some embodiments, the linker is a divalent linker (L 1 ) that links the ABD to the TLR7 agonist according to Formula IV.
  • the divalent linker (L 1 ) is created by the reaction between the linker (L) and the ADC for the conjugation.
  • Linkers (L 1 ) for use herein may be found, for example, in Antibody-Drug Conjugates and Immunotoxins, Phillips, G. L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates, Wang, J., Shen, W.-C., and Zaro, J. L., Eds.; Springer International Publishing, 2015.
  • the L 1 group for the ADCs provided herein is sufficiently stable to exploit the circulating half- life of the antigen binding domain and, at the same time, capable of releasing its payload after antigen-mediated internalization of the ADC.
  • Linker L 1 can be cleavable or non-cleavable.
  • Cleavable linkers for use as L 1 herein include linkers that are cleaved by intracellular metabolism following internalization, e.g., cleavage via hydrolysis, reduction, or enzymatic reaction.
  • Non-cleavable linkers for use as L 1 herein include linkers that release an attached payload via lysosomal degradation of the antigen binding domain following internalization.
  • Suitable L 1 linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction labile linkers, self-immolative linkers, and non- cleavable linkers. Suitable L 1 linkers also include, but are not limited to, those that are or comprise peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, and para-aminobenzyl (PAB) units. [0170] Any linker molecule or linker technology known in the art can be used as L 1 to create or construct an ADC provided herein.
  • L 1 linker is a cleavable linker. In other embodiments, the L 1 linker is a non-cleavable linker.
  • L 1 linkers that can be used in the ADCs provided herein include linkers that comprise or consist of e.g., MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), dipeptide site in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide site in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof.
  • MC maleimidocaproyl
  • MP maleimidopropanoyl
  • val-cit valine-citrulline
  • val-ala valine-alanine
  • L 1 linkers that can be used in the ADCs provided herein are disclosed, e.g., in U.S. Pat. No. 7,754,681 and in Ducry, Bioconjugate Chem., 2010, 21:5-13, and the references cited therein.
  • the L 1 linkers are stable in physiological conditions.
  • the L 1 linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value.
  • an L 1 linker comprises an enzyme-cleavable moiety.
  • enzyme-cleavable L 1 linkers include, but are not limited to, peptide bonds, ester linkages, and hydrazones.
  • the L 1 linker comprises a cathepsin-cleavable linker.
  • the L 1 linker comprises a non-cleavable moiety.
  • the L 1 linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L- or D- ⁇ -amino acids.
  • the L 1 linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or combination thereof.
  • one or more side chains of the amino acids is linked to a side chain group, described below.
  • the linker comprises valine and citrulline.
  • the L 1 linker comprises lysine, valine, and citrulline.
  • the L 1 linker comprises lysine, valine, and alanine. In some embodiments, the L 1 linker comprises valine and alanine. [0174] In some embodiments, the L 1 linker comprises a self-immolative group.
  • the self- immolative group can be any such group known to those of skill in the art.
  • the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof.
  • PAB p-aminobenzyl
  • PABC p-aminobenzyloxycarbonyl
  • the L 1 group can be modified with one or more enhancement groups.
  • the enhancement group can be linked to the side chain of any amino acid in L 1 .
  • amino acids for linking enhancement groups include lysine, asparagine, aspartate, glutamine, glutamate, and citrulline.
  • the link to the enhancement group can be a direct bond to the amino acid side chain, or the link can be indirect via a spacer and/or reactive group.
  • spacers and reactive groups include any described herein.
  • the enhancement group can be any group that imparts a beneficial effect to the payload, linker payload, or ADC including, but not limited to, biological, biochemical, synthetic, solubilizing, imaging, detecting, and reactivity effects, and the like.
  • the enhancement group is a hydrophilic group.
  • the enhancement group is a cyclodextrin.
  • the enhancement group is an alkyl, heteroalkyl, alkenyl, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, heteroalkenyl amine (e.g., quaternary amine), or heteroalkenyl sugar.
  • sugars include, without limitation, monosaccharides, disaccharides, and polysaccharides.
  • Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like.
  • sugars include sugar acids such as glucuronic acid, further including conjugated forms such as glucuronides (i.e., via glucuronidation).
  • Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like.
  • Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like.
  • the cyclodextrin can be any cyclodextrin known to those of skill.
  • the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or mixtures thereof.
  • the cyclodextrin is alpha cyclodextrin.
  • the cyclodextrin is beta cyclodextrin.
  • the cyclodextrin is gamma cyclodextrin.
  • the enhancement group is capable of improving solubility of the remainder of the ADC.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or non-substituted.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 ) 1-5 SO 3 H, –(CH 2 ) n –NH-(CH 2 ) 1-5 SO 3 H, –(CH 2 ) n –C(O)NH-(CH 2 ) 1-5 SO 3 H, – (CH 2 CH 2 O) m –C(O)NH-(CH 2 ) 1-5 SO 3 H, –(CH 2 ) n –N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , –(CH 2 ) n – C(O)N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , or
  • the alkyl or alkenyl sulfonic acid is —(CH 2 ) 1-5 SO 3 H.
  • the heteroalkyl or heteroalkenyl sulfonic acid is —(CH 2 ) n –NH-(CH 2 ) 1-5 SO 3 H, wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH 2 ) n –C(O)NH-(CH 2 ) 1-5 SO 3 H, wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 CH 2 O) m –C(O)NH-(CH 2 ) 1-5 SO 3 H, wherein m is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 ) n –N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 ) n – C(O)N((CH 2 ) 1-5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , wherein n is 1, 2, 3, 4, or 5.
  • the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH 2 CH 2 O) m –C(O)N((CH 2 ) 1- 5 C(O)NH(CH 2 ) 1-5 SO 3 H) 2 , wherein m is 1, 2, 3, 4, or 5.
  • L 1 contains a 3-thiosuccinimido group (resulting from conjugation of a maleimido group with a thiol, e.g., cysteine, of an antigen binding domain), an amido group (resulting from conjugation of a N-hydroxysuccinimido group with an amine, e.g., lysine, of an antigen binding domain) or a triazolocyclooctyl group (resulting from conjugation of a cyclooctynyl group using click chemistry with an antigen binding domain modified to contain an azido group).
  • a 3-thiosuccinimido group resulting from conjugation of a maleimido group with a thiol, e.g., cysteine, of an antigen binding domain
  • an amido group resulting from conjugation of a N-hydroxysuccinimido group with an amine, e.g., lysine, of an antigen binding domain
  • L 1 contains a 3-thiosuccinimido group. In such embodiments, the 3-thiosuccinimido group in L 1 results from reaction of a cysteine residue on an antigen binding domain with a maleimido group of L to form a carbon-sulfur bond.
  • L 1 is derived from L containing a maleimido group. In such embodiments, the maleimido group in L reacts with a cysteine residue on an antigen binding domain to form a carbon-sulfur bond.
  • L 1 is derived from L containing an N-hdroxysuccinimido ester group. In such embodiments, the N-hydroxysuccinimido ester group reacts with a lysine residue on an antigen binding domain to form an amide bond. [0180] In other embodiments, L 1 is derived from L containing an alkyne which can react via click chemistry with an azide, e.g., to form a click chemistry product. In some embodiments, the alkyne group reacts with an azide on a modified antigen binding domain.
  • L contains a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc.1961,357-396; Wang et al. J. Am. Chem. Soc.2003, 125(11), 3192-3193; and Agard et al. J. Am. Chem. Soc.2004, 126(46), 15046-15047).
  • the reactive group is an alkyne that is capable of undergoing a 1,3-cycloaddition reaction with an azide.
  • Alkynes that may be used in such embodiments include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • strained alkynes e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC)
  • cycloalkynes e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • Alkynes that may be used in such embodiments also include, but are not limited to, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted, e.g., fluorinated alkynes, aza-cycloalkynes and bicyclo[6.1.0]nonyne.
  • alkynes are useful for conjugating antibodies that have been functionalized with azido groups.
  • Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups.
  • such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, e.g., heavy chain Gln295, with a compound bearing an amino group and an azide group, in the presence of the enzyme transglutaminase.
  • L 1 contains an amido group.
  • the amido group in L 1 results from reaction of an N-hydroxysuccinimido ester group of L with a lysine residue on an antigen binding domain to form an amide bond.
  • L 1 contains a cyclic group which results from reaction of an alkyne via click chemistry with an azide, e.g., to form a click chemistry product.
  • the alkyne group reacts with an azide on a modified antigen binding domain.
  • an antigen-binding domain contains an alkyne group that reacts with an azide on L. See, e.g., click chemistry, Huisgen Proc. Chem. Soc.1961,357-396; Wang et al. J. Am. Chem. Soc. 2003, 125(11), 3192-3193; and Agard et al. J. Am. Chem. Soc. 2004, 126(46), 15046-15047.
  • the alkyne group is an alkyne that is capable of undergoing a 1,3-cycloaddition reaction with an azide.
  • Alkynes that may be used in such embodiments include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • strained alkynes e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC)
  • cycloalkynes e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • Alkynes that may be used in such embodiments also include, but are not limited to, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted, e.g., fluorinated alkynes, aza-cycloalkynes and bicycle[6.1.0]nonyne.
  • alkynes are useful for conjugating antibodies that have been functionalized with azido groups.
  • Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups.
  • such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, e.g., heavy chain Gln295, with a compound bearing an amino group and an azide group, in the presence of the enzyme transglutaminase.
  • L 1 is a group derived from 2-maleimido-1-ethyl, 2- maleimidoacetyl, 3-maleimidopropanoyl, , [0184]
  • L 1 is or contains a divalent group selected from:
  • L 1 is or contains a group selected from , O
  • L 1 is non-cleavable under physiological conditions. In some embodiments, L 1 is cleavable under physiological conditions. In some embodiments, L 1 is an acid-labile linker, a hydrolysis-labile linker, an enzymatically cleavable linker, a reduction labile linkers or a self-immolative linker. In some embodiments, L 1 is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit.
  • PEG polyethylene glycol
  • PAB para-aminobenzyl
  • L 1 comprises one or more amino acids. In some embodiments, L 1 comprises a self-immolative group. In some embodiments, L 1 comprises p-aminobenzyl (PAB) or p- aminobenzyloxycarbonyl (PABC). In some embodiments, L 1 comprises a maleimido, an N- hydroxysuccinimido ester or cyclooctynyl group.
  • PAB p-aminobenzyl
  • PABC p- aminobenzyloxycarbonyl
  • L 1 comprises a maleimido, an N- hydroxysuccinimido ester or cyclooctynyl group.
  • L 1 is a group derived from 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, , , O O O O N H N N H N H O O O O N N NH O , O NH 2 , O O O O O O H O O O O H N H N N H N N N N N N N N H H H H O O O O O O O O O O O O O O O OH N NH , H 2 , O
  • the ADC comprises ABD linked LP1-LP15.
  • B. Antigen-binding Domains (ABD) antigen-binding domains, i.e., ABD in Formula IV, for use in the ADCs provided herein include any molecule that specifically interacts with a particular antigen.
  • the ABD is an antibody or antigen-binding fragment of an antibody.
  • the ABD is an antibody.
  • the ABD is an antibody comprising an Fc region modified to enhance binding affinity to Fc ⁇ R.
  • ABD is an antibody with one or more mutations selected from F243L, R292P, Y300L, V305I, and P396L. In some embodiments, ABD is an antibody with one or more mutation selected from S239D and I332E. In some embodiments, ABD is an antibody with one or more mutations selected from S239D, I332E, and A330L. In some embodiments, ABD is an antibody with one or more mutations selected from S298A, E333A and K334A. In some embodiments, ABD is an antibody with one or more mutations selected from L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A.
  • ABD is an antibody with one or more mutations selected from D270E, K326D, A330M, and K334E.
  • ABD is an antibody with L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in one heavy chain and D270E, K326D, A330M, and K334E in the opposing heavy chain.
  • ABD is an antibody with one or more mutations selected from G236A, S239D, and I332E.
  • ABD is an antibody with one or more mutations selected from M252Y, S254T, and T256E.
  • ABD is an antibody with one or more mutations selected from M428L and N434S. In some embodiments, ABD is an antibody with one or more mutations selected from S267E and L328F. In some embodiments, ABD is an antibody with one or more mutations selected from N325S and L328F. [0191] In some embodiment, ABD is an antibody that comprises a glutamine residue. Antibodies comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain (glutaminyl-modified antibodies) are within the skill of the practitioners in the art. In other embodiments, ABD is an N297Q mutant antibody.
  • Z is an antibody that has one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites.
  • LLQG SEQ ID NO: 1
  • LLQGG SEQ ID NO: 2
  • LLQLLQG SEQ ID NO: 3
  • LLQYQG SEQ ID NO: 4
  • LLQGA S
  • the antibody is aglycosylated. In some embodiments, the antibody is glucosylated.
  • ABD is an antibody that is a monoclonal antibody, human antibody, humanized antibody, camelised antibody, or chimeric antibody.
  • ABD is an antibody of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
  • ABD has a molecular weight of at least 500, 600, 700, 800, 900, 1000, 10000, 50000 or 100000 Daltons.
  • antigen-binding domains that can be used in the ADCs provided herein include antibodies, antigen-binding fragments of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., (see, e.g., Boersma and Pluckthun, 2011, Curr. Opin.
  • DARPins e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc.
  • ABD comprises a scFv having binding specificity to a target antigen.
  • an antigen-binding domain includes polypeptides that bind a target antigen or a portion thereof with a K D of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured in a surface plasmon resonance assay.
  • the framework regions (FRs) of the antibodies or antigen- binding fragment thereof for use in the ADCs provided herein may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
  • the antigen-binding domains for use in the ADCs provided herein may comprise or consist of antigen-binding fragments of full antibody molecules.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments for use in the ADCs provided herein include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • an antigen-binding fragment of an antibody includes other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.
  • an antigen-binding fragment of an antibody will comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the V H and V L domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain V H -V H , V H -V L or V L -V L dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody for use in the ADCs provided herein include: (i) V H - C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H - C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2- C H 3; and (xiv) V L -C L .
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V H or V L domain (e.g., by disulfide bond(s)).
  • the antigen-binding domains used in the ADCs provided herein may comprise or consist of human antibodies and/or recombinant human antibodies, or antigen-binding fragments thereof.
  • the antigen-binding domains used in the ADCs provided herein may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof.
  • such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the antigen-binding domains used in the ADCs provided herein also include bispecific antigen-binding molecules, such as bispecific antibodies.
  • bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab 2 bispecific formats (see, e.g., Klein et al.
  • bispecific antigen binding molecules may comprise a first antigen-binding domain (also referred to herein as "D1"), and a second antigen-binding domain (also referred to herein as "D2").
  • D1 and D2 domains of a bispecific antibody are non-competitive with one another.
  • Non-competition between D1 and D2 means that, the respective monospecific antigen binding proteins from which D1 and D2 were derived do not compete with one another for binding to the target.
  • Exemplary antigen-binding protein competition assays are known in the art.
  • D1 and D2 bind to different (e.g., non-overlapping, or partially overlapping) epitopes on the target.
  • Bispecific antigen-binding molecules may be constructed using the antigen-binding domains of two separate monospecific antibodies. For example, a collection of monoclonal monospecific antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise against one another for cross-competition to the target protein.
  • a bispecific antigen- binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. Any antigen binding construct which has the ability to simultaneously bind two separate, non- identical epitopes of the target molecule is regarded as a bispecific antigen-binding molecule.
  • Bispecific antigen-binding molecules may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology) as will be known to a person of skill in the art.
  • bispecific antibodies are also provided wherein one arm of the bispecific antibody binds to an epitope on a first target protein, and the other arm of the bispecific antibody binds to a second epitope on a second target protein.
  • bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al.
  • Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. (Epub: Dec.4, 2012)).
  • the antigen binding domains for use in the ADCs provided herein also include antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art.
  • variants include variants of any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art having one or more conservative substitutions.
  • the antigen binding domains include antibodies or antigen binding fragments thereof having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc.
  • the antigen binding domains include antibodies or antigen binding fragments thereof also include variants having substantial sequence identity to any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • the antigen-binding domains for use in the ADCs provided herein encompass proteins having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind the target proteins.
  • Such variant antigen-binding domains comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence but exhibit biological activity that is essentially equivalent to that of the described antibodies.
  • Two antigen-binding domains are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses.
  • antigen-binding domains will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
  • two antigen-binding domains are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
  • two antigen-binding domains are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antigen-binding domains are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
  • Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antigen-binding domain or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) in a well- controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding domain.
  • Bioequivalent variants of antigen-binding domains for use in the ADCs provided herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • bioequivalent antigen- binding domains may include variants comprising amino acid changes which modify the glycosylation characteristics of the antigen-binding domain, e.g., mutations which eliminate or remove glycosylation.
  • the antigen-binding domains for use in the ADCs provided herein bind to a human target protein but not to target protein from other species. In other embodiments, the antigen-binding domains for use in the ADCs provided herein bind to a human target protein and to a target protein from one or more non-human species.
  • the antigen-binding domains for use in the ADCs provided herein may bind to a human target protein and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee target protein.
  • the antigen-binding domains specifically bind human target protein and cynomolgus monkey (e.g., Macaca fascicularis) target protein.
  • antigen-binding domains for use herein bind human target protein but do not bind, or bind only weakly, to cynomolgus monkey target protein.
  • ABD Sequences [0215] In some embodiments, the ABD comprises the heavy chain and light chain of an antibody. [0216] In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. 2. Linkage Sites [0217] The ABD can be linked to the TLR7 agonist directly or indirectly via a linker, through an attachment at a particular amino acid within the ABD.
  • Exemplary amino acid attachments that can be used in the context of this embodiment of the disclosure include, e.g., lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358- 361; WO 2005/089808; US 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al., Proc.
  • lysine see, e.g., US 5,208,020; US 2010/0129314; Hollander e
  • Linkers can also be conjugated to an ABD via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130, and Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997).
  • ABD is bonded to the linker through a lysine residue.
  • the antibody or antigen binding molecule is bonded to the linker through a cysteine residue, lysine residue, or glutamine residue.
  • the ABD is bonded to the linker through a cysteine residue.
  • a linker maleimide moiety bonds to an antibody cysteine residue.
  • the ABD is bonded to the linker through a lysine residue.
  • a linker N-hydroxysuccinimide moiety bonds to an antibody lysine residue to form an amide linkage.
  • the ABD is bonded to the linker through a glutamine residue (see, e.g., Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997 and Dennler et al., Bioconjugate Chem. 2014, 25:569-578).
  • Antibodies comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues.
  • antibodies or antigen binding molecules are engineered by mutations, for example insertions or deletions to facilitate reaction via transglutaminase.
  • antibodies or antigen binding molecules are engineered to remove one or more glycosylation sites.
  • antibodies or antigen binding molecules are engineered to add one or more glutamine residues.
  • glutamine residues are added within a TGase recognition tag, as described herein. Techniques for engineering glutamine residues into an antibody polypeptide chain (glutaminyl-modified antibodies or antigen binding molecules) are within the skill of the practitioners in the art.
  • the antibody is aglycosylated.
  • ABD comprises at least one glutamine residue in at least one polypeptide chain sequence.
  • ABD comprises two heavy chain polypeptides, each with one Gln295 or Q295 residue.
  • ABD comprises one or more glutamine residues at a site other than a heavy chain 295. Included herein are antibodies of this section bearing N297Q mutation(s) described herein. In certain embodiments, a glutamine residue is added at the heavy chain C-terminus.
  • the glutamine is polypeptide engineered with a glutamine- containing tag (e.g., glutamine-containing peptide tags, Q-tags or TGase recognition tag).
  • a glutamine-containing tag e.g., glutamine-containing peptide tags, Q-tags or TGase recognition tag.
  • the term “TGase recognition tag” or “Q-Tag” refers to a sequence of amino acids comprising a glutamine residue that when incorporated into (e.g., appended to) a polypeptide sequence, under suitable conditions, is recognized by a transglutaminase (“TGase”) and leads to cross- linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reactive group.
  • the recognition tag may be a peptide sequence that is not naturally present in the polypeptide.
  • the TGase recognition tag comprises at least one glutamine.
  • the TGase recognition tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., conventional amino acid Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, He, Met, Pro, Thr, Lys, or Trp or nonconventional amino acid).
  • the TGase recognition tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 2), LLQG (SEQ ID NO: 1), LSLSQG (SEQ ID NO: 17), GGGLLQGG (SEQ ID NO: 18), GLLQG (SEQ ID NO: 19), LLQ, GSPLAQSHGG (SEQ ID NO: 20), GLLQGGG (SEQ ID NO: 21), GLLQGG (SEQ ID NO: 22), GLLQ (SEQ ID NO: 23), LLQLLQGA (SEQ ID NO: 3), LLQGA (SEQ ID NO: 5), LLQYQGA (SEQ ID NO: 12), LLQGSG (SEQ ID NO: 6), LLQYQG (SEQ ID NO: 4), LLQLLQG (SEQ ID NO: 3), SLLQG (SEQ ID NO: 7), LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 2
  • ABD includes an antibody heavy chain and further includes a TGase recognition tag at the C-terminus of the antibody heavy chain.
  • ABD includes an antibody heavy chain and further includes a TGase recognition tag at the C- terminus of the antibody heavy chain, wherein the TGase recognition tag is the pentapeptide sequence LLQGA (SEQ ID NO: 5).
  • ABD includes two antibody heavy chains and further includes a TGase recognition tag at the C-terminus of each antibody heavy chain.
  • ABD includes two antibody heavy chains and further includes a TGase recognition tag at the C-terminus of each antibody heavy chain, wherein the TGase recognition tag is the pentapeptide sequence LLQGA (SEQ ID NO: 5).
  • ABD can be also modified at one or more glutamine residues via transglutaminase (see, e.g., Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997 and Dennler et al., Bioconjugate Chem. 2014, 25:569-578).
  • one or more glutamine residues of an antibody can be coupled to a primary amine compound to provide a moiety capable of reacting with a reactive group on a linker-payload.
  • the primary amine compound provides a diene or dienophile.
  • the primary amine compound provides a diene or dienophile
  • the linker- payload provides a complementary dienophile or diene, respectively, for conjugation via a Diels-Alder reaction.
  • the primary amine compound provides an azido group.
  • the primary amine compound provides an azido group
  • the linker-payload provides a complementary alkyne, for conjugation via a click reaction.
  • the ABD comprises a heavy chain and the heavy chain is linked to ABD directly or indirectly via a linker.
  • the ABD comprises a light chain and the light chain is linked to ABD directly or indirectly via a linker.
  • the ABD comprises a heavy chain and the C-terminus of the heavy chain is linked to ABD directly or indirectly via a linker.
  • the ABD comprises a light chain and the C-terminus of the light chain is linked to ABD directly or indirectly via a linker.
  • the ABD comprises two heavy chains and each of the two heavy chains is linked to ABD directly or indirectly via a linker. In some embodiments, the ABD comprises two light chains and each of the two light chains is linked to ABD directly or indirectly via a linker. [0227] In some embodiments, the ABD comprises two heavy chains and C-terminus of each of the two heavy chains is linked to ABD directly or indirectly via a linker. In some embodiments, the ABD comprises two light chains and C-terminus of each of the two light chains is linked to ABD directly or indirectly via a linker. 3.
  • the epitope to which the antigen-binding domains bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a target protein.
  • the relevant epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of the target protein.
  • the epitope is located on or near the binding domain of the target protein. In other embodiments, the epitope is located outside of the binding domain of the target protein.
  • Various techniques known to persons of ordinary skill in the art can be used to determine the epitope with which the antigen-binding domains used in the ADCs provided herein interact.
  • Exemplary techniques that can be used to determine an epitope or binding domain of a particular antigen-binding domain include, e.g., point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis.
  • the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen-binding domain to the deuterium-labeled protein. Next, the protein/antigen-binding domain complex is transferred to water to allow hydrogen- deuterium exchange to occur at all residues except for the residues protected by the antigen- binding domain (which remain deuterium-labeled).
  • the target protein After dissociation of the antigen-binding domain, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antigen-binding domain interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystal structure analysis can also be used to identify the amino acids within a polypeptide with which an antigen-binding domain interacts. 4. Synthesis of ABDs [0230] In one embodiment, the antibodies for use in the ADCs provided herein are fully human antibodies.
  • Methods for generating monoclonal antibodies, including fully human monoclonal antibodies are known in the art. Any such known methods can be used in the context of the present disclosure to make human antibodies that specifically bind to a human protein target.
  • VELOCIMMUNETM technology for example, or any other similar known method for generating fully human monoclonal antibodies
  • high affinity chimeric antibodies to a human protein target are initially isolated having a human variable region and a mouse constant region.
  • the antibodies are characterized and selected for desirable characteristics, including affinity, ligand blocking activity, selectivity, epitope, etc.
  • mouse constant regions are replaced with a desired human constant region, for example wild-type or modified IgG1 or IgG4, to generate a fully human antibody.
  • Monoclonal antibodies can be generated by any techniques with which those having ordinary skill in the art will be familiar. Such methods include, but are not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B-cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein.
  • EBV Epstein Barr Virus
  • Fully human monoclonal antibodies can be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • Methods for obtaining fully human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun.6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol.8:455-58; Jakobovits et al., 1995 Ann. N. Y. Acad. Sci.764:525-35.
  • human immunoglobulin transgenes may be mini-gene constructs, or transloci on yeast artificial chromosomes, which undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue.
  • Fully human monoclonal antibodies may be obtained by immunizing the transgenic mice, which may then produce human antibodies specific for a target antigen.
  • Lymphoid cells of the immunized transgenic mice can be used to produce human antibody-secreting hybridomas according to the methods described herein. Polyclonal sera containing fully human antibodies may also be obtained from the blood of the immunized animals.
  • Another method for generating human antibodies of the present disclosure includes immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Patent No. 4,464,456. Such an immortalized B-cell line (or lymphoblastoid cell line) producing a monoclonal antibody that specifically binds to a target antigen can be identified by immunodetection methods as provided herein, for example, an ELISA, and then isolated by standard cloning techniques.
  • the stability of the lymphoblastoid cell line producing an antibody against a target antigen can be improved by fusing the transformed cell line with a murine myeloma to produce a mouse-human hybrid cell line according to methods known in the art (see, e.g., Glasky et al., Hybridoma 8:377-89 (1989)).
  • Still another method to generate human monoclonal antibodies is in vitro immunization, which includes priming human splenic B-cells with a target antigen, followed by fusion of primed B-cells with a heterohybrid fusion partner. See, e.g., Boerner et al., 1991 J. Immunol.147:86-95.
  • a B-cell that is producing an antibody against a target antigen is selected and the light chain and heavy chain variable regions are cloned from the B-cell according to molecular biology techniques known in the art (WO 92/02551; U.S. Patent 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein.
  • B-cells from an immunized animal may be isolated from the spleen, lymph node, or peripheral blood sample by selecting a cell that is producing an antibody that specifically binds to a target antigen.
  • B-cells may also be isolated from humans, for example, from a peripheral blood sample.
  • Methods for detecting single B-cells that are producing an antibody with the desired specificity are well known in the art, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibody, and the like.
  • Methods for selection of specific antibody-producing B-cells include, for example, preparing a single cell suspension of B-cells in soft agar that contains a target antigen. Binding of the specific antibody produced by the B-cell to the antigen results in the formation of a complex, which may be visible as an immune-precipitate.
  • the methods for obtaining antibodies of the present disclosure can also adopt various phage display technologies known in the art. See, e.g., Winter et al., 1994 Annu.
  • Human or murine immunoglobulin variable region gene combinatorial libraries may be created in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind specifically to a target antigen or variant or fragment thereof. See, e.g., U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci.
  • a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments may be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with the sequence encoding a phage coat protein.
  • a fusion protein may be a fusion of the coat protein with the light chain variable region domain and/or with the heavy chain variable region domain.
  • immunoglobulin Fab fragments may also be displayed on a phage particle (see, e.g., U.S. Patent No.5,698,426).
  • Antibody fragments fused to another protein, such as a minor coat protein can be also used to enrich phage with antigen. Then, using a random combinatorial library of rearranged heavy (V H ) and light (V L ) chains from mice immune to the target antigen (e.g., HBV sAg, tumor specific antigen), diverse libraries of antibody fragments are displayed on the surface of the phage.
  • Heavy and light chain immunoglobulin cDNA expression libraries may also be prepared in lambda phage, for example, using ⁇ lmmunoZap TM (H) and ⁇ ImmunoZap TM (L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B-cell population, and used to create heavy and light chain immunoglobulin cDNA expression libraries in the ⁇ ImmunoZap(H) and ⁇ ImmunoZap(L) vectors.
  • variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers. These primers may be synthesized by one of ordinary skill in the art or may be purchased from commercially available sources.
  • the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein.
  • the antibodies produced therefrom may be sequenced and the CDRs identified and the DNA coding for the CDRs may be manipulated as described previously to generate other antibodies according to the disclosure.
  • the binding agents of the present disclosure preferably modulate activity of the target antigen in the cell-based assay described herein and/or the in vivo assay described herein and/or bind to one or more of the domains described herein and/or cross-block the binding of one of the antibodies described in this application and/or are cross-blocked from binding the target antigen by one of the antibodies described in this application. Accordingly, such binding agents can be identified using the assays described herein.
  • antibodies are generated by first identifying antibodies that bind to one or more of the domains provided herein and/or neutralize in the cell-based and/or in vivo assays described herein and/or cross-block the antibodies described in this application and/or are cross-blocked from binding a target antigen by one of the antibodies described in this application.
  • the CDR regions from these antibodies are then used to insert into appropriate biocompatible frameworks to generate binding agents against the target antigen.
  • the non-CDR portion of the binding agent may be composed of amino acids or may be a non- protein molecule.
  • the assays described herein allow the characterization of binding agents.
  • the binding agents of the present disclosure are antibodies as defined herein.
  • CDRs complementarity determining regions
  • the antigen binding proteins also may be employed in purifying a target antigenby immunoaffinity chromatography.
  • human, partially human, or humanized antibodies will be suitable for many applications, particularly those involving administration of the antibody to a human subject, other types of antigen binding proteins will be suitable for certain applications.
  • Non-human antibodies of the present disclosure can be, for example, derived from any antibody-producing animal, such as mouse, rat, rabbit, goat, donkey, or non-human primate (such as monkey (e.g., cynomolgus or rhesus monkey) or ape (e.g., chimpanzee)).
  • An antibody from a particular species can be made by, for example, immunizing an animal of that species with the desired immunogen (e.g., HBV sAg, tumor specific antigen or using an artificial system for generating antibodies of that species (e.g., a bacterial or phage display-based system for generating antibodies of a particular species), or by converting an antibody from one species into an antibody from another species by replacing, e.g., the constant region of the antibody with a constant region from the other species, or by replacing one or more amino acid residues of the antibody so that it more closely resembles the sequence of an antibody from the other species.
  • the antibody is a chimeric antibody comprising amino acid sequences derived from antibodies from two or more different species.
  • Antigen binding proteins may be prepared, and screened for desired properties, by any of a number of conventional techniques. Certain of the techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of an antigen binding protein of interest (e.g., an anti-HBV sAg antibody, a tumor specific antigen), and manipulating the nucleic acid through recombinant DNA technology.
  • the nucleic acid may be fused to another nucleic acid of interest, or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues, for example.
  • the antigen binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems, using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). [0247] Any expression system known in the art can be used to make the recombinant polypeptides of the present disclosure. Expression systems are detailed comprehensively above.
  • host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired polypeptide.
  • host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells.
  • Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacilli.
  • Higher eukaryotic cells include insect cells and established cell lines of mammalian origin.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al.
  • an antibody of the present disclosure may have at least one amino acid substitution, providing that the antibody retains binding specificity. Therefore, modifications to the antibody structures are encompassed within the scope of the present disclosure. These may include amino acid substitutions, which may be conservative or non- conservative that do not destroy the target binding capability of an antibody. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
  • a conservative amino acid substitution may also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid mimetics for a member from another class with different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.
  • one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue.
  • variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations. [0251] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques.
  • one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. [0252] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins.
  • variants of antibodies include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide.
  • variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline.
  • the substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.
  • Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence.
  • Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired.
  • preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).
  • a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence.
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991), which are each incorporated herein by reference.
  • antibodies of the present disclosure may be chemically bonded with polymers, lipids, or other moieties.
  • the binding agents may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure.
  • the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to display one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region.
  • Such structures can be a naturally occurring polypeptide or polypeptide “fold” (a structural motif), or can have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally occurring polypeptide or fold.
  • These scaffolds can be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus.
  • the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains.
  • suitable binding agents include portions of these antibodies, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3.
  • the non-CDR portion of the antibody may be a non-protein molecule, wherein the binding agent cross-blocks the binding of an antibody disclosed herein to a target antigen.
  • the non-CDR portion of the antibody may be a non-protein molecule in which the antibody exhibits a similar binding pattern to a target antigen in a competition binding assay as that exhibited by at least one of antibodies disclosed herein.
  • the non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to a target antigen and/or neutralizes a target antigen.
  • the non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant antibody, and the recombinant antibody exhibits a similar binding pattern to a target antigen in the target epitope competition binding assay (described hereinbelow) as that exhibited by at least one of the antibodies disclosed herein, and/or neutralizes the target antigen.
  • an antibody comprises one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as described above, it may be obtained by expression from a host cell containing DNA coding for these sequences.
  • a DNA coding for each CDR sequence may be determined on the basis of the amino acid sequence of the CDR and synthesized together with any desired antibody variable region framework and constant region DNA sequences using oligonucleotide synthesis techniques, site-directed mutagenesis and polymerase chain reaction (PCR) techniques as appropriate.
  • DNA coding for variable region frameworks and constant regions is widely available to those skilled in the art from genetic sequences databases such as GenBank®.
  • the heavy chain and the light chain of the antibody are expressed from a single DNA construct. In some embodiments, the heavy chain and the light chain of the antibody are expressed from two or more separate DNA constructs.
  • the DNA encoding an antibody of the present disclosure or fragment thereof may be propagated and expressed according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection using any number of known expression vectors.
  • expression of an antibody fragment may be preferred in a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol.178:497-515).
  • expression of the antibody or a fragment thereof may be preferred in a eukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells) or plant cells.
  • yeast e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris
  • animal cells including mammalian cells
  • suitable animal cells include, but are not limited to, myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells.
  • plant cells include tobacco, corn, soybean, and rice cells.
  • One or more replicable expression vectors containing DNA encoding an antibody variable and/or constant region may be prepared and used to transform an appropriate cell line, for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacteria, such as E. coli, in which production of the antibody will occur.
  • an appropriate cell line for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacteria, such as E. coli, in which production of the antibody will occur.
  • the DNA sequence in each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operatively linked to the variable domain sequence.
  • Particular methods for producing antibodies in this way are generally well-known and routinely used. For example, basic molecular biology procedures are described by Maniatis et al.
  • DNA sequencing can be performed as described in Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International plc sequencing handbook, and site directed mutagenesis can be carried out according to methods known in the art (Kramer et al., Nucleic Acids Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci.
  • the antigen-binding domains for use in the ADCs provided herein encompass proteins having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind the target proteins.
  • Such variant antigen-binding domains comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies.
  • Two antigen-binding domains are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple doses.
  • antigen-binding domains will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
  • two antigen-binding domains are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
  • two antigen-binding domains are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antigen-binding domains are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
  • Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antigen-binding domain or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) in a well- controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding domain.
  • Bioequivalent variants of antigen-binding domains for use in the ADCs provided herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • bioequivalent antigen- binding domains may include variants comprising amino acid changes which modify the glycosylation characteristics of the antigen-binding domain, e.g., mutations which eliminate or remove glycosylation. 6.
  • the antigen-binding domains for use in the ADCs provided herein bind to a human target protein but not to target protein from other species. In other embodiments, the antigen-binding domains for use in the ADCs provided herein bind to a human target protein and to a target protein from one or more non-human species.
  • the antigen-binding domains for use in the ADCs provided herein may bind to a human target protein and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee target protein.
  • the antigen-binding domains specifically bind human target protein and cynomolgus monkey (e.g., Macaca fascicularis) target protein.
  • antigen-binding domains for use herein bind human target protein but do not bind, or bind only weakly, to cynomolgus monkey target protein. 7.
  • the antigen binding domain (ABD) for use in the ADCs provided herein can have binding specificity for any antigen (target protein) deemed suitable to those of skill in the art.
  • the antigen is a transmembrane molecule (e.g., receptor) or a surface protein.
  • HBV Hepatitis B virus
  • HBV sAg HBV surface antigen
  • Current therapy with nucleoside analogs reduces HBV load in plasma but rarely is accompanied by HBV sAg loss. Thus, the nucleoside therapy needs to be given lifelong to prevent viral rebound.
  • Some embodiments of the present disclosure relate to an ADC targeting an HBV antigen.
  • the ADC can be used for treatment of Hepatitis B.
  • the ADC comprises an ABD specific to HBV sAg, where HBV sAg could relate to a non-infectious HBV sAg particle, infectious HBV virion, or HBV sAg expressing cells.
  • the ABD comprises the heavy chain and light chain of an antibody specific to HBV sAg.
  • the ABD comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of an antibody specific to HBV sAg .
  • the ABD is an antibody specific to HBV sAg .
  • the HBV antigen is HBV sAg available from Prospect Bio with Cat No. HBS-872.
  • ABD against a tumor antigen Some embodiments of the present disclosure relate to an ADC targeting a tumor antigen.
  • the ADC can be used for treatment of cancer.
  • the ADC comprises an ABD specific to a tumor antigen.
  • the antigen is expressed on a tumor.
  • the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed, or modified on a particular type of tumor.
  • the antigen is expressed on solid tumors.
  • antigens include, but are not limited to, lipoproteins; alpha1-antitrypsin; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD
  • the antigen is PRLR or HER2. In some embodiments, the antigen is HER2. In some embodiments, the antigen is human HER2. In some embodiments, the antigen is STEAP2. In some embodiments the antigen is human STEAP2. In some embodiments, the MAGE proteins are selected from MAGE-1, -2, -3, -4, - 6, and -12. In some embodiments, the GAGE proteins are selected from GAGE-1 and GAGE- 2.
  • the antibody comprises a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In the present disclosure, this position is referred to as glutamine 295, or as Gln295, or as Q295.
  • the antibody can be engineered to comprise a glutamine residue.
  • the antibody comprises one or more N297Q mutations. Techniques for modifying an antibody sequence to include a glutamine residue are within the skill of those in the art (see, e.g., Ausubel et al. Current Protoc. Mol. Biol. (John Wiley & Sons)).
  • the antibody contains a Q295 residue, an N297Q mutation, or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites, a payload of Formula I where R 3 is -alkylene-Y, - alkylene-arylene-Y, -heteroalkylene-Y
  • R 10 is -alkylene-NH-, -heteroalkylene-NH- or -heteroalkylene-arylene-NH-.
  • R 10 is -alkylene-NH-.
  • R 10 is -heteroalkylene-NH-.
  • R 10 is -heteroalkylene-arylene-NH-.
  • R 10 is -CH 2 -NH-, -CH 2 -O- heteroalkylene-NH-, or -CH 2 -O-alkylene-NH-.
  • R 10 is - CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -(4- NH-1-phenyl), -CH 2 OCH(NH-)CH 2 OH or -CH 2 NH-.
  • R 10 is -CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -(4- NH-1-phenyl) or -CH 2 NH-.
  • the ADC provided herein has the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, or ABD- P34, wherein ABD is attached to the payload (i.e., TLR7 agonist) on the amino group of R 3 .
  • ABD is attached to the payload (i.e., TLR7 agonist) on the amino group of R 3 .
  • the ADCs provided herein for use in the compositions and methods provided herein are prepared from linker-TLR7 agonist of Formula III and have Formula VI: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , L 1 , ABD and x are as defined elsewhere for Formula III; and k is an integer from one to thirty.
  • the ADC provided herein has the formula ABD-L 1 -P1, ABD- L 1 -P2, ABD-L 1 -P6, ABD-L 1 -P8, ABD-L 1 -P17, ABD-L 1 -P18, ABD-L 1 -P19, ABD-L 1 -P20, ABD-L 1 - P23, ABD-L 1 -P27, ABD-L 1 -P29, ABD-L 1 -P32, ABD-L 1 -P33, ABD-L 1 -P37, or ABD-L 1 -P39, where ABD-L 1 is attached to the payload (i.e., TLR7 agonist).
  • the payload i.e., TLR7 agonist
  • ADCs comprising an ABD, linker and TLR7 agonist.
  • Each component of the ADCs i.e., ABD, linker, and TLR7 agonist
  • the method involves the step of partial reduction of an antigen-binding domain with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced cysteine residues with maleimide functionalized linker- payload (i.e., TLR7 agonist).
  • TCEP tris(2-carboxyethyl)phosphine
  • an antigen-binding domain is partially reduced via addition of 1.5 - 3.0- fold molar excess of TCEP in PBS pH 7.4 and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 h at 37 °C.
  • the reduced antigen-binding domain may be buffer exchanged into PBS with 1% w/v polysorbate 20.
  • Linker-payloads can be added at a linker-payload / antigen-binding domain molar ratio of 5 - 10 and reacted for an additional 2 h at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO).
  • the mixture may be purified, e.g., via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase) to afford the ADCs provided herein.
  • SEC size exclusion chromatography
  • the reaction of ABD and the payload i.e., TLR7 agonist
  • the transglutaminase enzyme is a murine transglutaminase enzyme.
  • the tetrazine-linkers were designed to have two functions: (1) tetrazine-linker as a handle that has an additional chemical moiety (such as an amine) to be attached with an antibody while the tetrazine-moiety can react with a linker-payload to generate an ADC; (2) tetrazine-linker as the linker of the linker-payload that can be attached with an antibody-handle (Titas Deb, et al., Chem. Rev. 2021, 121, 12, 6850–6914; Astrid- Caroline Knall and Christian Slugovc. Chem. Soc. Rev., 2013, 42, 5131).
  • the ADCs provided herein are selected from those in Table 3: VIII.
  • Pharmaceutical Compositions [0290] In one aspect, the present disclosure provides a pharmaceutical composition comprising an ADC described herein a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a target antigen (e.g., an HBV sAg). In some embodiments, the pharmaceutical composition comprises an immunocomplex (IC) of the ADC and the HBV sAg. [0291] The TLR7 agonists or ADCs can be formulated into suitable pharmaceutical preparations.
  • the TLR7 agonists or ADCs described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).
  • effective concentrations of one or more TLR7 agonists or ADCs or pharmaceutically acceptable salts is (are) mixed with a suitable pharmaceutical carrier.
  • the concentrations of the TLR7 agonists or ADCs in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder disclosed herein.
  • the compositions are formulated for single dosage administration.
  • the weight fraction of a TLR7 agonists or ADC is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved or ameliorated.
  • Pharmaceutical carriers suitable for administration of the TLR7 agonists or ADCs provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the TLR7 agonist or ADC is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems described herein and well known to those of skill in the art, and then extrapolated therefrom for dosages for humans.
  • the ADC is administered in a method to achieve a therapeutically effective concentration of the payload.
  • a companion diagnostic see, e.g., Olsen D and Jorgensen J T, Front. Oncol., 2014 May 16, 4:105, doi: 10.3389/fonC.2014.00105
  • a companion diagnostic see, e.g., Olsen D and Jorgensen J T, Front. Oncol., 2014 May 16, 4:105, doi: 10.3389/fonC.2014.00105
  • the concentration of TLR7 agonist or ADC in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation and excretion rates of the TLR7 agonist or ADC, the physicochemical characteristics of the TLR7 agonist or ADC, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to ameliorate one or more of the symptoms of a disease or disorder disclosed herein.
  • the compositions may be administered at once or may be divided into a number of smaller doses to be administered at intervals of time.
  • compositions may include other active compounds to obtain desired combinations of properties.
  • the TLR7 agonists or ADCs provided herein, or pharmaceutically acceptable salts thereof as described herein, may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the general art to be of value in treating one or more of the diseases or medical conditions referred to herein. It is to be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.
  • the pharmaceutical composition can be in any form appropriate for human or veterinary medicine, including a liquid, an oil, an emulsion, a gel, a colloid, an aerosol or a solid.
  • the pharmaceutical composition can be formulated for administration by any route of administration appropriate for human or veterinary medicine, including enteral and parenteral routes of administration.
  • the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.
  • IX. Dosing The compounds and pharmaceutical compositions provided herein may be dosed in certain therapeutically or prophylactically effective amounts, certain time intervals, certain dosage forms, and certain dosage administration methods as described below.
  • the methods provided herein encompass treating a patient regardless of subject's age, although some diseases or disorders are more common in certain age groups.
  • the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, can be administered repeatedly if necessary, for example, until the subject experiences stable disease or regression, or until the subject experiences disease progression or unacceptable toxicity.
  • the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID).
  • the administration can be continuous (i.e., daily for consecutive days or every day), intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug).
  • the term "daily” is intended to mean that a therapeutic compound, such as the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administered once or more than once each day, for example, for a period of time.
  • continuous is intended to mean that a therapeutic compound, such as the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administered daily for an uninterrupted period of at least 10 days to 52 weeks.
  • intermittent or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals.
  • intermittent administration of the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • cycling as used herein is intended to mean that a therapeutic compound, such as the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administered daily or continuously but with a rest period. In some such embodiments, administration is once a day for two to six days, then a rest period with no administration for five to seven days.
  • a method of treating a subject with a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof is provided. In some embodiments, a method of treating a subject with a pharmaceutical composition comprising a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is provided.
  • the pharmaceutical composition comprises any of the TLR7 agonists or ADCs disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the TLR7 agonists or ADCs provided herein are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by expression, signaling or activity of the target protein of the antigen-binding domain.
  • the ADCs provided herein are used to treat primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye.
  • the TLR7 agonists or ADCs provided herein are used to treat one or more of the following cancers: acute myelogenous leukemia, adult T-cell leukemia, astrocytomas, bladder cancer, breast cancer, PRLR positive (PRLR+) breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastomata, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), Kaposi's sarcoma, kidney cancer, leiomyosarcomas, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC)), lymphomas, malignant gliomas, malignant mesothelioma, melanoma, mesothelioma, malignant mesothelioma, MF
  • the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. [0308] In some embodiments, the subject has chronic hepatitis B. In some embodiments, the ADCs provided herein are used to treat chronic hepatitis B. [0309] In some embodiments, the subject is diagnosed to have chronic hepatitis B. In some embodiments, the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or the pharmaceutical composition. In some embodiments, the treatment method provided herein further comprises the step of measuring circulating HBV DNA or HBV sAg in serum of the subject before administration of the ADC or the pharmaceutical composition.
  • the treatment method provided herein further comprises the step of measuring circulating HBV DNA or HBV sAg in serum of the subject to assess therapeutic efficacy of the ADC or the pharmaceutical composition after administration.
  • the TLR7 agonists or ADCs may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein). VII.
  • the TLR7 agonists or ADCs provided herein may be co-formulated with and/or administered in combination with one or more additional therapeutically active component(s) selected from a MET antagonist (e.g., an anti-MET antibody (e.g., onartuzumab, emibetuzumab, and H4H14639D) or small molecule inhibitor of MET), an EGFR antagonist (e.g., an anti-EGFR antibody (e.g., cetuximab or panitumumab) or small molecule inhibitor of EGFR (e.g., gefitinib or erlotinib)), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 (e.g., trastuzumab or T-DM1 ⁇ KADCYLA® ⁇ ), anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, Er
  • a PD-1 inhibitor such as an anti-PD-1 antibody can be combined with a TLR7 agonist or ADC as described herein.
  • the TLR7 agonists or ADCs provided herein may also be administered and/or co- formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and/or NSAIDs.
  • the additional therapeutically active component(s) e.g., any of the agents listed above or derivatives thereof, may be administered just prior to, concurrent with, or shortly after the administration of a TLR7 agonist or ADC provided herein.
  • the term “in combination” includes the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). However, the use of the term “in combination” does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject with a disease or disorder.
  • a first therapy (e.g., an ADC provided herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent) to the subject.
  • a second therapy e.g., a prophylactic or therapeutic agent
  • R 1 is halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 ;
  • R 2 is halo, or alkoxy;
  • R 3 is -CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y;
  • R 23 is H, alkyl or aryl;
  • R 4 is alkyl optionally substituted with alkoxy or heteroalkyl;
  • X is CH or N;
  • Y is -OH, Gly, -NR 5 R 6 or -COZ;
  • Z is -OH or -NR 7 R 8 ;
  • R 5 and R 6 are selected from (i), (ii), and (iii): (
  • Embodiment 3 The compound of embodiment 1 or 2 selected with the proviso that R 4 is not substituted with hydroxyl.
  • Embodiment 4. The compound of any one of embodiments 1-3 selected with the proviso that the alkylene and heteroalkylene portions of R 3 are not substituted with oxo.
  • Embodiment 7 The compound of any one of embodiments 1-6, wherein R 1 is halo.
  • Embodiment 8 The compound of any one of embodiments 1-6, wherein R 1 is -NHR 4 .
  • Embodiment 9. The compound of any one of embodiments 1-6, wherein R 1 is -OR 4 .
  • Embodiment 10. The compound of any one of embodiments 1-6, wherein R 1 is -NH-OR 4 .
  • Embodiment 11 The compound of any one of embodiments 1-6, wherein R 1 is -R 4 .
  • R 3 is -C(Me) 2 OH, -CO 2 H -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-
  • Embodiment 33 The compound of any one of embodiments 1-22, wherein R 3 is -C(Me) 2 OH, -CO 2 H, -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OH, - CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 NHC(O)CH 2 NH 2
  • Embodiment 34 The compound of any one of embodiments 1-33, wherein R 4 is n-butyl, n- pentyl, n-hexyl or ethoxyethyl.
  • Embodiment 35 The compound of any one of embodiments 1-33, wherein R 4 is n-butyl.
  • Embodiment 36 The compound of any one of embodiments 1-33, wherein R 4 is n-pentyl.
  • Embodiment 37 The compound of any one of embodiments 1-33, wherein R 4 is n-hexyl.
  • Embodiment 38 The compound of any one of embodiments 1-33, wherein R 4 is ethoxyethyl.
  • Embodiment 39 The compound of any one of embodiments 1-33, wherein R 4 is ethoxyethyl.
  • Embodiment 45 The compound of any one of embodiments 1-42, wherein Y is Gly.
  • Embodiment 45 The compound of any one of embodiments 1-42, wherein Y is -NR 5 R 6 .
  • Embodiment 46 The compound of any one of embodiments 1-42, wherein Y is -COZ.
  • Embodiment 47 The compound of any one of embodiments 1-42, wherein Y is -OH, Gly, - NH 2 , 1-piperazinyl, -COOH, -COOEt, -CONPr 2 or -CO-1-piperazinyl.
  • Embodiment 48 The compound of any one of embodiments 1-47, wherein Z is -OH.
  • Embodiment 49 The compound of any one of embodiments 1-47, wherein Z is -OH.
  • Embodiment 50 The compound of any one of embodiments 1-47, wherein Z is alkoxy.
  • Embodiment 50 The compound of any one of embodiments 1-47, wherein Z is -NR 7 R 8 .
  • Embodiment 51 The compound of any one of embodiments 1-47, wherein Z is -OH, ethoxy, -N-n-Pr 2 or 1-piperazinyl.
  • Embodiment 52 The compound of any one of embodiments 1-47, wherein Z is -OH or 1- piperazinyl.
  • Embodiment 53 The compound of any one of embodiments 1-52, wherein R 7 and R 8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1- piperazinyl.
  • Embodiment 54 The compound of any one of embodiments 1-52, wherein R 7 and R 8 , together with the N to which they are attached, form 1-piperazinyl.
  • Embodiment 55 A compound selected from:
  • Embodiment 56 A compound of Formula II: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 and X are as defined for Formula I in embodiment 1; R 9 is a divalent group formed by removal of a terminal hydrogen (i.e., a hydrogen distal from the phenyl group to which R 9 is attached) from an R 3 group, as defined in embodiment 1; and L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; with the proviso that the compound is not a compound of the formula: Embodiment 57.
  • R 1 , R 2 and X are as defined for Formula I in embodiment 1
  • R 9 is a divalent group formed by removal of a terminal hydrogen (i.e., a hydrogen distal from the phenyl group to which R 9 is attached) from an R 3 group, as defined in embodiment 1
  • L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; with the proviso that
  • R 9 is -C(Me) 2 O-, C(O)-, - CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, - CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazin-4-yl-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -((4-NH-)-1-phenyl), -CH 2 OCH 2 COO-, -CH 2 OCH 2 CH 2 OCH 2 CO-,
  • Embodiment 66 The compound of any one of embodiments 55-65, wherein Y 1 is -O-.
  • Embodiment 67 The compound of any one of embodiments 55-65, wherein Y 1 is Gly.
  • Embodiment 68 The compound of any one of embodiments 55-65, wherein Y 1 is -NR 5 -.
  • Embodiment 69 The compound of any one of embodiments 55-65, wherein Y 1 is -COZ 1 .
  • Embodiment 70 The compound of any one of embodiments 55-65, wherein Y 1 is -O-, Glycine, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl.
  • Embodiment 71 The compound of embodiment 69, wherein Z 1 is -O-.
  • Embodiment 72 The compound of embodiment 69, wherein Z 1 is -NR 7 -.
  • Embodiment 73 The compound of embodiment 69, wherein Z 1 is -O- or 1-piperazin-4-yl.
  • Embodiment 74 The compound of embodiment 68, wherein R 5 is H.
  • Embodiment 75 The compound of embodiment 68, wherein R 5 is alkyl.
  • Embodiment 76 The compound of any one of embodiments 55-75, wherein L is non- cleavable under physiological conditions.
  • Embodiment 77 The compound of any one of embodiments 55-75, wherein L is cleavable under physiological conditions.
  • Embodiment 78 The compound of embodiment 77, wherein L is an acid-labile linker, a hydrolysis-labile linker, an enzymatically cleavable linker, a reduction labile linkers or a self- immolative linker.
  • Embodiment 79 The compound of any one of embodiments 55-78, wherein L is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit.
  • PEG polyethylene glycol
  • PAB para-aminobenzyl
  • Embodiment 81 The compound of any one of embodiments 55-80, wherein L comprises a self-immolative group.
  • Embodiment 82 The compound of any one of embodiments 55-81, wherein L comprises p- aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC).
  • Embodiment 83 The compound of any one of embodiments 55-82, wherein L comprises a maleimido, an N-hydroxysuccinimido ester or cyclooctynyl group.
  • Embodiment 84 The compound of any one of embodiments 55-83, wherein L is a group selected from 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, , OH ,
  • Embodiment 85 A compound selected from:
  • Embodiment 86 A compound of Formula III: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 and X are as defined for Formula I in embodiment 1; L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or
  • Embodiment 87 The compound of embodiment 86, wherein R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or 6- membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 11 or R 14 to form a 4-, 5-, or 6-membere
  • Embodiment 88 The compound of embodiment 86 or embodiment 87, wherein the TLR7 agonist used in preparing the compound is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, P39, P41, P42, or P43.
  • Embodiment 89 An antibody-drug-conjugate (ADC), comprising the compound of any one of embodiments 1-88 or compounds of the formulae: Embodiment 90.
  • ADC antibody-drug-conjugate
  • R 1 , R 2 and X are as defined for Formula I in embodiment 1;
  • R 10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG- NH-;
  • ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQ
  • Embodiment 93 The ADC of embodiment 92, wherein R 10 is -alkylene-NH-, -heteroalkylene- NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene- NH-, or -alkylene-PEG-NH-.
  • Embodiment 94 The ADC of embodiment 92 or embodiment 93, wherein R 10 is -alkylene- NH-.
  • Embodiment 95 The ADC of embodiment 92 or embodiment 93, wherein R 10 is - heteroalkylene-NH-.
  • Embodiment 96 The ADC of embodiment 92 or embodiment 93, wherein R 10 is - heteroalkylene-NH-.
  • ADC of any one of embodiments 89-102 having the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, ABD-P34 or ABD-P41, wherein ABD is attached to the payload (i.e., TLR7 agonist) on the amino group of R 3 .
  • Embodiment 104 Embodiment 104.
  • Embodiment 105 is a divalent linker; R 1 , R 2 , R 16 , R 11 , R 12 , R 13 , R 14 , R 15 , X, and x are as defined for Formula III in embodiment 86; and k is an integer from one to thirty.
  • the ADC of embodiment 104 having the formula ABD-L 1 -P1, ABD-L 1 -P2, ABD-L 1 -P6, ABD-L 1 -P8, ABD-L 1 -P17, ABD-L 1 -P18, ABD-L 1 -P19, ABD-L 1 -P20, ABD-L 1 -P23, ABD-L 1 -P27, ABD-L 1 -P29, ABD-L 1 -P32, ABD-L 1 -P33, ABD-L 1 -P37, ABD-L 1 -P39, or ABD-L 1 - P42, where ABD-L 1 is attached to the payload (i.e., TLR7 agonist) on the alcohol group of R 3 .
  • the payload i.e., TLR7 agonist
  • Embodiment 106 The ADC of any one of embodiments 89-105, wherein ABD has binding specificity for a transmembrane molecule (e.g., receptor) expressed on a tumor.
  • Embodiment 107. A pharmaceutical composition, comprising a compound of any one of embodiments 1-88 or an ADC of any one of embodiments 89-106, and a pharmaceutically acceptable carrier.
  • Embodiment 108. A method of treating or diagnosing disease, comprising administering to a subject a compound of any one of embodiments 1-88 or an ADC of any one of embodiments 89-106 or a pharmaceutical composition of embodiment 107.
  • Embodiment 109. The method of embodiment 108, wherein the method treats a disease.
  • An antibody-drug-conjugate comprising a. an antigen-binding domain (ABD) having binding specificity to a hepatitis B virus surface antigen (HBV sAg); and b. a Toll-like receptor 7 (TLR7) agonist.
  • ADC antigen-binding domain
  • HBV sAg hepatitis B virus surface antigen
  • TLR7 Toll-like receptor 7
  • ADC of clause 2 wherein the ADC is according to Formula IV: or a pharmaceutically acceptable salt thereof, wherein: L 1 is a divalent linker; R 1 is H, halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4 ; R 2 is H, halo, or alkoxy; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 9 is a divalent group formed by removal of a hydrogen from R 3 , R 3 being a group attached to the phenyl group at the position of R 9 ; R 3 is -COOH, -CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R 23 is H, al
  • R 3 is -C(Me) 2 OH, -CO 2 H, -CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 CH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH 2 , -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazinyl, -CH 2 OCH 2 NHC(O)CH 2 NH 2 , -CH 2 OCH 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH,-CH 2 OCH 2 COOEt, -CH 2
  • Clause 33 The ADC of any one of clauses 3-32, wherein R 4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl.
  • Clause 34 The ADC of clause 33, wherein R 4 is n-butyl.
  • Clause 35 The ADC of clause 33, wherein R 4 is n-pentyl.
  • Clause 36 The ADC of clause 33, wherein R 4 is n-hexyl.
  • Clause 37 The ADC of clause 33, wherein R 4 is ethoxyethyl.
  • Clause 56 The ADC of any one of clauses 3-54, wherein R 9 is -alkylene-Y 1 -, - heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , - (amino)heteroalkylene-Y 1 , or -alkylene-PEG-Y 1 -.
  • Clause 58 The ADC of clause 56, wherein R 9 is -heteroalkylene-Y 1 -.
  • Clause 59 The ADC of any one of clauses 3-54, wherein R 9 is -alkylene-Y 1 -, - heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , - (a
  • R 9 is -C(Me) 2 O-, C(O)-, -CH 2 OCH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -1-piperazin-4-yl-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -((4-NH-)-1-phenyl), -CH 2 OCH 2 COO-, -CH 2 OCH 2 CH 2 OCH 2 CO-, -CH 2
  • Clause 65 The ADC of any one of clauses 56-64, wherein Y 1 is -O-.
  • Clause 66. The ADC of any one of clauses 56-64, wherein Y 1 is Gly.
  • Clause 67. The ADC of any one of clauses 56-64, wherein Y 1 is -NR 5 -.
  • Clause 68. The ADC of any one of clauses 56-64, wherein Y 1 is -COZ 1 , wherein Z 1 is -O-, -NR 7 -, -O-alkylene-, or 1-piperazin-4-yl.
  • L 1 is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit.
  • PEG polyethylene glycol
  • PAB para-aminobenzyl
  • L 1 comprises p-aminobenzyl (PAB) or p- aminobenzyloxycarbonyl (PABC).
  • PAB p-aminobenzyl
  • PABC p- aminobenzyloxycarbonyl
  • L 1 comprises a maleimido, an N- hydroxysuccinimido ester or cyclooctynyl group.
  • Clause 85 The ADC of any one of clauses 3-76, wherein L 1 is a group derived from 2- maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, , O O O O O O N H N N H N O O H O O N N N NH O , O NH 2 , ,
  • R 1 , R 2 and X are as defined for Formula I in clause 3;
  • L is any group or moiety that links to ABD;
  • R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl;
  • R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl;
  • R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein
  • R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or 6-membered heterocyclyl;
  • R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 4-, 5-, or 6-membered heterocyclyl;
  • R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 11 or R 14 to form a 4-, 5-, or 6-membered
  • ADC of clause 3 wherein the ADC is according to Formula V: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , and X are as defined for Formula In in clause 3; R 10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or - alkylene-PEG-NH-;
  • ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6),
  • Clause 92 The ADC of clause 91, wherein R 10 is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or - alkylene-PEG-NH-.
  • Clause 93 The ADC of clause 92, wherein R 10 is -alkylene-NH-.
  • Clause 94 The ADC of clause 92, wherein R 10 is -heteroalkylene-NH-.
  • Clause 95 The ADC of clause 92, wherein R 10 is -heteroalkylene-arylene-NH-.
  • Clause 96 The ADC of clause 92, wherein R 10 is -(hydroxy)heteroalkylene-NH-. Clause 97. The ADC of clause 92, wherein R 10 is -(amino)heteroalkylene-NH-. Clause 98. The ADC of clause 92, wherein R 10 is -alkylene-PEG-NH-. Clause 99. The ADC of clause 91 or 92, wherein R 10 is -CH 2 -NH-, -CH 2 -O-heteroalkylene- NH-, or -CH 2 -O-alkylene-NH-. Clause 100.
  • R 10 is -CH 2 OCH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -(4-NH-1-phenyl)-, -CH 2 OCH(NH-)CH 2 OH or -CH 2 NH-.
  • R 10 is -CH 2 OCH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 CH 2 CH 2 NH-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, - CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 NH-, -CH 2 OCH 2 NHC(O)CH 2 NH-, -CH 2 OCH 2 -(4-NH-1-phenyl)- or -CH 2 NH-.
  • the ADC of clause 3 comprising ABD linked to a compound selected from P4, P5, P7, P9, P11, P12, P19, P21, P24, P30, and P34 via an amino group of R 3 .
  • Clause 103 The ADC of clause 2, wherein the ADC is according to Formula VI:
  • L 1 is a divalent linker
  • R 1 is H, halo, -NHR 4 , -OR 4 , -NH-OR 4 or -R 4
  • R 2 is H, halo, or alkoxy
  • R 4 is alkyl optionally substituted with alkoxy or heteroalkyl
  • R 11 and R 12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl
  • R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R 13
  • Clause 104 The ADC of clause 103, comprising ABD-L 1 linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39.
  • Clause 105 The ADC of clause 103 or 104, wherein k is 1, 2, 3, 4, or 5.
  • Clause 106 The ADC of clause 103, wherein k is 2.
  • Clause 107 The ADC of clause 103, wherein ABD comprises a heavy chain and C-terminus of the heavy chain is conjugated to L 1 .
  • Clause 118. The ADC of any one of clauses 1-114, wherein said ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 25, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 29.
  • CDRs heavy chain complementarity determining regions
  • LCVR light chain complementarity determining regions
  • HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26
  • HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27
  • HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28
  • LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 30
  • LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31
  • LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32.
  • Clause 122 The ADC of clause 118 or 119, wherein said LCVR comprises the amino acid sequence of SEQ ID NO: 29.
  • Clause 123 The ADC of claim 120, wherein said LCVR is a component of a light chain comprising the amino acid sequence of SEQ ID NO: 34.
  • Clause 124. The ADC of any one of clauses 118-123, wherein said ABD is a component of an antibody or antigen-binding fragment thereof. Clause 125.
  • the ADC of clause 1, wherein the TLR7 agonist with a divalent linker is any one of LP1-5, LP6A-6B, LP7A-7E, LP8A-8B, LP9, LP10A-10B, LP11A-11D, and LP12-15
  • a pharmaceutical composition comprising the ADC of any one of clauses 1- 117 and one or more pharmaceutically acceptable carriers, excipients, or diluents.
  • Clause 127. A method of treatment, comprising administering to a subject in need thereof an effective amount of the ADC of any one of clauses 1-117 or the pharmaceutical composition of clause 118. Clause 128. The method of clause 127, wherein the subject has Hepatitis B. Clause 129.
  • Clause 134 The method of clause 133, wherein the step of administrating the ADC or the pharmaceutical composition is repeated twice, three times, or more. Clause 135. The method of clause 133 or 134, wherein the step of administrating the ADC or the pharmaceutical composition is repeated at least at 1-week intervals, at 2-week intervals, at 3-week intervals, or at 4-week intervals. Clause 136. The method of clause 133 or 134, wherein the step of administrating the ADC or the pharmaceutical composition is repeated at 1-week intervals, at 2-week intervals, at 3- week intervals, or at 4-week intervals. Clause 137.
  • Payload P1 (4- ⁇ [2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)methanol (P1) [0382] Following the general procedure V starting from intermediate Aa (65 mg, 0.16 mmol), payload P1 (35 mg, 43% yield, TFA salt) was obtained as a white solid. ESI m/z: 389.3 (M + H) + .
  • Payload P20 [0384] (4- ⁇ [2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)methanol (P20) [0385] Following the general procedure V starting from intermediate Ba (0.15 g, 0.38 mmol), payload P1 (0.13 g, 72% yield, TFA salt) was obtained as a white solid. ESI m/z: 372.5 (M + H) + .
  • Payload P23 [0387] [4-( ⁇ 2-Amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl ⁇ methyl)-3- methoxyphenyl]methanol (P23) [0388] Following the general procedure V starting from intermediate Ca (30 mg, 75 ⁇ mol), payload P23 (15 mg, 41% yield, TFA salt) was obtained as a white solid. ESI m/z: 372.3 (M + H) + .
  • Payload P32 (4- ⁇ [2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl ⁇ phenyl)methanol (P32) [0397] Following the general procedure V starting from intermediate Bb (82 mg, 0.22 mmol), payload P32 (70 mg, 69% yield, TFA salt) was obtained as a white solid. ESI m/z: 342.4 (M + H) + .
  • Payload P3 [0413] 5-( ⁇ 2-Methoxy-4-[(piperazin-1-yl)methyl]phenyl ⁇ methyl)-N 4 -pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P3) [0414] Following the general procedure VI starting from P1, payload P3 (0.23 g, 31% yield, TFA salt) was obtained as a white solid. ESI m/z: 438.4 (M + H) + .
  • Payload P22 [0430] 4- ⁇ [2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxybenzamide (P22) [0431] Following the general procedure VII starting from Ba, payload P22 (82 mg, 28% yield, TFA salt) was obtained as a white solid. ESI m/z: 385.5 (M + H) + .
  • EXAMPLE 13 [0443] Payload P21 [0444] 5- ⁇ [4-(Aminomethyl)-2-methoxyphenyl]methyl ⁇ -N 4 -butoxy-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P21) [0445] To a stirred suspension of lithium aluminum tetrahydride (39 mg, 1.0 mmol) in anhydrous THF (6 mL) was added dropwise a solution of compound P22 (65 mg, 0.17 mmol) in anhydrous THF (4 mL) over 10 minutes at 0 o C under nitrogen protection. The reaction mixture was stirred at 65 o C for 4 hours which was monitored by LCMS.
  • EXAMPLE 16 [0452] Payload P34 [0453] 5- ⁇ [4-(Aminomethyl)phenyl]methyl ⁇ -N 4 -(2-ethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine- 2,4-diamine (P34) [0454] Following the similar procedure as Payload P30 except substituting Cc for Bc, payload P34 (0.10 g, 60% yield, TFA salt) was obtained as a white solid. ESI m/z: 341.3 (M + H) + .
  • the resulting suspension was stirred at -15 to 0 o C under nitrogen protection for 10 minutes.
  • To the stirred mixture were then added tetrabutylammonium iodide (TBAI) (0.05 equiv.) and benzyl halide (1.0 equiv.), and the reaction mixture was stirred at -15 to 0 o C for 30 minutes, which was monitored by LCMS.
  • the resulting mixture was quenched with methanol.
  • the mixture (with Boc protection or propylidene protection) was directly used for the next step without further purification.
  • the mixture was neutralized with TFA (to pH 6-7) and then concentrated in vacuo.
  • the residual mixture was purified by prep-HPLC (5-95% acetonitrile in aq.
  • Payload P4 [0463] 5-( ⁇ 4-[(2-Aminoethoxy)methyl]-2-methoxyphenyl ⁇ methyl)-N 4 -pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P4) [0464] Following the general procedures VIII and IX subsequently starting from benzyl bromide P4-1 reacting with N-Boc-aminoethanol P4-2, payload P4 (13 mg, 15% yield, di-TFA salt) was obtained as a white solid.
  • EXAMPLE 23 [0468] Payload P6 [0469] 2- ⁇ 2-[(4- ⁇ [2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)methoxy]ethoxy ⁇ ethan-1-ol (P6) [0470] Following the general procedure VIII starting from benzyl bromide P4-1 reacting with diethylene glycol P6-2, payload P6 (12 mg, 23% yield, TFA salt) was obtained as an off-white solid. ESI m/z: 458.1 (M + H) + .
  • Payload P44 [0475] 1-(4- ⁇ [2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-oic acid (P44) [0476] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with Hydroxy-PEG 3 -CH 2 CO 2 t Bu (CAS: 518044-31-0), payload P44 (4.6 mg, 13% yield) was obtained as a white solid (tBu group was lost during the reaction). ESI m/z: 560.3 (M + H) + .
  • Payload P45 2-(2- ⁇ 2-[(4- ⁇ [2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)methoxy]ethoxy ⁇ ethoxy)acetic acid (P45) [0479] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with Hydroxy-PEG 2 -CH 2 CO 2 t Bu (CAS: 149299-82-1), payload P45 (3.9 mg, 15% yield) was obtained as a white solid (tBu group was lost during the reaction). ESI m/z: 516.3 (M + H) + .
  • Payload P46 [0481] 2- ⁇ 2-[(4- ⁇ [2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)methoxy]ethoxy ⁇ acetic acid (P46) [0482] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with tert-butyl 2-(2-hydroxyethoxy)acetate (CAS: 287174-32-7), payload P46 (3.2 mg, 12% yield) was obtained as a white solid (tBu group was lost during the reaction). ESI m/z: 472.3 (M + H) + .
  • Payload P48 2-[(4- ⁇ [2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl ⁇ -3- methoxyphenyl)methoxy]ethan-1-ol (P48) [0485] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with ethylene glycol (CAS: 107-21-1), payload P48 (5 mg, 10% yield, TFA salt) was obtained as an white solid. ESI m/z: 414.3 (M + H) + .
  • Boc-LP3- 2 (45 mg, ESI m/z: 581.5 (M + H) + ) as a light yellow solid, which was dissolved in DCM (5 mL).
  • hydrochloride in dioxane (4 N, 5 mL) and the solution was stirred at room temperature for an hour until Boc was totally removed according to LCMS.
  • the volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq.
  • linker-payload LP7B (40 mg, 36% yield) as a white solid.
  • linker-payload LP8A 9.2 mg, 17% yield from P9, TFA salt
  • ESI m/z 501.0 (M/2 + H) + .
  • ADCs antibody drug conjugates
  • TCEP tris(2-carboxyethyl)phosphine
  • EDTA ethylenediaminetetraacetic acid
  • Linker-payloads were added at a linker- payload / antibody molar ratio of 5 - 10 and reacted for an additional 2 h at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO).
  • the reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80 °C.
  • the protein concentration was determined via UV spectrophotometer.
  • ADC monomer purity was >90% by SEC.
  • the ADCs were further characterized via hydrophobic interaction chromatography (HIC), and liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) to calculate drug-antibody ratio (DAR).
  • HIC hydrophobic interaction chromatography
  • LC-ESI MS liquid chromatography electrospray ionization mass spectrometry
  • DAR drug-antibody ratio
  • EXAMPLE 64 Conjugation of TLR7 Agonist to an Antibody [0701] In a specific example shown in the conjugation scheme of FIG.5, 15 mg/mL anti- Her2 human IgG antibody in PBS was partially reduced via addition of 2.5-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP) and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 h at 37 °C.
  • TCEP tris(2-carboxyethyl)phosphine
  • EDTA ethylenediamine
  • the reduced antibody was buffer exchanged into PBS with 1% w/v polysorbate 20.
  • Linker-payload was added at a linker-payload / antibody molar ratio of 6 and reacted for an additional 2 h at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80°C.
  • the protein concentration was determined via UV spectrophotometer.
  • ADC monomer purity was 99.7% by SEC.
  • EXAMPLE 65 [0702] Purification method for antibody-LP11A conjugate through preparative size- exclusion chromatography (SEC).
  • DTT dithiothreitol
  • HIC Analytical Hydrophobic interaction Chromatography
  • TLR-7 is an endosomal localized pattern recognition receptor that responds to pathogen-associated single stranded ribonucleic acid (ssRNA) and plays a key role in innate immune responses.
  • TLR-7 activation induces proinflammatory cytokine and type I interferon (IFN) expression via the activation of nuclear factor kappa light chain enhancer of activated B cells (NF- ⁇ B) and interferon regulatory factor (IRF) signaling pathways.
  • IFN interferon
  • HEK-Blue hTLR7 cells were utilized.
  • HEK-Blue TLR7 cells are a human embryonic kidney HEK293 cell line expressing human TLR7 and a secreted alkaline phosphatase (SEAP) reporter gene under the control of the interferon-beta (IFN- ⁇ ) minimal promoter fused to five NF- ⁇ B and AP-1 binding sites.
  • SEAP secreted alkaline phosphatase
  • a cytotoxicity assay was also performed.
  • the test compounds and a reference compound, resiquimod were aliquoted at 10 concentrations with a serial of 3-fold dilution in 96 well plates. Subsequently, 50,000 cells/well were seeded into 96-well plates containing test compounds and then the plates were incubated at 37 °C, 5% CO 2 for 24 hours.
  • EXAMPLE 68 [0718] To assess the plasma stability of representative mAb2 TLR7 antibody drug conjugates (ADCs) containing linker payloads provided herein, ncADCs were incubated in vitro with plasma from different species and the DAR was evaluated after incubation at 37 °C for up to 13 days.
  • ADCs antibody drug conjugates
  • ncADC sample (anti-HER2 Ab-LP1 ADC (comparator), anti-HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC and anti-HER2 Ab-LP7A ADC) diluted in PBS buffer (Irvine Scientific, Cat#9236) was added to pooled mouse plasma (BioIVT, Cat#MSE01PLK2P2N) and IgG depleted human plasma (BiochemMed), independently, at a final concentration of 50 ⁇ g/mL, and subsequently incubated at 37 °C. A 100- ⁇ L aliquot was removed at the time 0, 24, 36, 72, 168 and 312 hours and then immediately stored frozen at -80 °C until analysis.
  • Affinity capture of the ncADCs from the plasma samples was carried out on a KingFisher Apex 96 magnetic particle processor (Thermo Electron).
  • biotinylated anti- human Fc antibody (Regeneron generated reagent) was immobilized on Dynabeads M280 streptavidin paramagnetic beads (Invitrogen, Cat#60210).
  • Each plasma sample containing TLR7 ncADCs was mixed with 0.5 mg of the beads (Regeneron generated reagent immobilized bead) at room temperature for 2 hours in a 96 well plate.
  • the beads were then washed three times with 500 ⁇ L of HBS-EP (GE healthcare, Cat#BR100188), once with 500 ⁇ L of water, and then once with 500 ⁇ L of 8% acetonitrile in water (VWR Chemicals, Cat#BDH83640.100E). Following the washes, the ncADCs were eluted by incubating the beads with 70 ⁇ L of 1% formic acid in 30% acetonitrile / 70% water for 20 minutes at room temperature. Fifty ⁇ L eluted samples were further reduced by adding 50 ⁇ L 10 mM TCEP (Sigma, Cat 646547-10X1ML) and incubated at 37 °C for 20 minutes in ThermoMixer C.
  • antibodies were partially reduced via addition of 1.5 - 3.0-fold molar excess of TCEP in PBS pH 7.4 and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 hours at 37 °C.
  • the reduced antibodies were buffer exchanged into PBS with 1% w/v polysorbate 20.
  • Linker-payloads were added at a linker-payload / antibody molar ratio of 5 - 10 and reacted for an additional 2 hours at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80 °C.
  • SEC size exclusion chromatography
  • the protein concentration was determined via UV spectrophotometer.
  • ADC monomer purity was >90% by SEC.
  • the ADCs were further characterized via hydrophobic interaction chromatography (HIC), and liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) to calculate drug-antibody ratio (DAR). Results are shown in Tables 6 and 7 and FIG.6, FIG.7, and FIG.8.
  • Example (anti-HER2 Ab-LP1 ADC) [0728]
  • 15 mg/mL of anti-PSMA antibody in PBS was partially reduced via addition of 2.5-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP) and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 hours at 37 °C.
  • TCEP tris(2-carboxyethyl)phosphine
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged into PBS with 1% w/v polysorbate 20.
  • a linker-payload LP1 was added at a linker- payload / antibody molar ratio of 6 and reacted for an additional 2 hours at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80 °C.
  • SEC size exclusion chromatography
  • the protein concentration was determined via UV spectrophotometer.
  • ADC monomer purity was 99.7% by SEC.
  • Table 6 TLR7 antibody drug conjugates
  • HEK-Blue TLR7 cells are a human embryonic kidney HEK293 cell line expressing human TLR7 and a secreted alkaline phosphatase (SEAP) reporter gene under the control of the interferon-beta (IFN- ⁇ ) minimal promoter fused to five NF- ⁇ B and AP-1 binding sites.
  • SEAP secreted alkaline phosphatase
  • HEK-Blue TLR7/h-antigen2 cells were also utilized.
  • HEK-Blue TLR7 cells were engineered to overexpress full-length human antigen 2 via lentiviral mediated transduction and are herein referred to as HEK-Blue TLR7/h-antigen2 cells.
  • HEK-Blue TLR7/h-antigen2 cells were grown for at least 2 weeks under G418 selective pressure to enrich for antigen2 positive cells and surface expression was validated via flow cytometry with mAb2.
  • 40,000 cells were seeded into 96 well plates in HEK-Blue Detection media. Three-fold serial dilutions of free payloads or LPs were prepared in 100% DMSO, transferred to fresh assay media, and added to the cells at a final constant DMSO concentration of 0.2%. The last well in the plate served as a blank control containing only the assay media and 0.2% DMSO (untreated well) and was plotted as a continuation of the 3-fold serial dilution.
  • SEAP activity was determined in a colorimetric assay according to manufacture instructions in which the optical density at 650 nm (OD 650 ) was measured on an Envision luminometer (PerkinElmer) and EC 50 values were determined using a four-parameter logistic equation over a 10-point dose response curve (GraphPad Prism).
  • the signal to noise (S/N) was determined by taking the ratio of the highest OD 650 value on the dose response curve to the OD 650 value in the untreated wells.
  • ncADCs for TLR-dependent reporter activity, HEK-Blue hTLR7 or HEK- Blue hTLR7/h-antigen2 cells were seeded in complete growth medium at 40,000 cells per well of a 96-well plate and grown overnight. Three-fold serial dilutions of ncADCs, unconjugated antibodies, or isotype control ncADCs were prepared in dilution media (Opti-Mem TM + 0.1% BSA) and added to cells for a final assay medium of 20% dilution medium + 80% growth medium (concentrations were corrected for the DAR (drug antibody ratio) and dosed based on the effective payload concentration).
  • Opti-Mem TM + 0.1% BSA dilution media
  • concentration concentration were corrected for the DAR (drug antibody ratio) and dosed based on the effective payload concentration.
  • P9 did not increase human TLR8 within the tested dose range.
  • a known TLR7 payload, P3, increased human and mouse TLR7-dependent activity with EC 50 values of 6.98 nM and 7.27 nM, respectively, and S/N values of 2.7 and 3.2 respectively.
  • P3 increased human TLR8 activity with an EC 50 value of 826 nM and S/N of 4.9.
  • P3 with an attached linker (LP1) increased human and mouse TLR7-dependent activity with EC 50 values of 25.7 nM and 107 nM, respectively, and S/N values of 5.8 and 2.5 respectively.
  • LP1 increased human TLR8 activity with an EC 50 value of 746 ⁇ M and S/N of 2.1.
  • a known TLR8 agonist increased human TLR8 activity with an EC 50 value of 4.7 nM and S/N of 5.2, but was weakly agonistic on human TLR7 and mouse TLR7 with EC 50 values > 10 uM and S/N less than or equal to 1.3.
  • a known dual TLR7/8 agonist increases human TLR8 and mouse TLR7 activity with EC 50 values of 311 nM and 335 nM, respectively, and S/N values of 4.7 and 3.0 respectively. None of the tested payloads increased TLR3 or TLR9 activity within the tested dose ranges and were inactive in HEK-Blue null cells.
  • P9 increased human and mouse TLR7-dependent activity.
  • LPs generated from P9 (LP6A, LPA7, LP8A and LP11A) increased human TLR7 reporter activity.
  • P9-derived LPs also increased mouse TLR7 reporter activity.
  • P9 and associated LPs did not activate human TLR8 within the tested dose range.
  • LP1 increased hTLR7 and mTLR7 activity.
  • LP1 was weakly agonistic toward human TLR8. None of the test articles were active in HEK-Blue null cells within the tested dose range.
  • the mAb2-TLR7 ncADCs increased human TLR7 reporter activity in HEK-Blue hTLR7/HER2 cells with EC 50 s ranging from 13.2 nM to 16.2 nM and S/N values from 2.2 to 4.9. These same ncADCs were weakly agonistic in HEK-Blue hTLR7 cells with EC 50 s > 1.0 uM and S/N values less than or equal to 1.3.
  • a known anti-HER2 ncADC increased TLR7 reporter activity in HEK-Blue hTLR7/h-HER2 and HEK-Blue hTLR7 cells with EC 50 values of 9.59 nM and 465 nM, respectively, and S/N values of 6.4 and 2.9, respectively.
  • the free payload of anti- HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC, anti-HER2 Ab-LP7A ADC, and anti-HER2 Ab-LP8A ADC increased TLR7 activity in HEK-Blue hTLR7/h-HER2 and HEK-Blue hTLR7 cells with EC 50 values of 234nM and 153 nM, respectively and S/N values of 8.3 and 6.9, respectively.
  • the known payload, P3 was agonistic in HEK-Blue hTLR7/h-HER2 and HEK- Blue hTLR7 cells with EC 50 values of 21.8 nM and 8.16 nM, respectively, and S/N values of 9.1 and 7.5, respectively.
  • the known LP, LP1 increased hTLR7 activity in HEK-Blue hTLR7/h-HER2 and HEK-Blue hTLR7 cells with EC 50 values of 115 nM and 71.8 nM, respectively, and S/N values of 8.2 and 7.0, respectively.
  • Non-binding isotype controls (mAb1) conjugated to TLR7 LPs were weakly cytotoxic in all tested cells with EC 50 s > 1.0 ⁇ M and S/N less than or equal to 2.0.
  • EXAMPLE 71 [0740] Protocol for human PBMC TLR7 IFN- ⁇ release assay [0741] Protocol: 1.
  • Protocol for human PBMC TLR7 TNF- ⁇ release assay 1. Thaw frozen human PBMC and put all cells into 40 mL assay media (RPMI1640 supplemented with 10% HI-FBS and 1% penicillin-streptomycin) in 50 mL conical tubes. 2. Spin human PBMC at 1200 rpm for 4 minutes. 3. Remove supernatant by aspiration and resuspend pellet in assay media to 1.5625 ⁇ 10 6 cells/mL. 4. Add 80 ⁇ L cells to each well (1.25 ⁇ 10 5 cells/well). 5. Incubate the plate at 37 o C 5% CO 2 incubator for 24 hours. 6.
  • EXAMPLE 72 [0745] The ADCs provided herein were examined for their anti-tumor efficacy using a series of in vivo mouse models. [0746] Growth and Implantation of N87 Tumor Cells into NSGTM Mice [0747] The N87 gastric carcinoma tumor cell line was expanded in T225 flasks in RPMI1640 culture media supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection.
  • N87 cells were then washed twice and an aliquot of cells was collected for determining cell viability and counts using ViaStainTM, a solution containing acridine orange and propidium iodide (AOPI), in combination with the Nexcelom Cellaca MX cell counter. The remaining cells were resuspended in Matrigel Basement Membrane Matrix (50%) prepared in sterile solution and 4-5x10 6 N87 cells were implanted subcutaneously into immunodeficient Fox Chase SCID ® Beige mice across studies. In this model, a single dose (5 mg/kg) of ADCs provided herein eradicated tumors (FIG.10 and FIG.11).
  • ViaStainTM a solution containing acridine orange and propidium iodide (AOPI)
  • AOPI acridine orange and propidium iodide
  • FIG.10 shows that tumor regression was observed after treatment with 5 mg/kg (gray circle) of anti-HER2 Ab- LP6A ADC, while treatment with 1 mg/kg (gray square) anti-HER2 Ab-LP6A ADC resulted in tumor stasis, when compared to saline treated animals (open circle).
  • Regression of N87 gastric tumors was not observed in the N87 xenograft mice treated with 5 mg/kg of isotype control Ab-LP6A ADC (Table 3) (black circle), 0.5 mg/kg (gray triangle) anti-HER2 Ab-LP6A ADC, or 0.1 mg/kg (gray diamond) anti-HER2 Ab-LP6A ADC when compared to saline treated animals (open circle).
  • JIMT-1 epithelial breast tumor cell line was expanded in T225 flasks in DMEM culture media supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection.
  • JIMT-1 cells were then washed twice and an aliquot of cells was collected for determining cell viability and counts using ViaStainTM, a solution containing acridine orange and propidium iodide (AOPI), in combination with the Nexcelom Cellaca MX cell counter. The remaining cells were resuspended in Matrigel Basement Membrane Matrix (50%) prepared in sterile solution and 2.5x10 6 JIMT-1 cells were implanted subcutaneously into immunodeficient Fox Chase SCID ® Beige mice across studies.
  • ViaStainTM a solution containing acridine orange and propidium iodide (AOPI)
  • AOPI propidium iodide
  • ADCs provided herein were found to delay growth relative to an isotype control ADC, and when combined with a non-competing anti-HER2 antibody, pertuzumab, resulted in tumor regression, suggesting that FcR clustering can be used to enhance therapeutic efficacy (FIG.12).
  • MC38 Tumor Cells Engineered to Express CD20 into C57BL/6J Mice [0751] The MC38 melanoma tumor cell line expressing a defined tumor was expanded in T225 flasks in DMEM culture media supplemented with penicillin, streptomycin, L-glutamine, sodium pyruvate, 1% HEPES, 1% non-essential amino acids and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection.
  • Tumor cells were then washed twice and an aliquot of cells was collected for determining cell viability and counts using ViaStainTM, a solution containing acridine orange and propidium iodide (AOPI), in combination with the Nexcelom Cellaca MX cell counter.
  • ViaStainTM a solution containing acridine orange and propidium iodide (AOPI)
  • AOPI propidium iodide
  • Tumor cells were resuspended in Hanks Balanced Salt Solution and 1e6 tumor cells were subcutaneously implanted into each C57BL/6J mouse.
  • An anti-human CD20 ADC was shown to mediate tumor regression in this model (FIG.13).
  • N87 cells were cultured in RMPI, 10% FBS, P/S/G, before implantation, cells were mixed with an equal volume of Matrigel, and 100ul of the mixture (5e6 cells) were implanted subcutaneously into the right flank of 6-8-week-old female SCID-Beige mice (Charles River).
  • JIMT-1 cells were cultured in DMEM, 10% FBS, P/S/G. Before implantation, cells were mixed with an equal volume of Matrigel, and 200 uL of the mixture (2.5e6 cells) were implanted subcutaneously into the right flank of 6-8-week-old female SCID-Beige mice (Charles River).
  • MC38.hTAA Pos cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200 uL of 1e6 cells were implanted subcutaneously into the right flank of 6-8-week- old female C57BL/6J mice (The Jackson Laboratory).
  • MC38 cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200 uL of 5e5 cells were implanted subcutaneously into the left flank of 6-8-week-old female C57BL/6J mice for the rechallenge study (The Jackson Laboratory).
  • MC38.hTAA Pos cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200 uL of 1e6 cells were implanted subcutaneously into the right flank of 6-8-week- old female hIFNAR mice (Velocigene).
  • MC38h.TAA Pos cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200ul of 1e6 cells were implanted subcutaneously into the right flank of 6-8-week- old female mice having been humanized for TAA and hCD3 mice (Velocigene).
  • FIG.16 depicts results following parental MC38 tumor cell rechallenge in mice having initially cleared MC38.hTAA Pos engrafted tumors (the same as used in Fig. 13).
  • mice On day 60 after MC38.hTAA Pos tumor cell inoculation, tumor free mice (black square) were rechallenged with parental MC38 cells without overexpression of human TAA. Compared to control na ⁇ ve mice (open circle), mice previously treated with anti-CD20 -LP6A conjugate are protected against tumor rechallenge. Data represent mean tumor volumes (mean+/-SEM) over time (post-rechallenge). [0762] FIG.
  • FIG. 17 depicts results following treatment of mice having been inoculated with MC38.hTAA Pos tumor cells with 3 doses every seven days of anti-CD20-LP11A conjugate in wild type mice (closed symbols with solid lines) and in humanized IFNAR mice (open symbols with dashed lines) that lack the ability to respond to murine type I IFN. Regression of tumor was observed after treatment with 5 mg/kg of anti-CD20-LP11A conjugate (closed triangle) when compared to saline treated animals (closed circle) and isotype control antibody conjugate (closed square).
  • FIG. 18 depicts results following treatment of mice having been inoculated with MC38.hTAA Pos tumor cells with 3 doses every seven days of anti-CD20-LP6A conjugate with or without 5 doses every four days of anti-CD20 x anti-hCD3 bispecific antibody in mice humanized for TAA and human CD3.
  • FIG.19 depicts the ring opening of the imide bond of the antibody-drug conjugates from the conjugation of the cysteine thiol with the maleimide of the linker-payload. Ring- opening of the imide bond under physiological conditions affords two regio-isomers that one is the thiol attached to the alpha carbon and the other is the thiol attached to the beta carbon to the carboxylic acid group, respectively.
  • HBV Hepatitis B virus
  • AAV Adeno-Associated Virus
  • CHB chronic hepatitis B
  • This AAV- HBV mouse model can be used to assess different therapeutic interventions to look at sustained HBV sAg reduction of treatment (functional cure) or complete elimination of HBV infected hepatocytes.
  • mice were then treated with an anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS three times, two weeks apart subcutaneously (SC). MAb doses used are outlined in Table 12 from two independent experiments. Mice were then bled weekly or biweekly to measure sAg levels before, during and after mAb treatments. As shown in FIG. 14 and FIG.15, CHB mice treated with the anti-sAg-TLR7 ADCs displayed rapid and sustained reduction in circulating sAg levels compared to anti-sAg or TLR7 agonist treated mice.
  • Table 13 shows sAg concentrations at study end for each treatment group. Mice treated with anti- sAg-TLR7 ADCs displayed lower sAg levels than anti-mAb or TLR7 agonist treated mice in both experimental studies. Furthermore, the majority of anti-sAg-TLR7 ADC-treated mice at the lower dosed arms had undetectable sAg compared to all other treatment groups even 67 days after the last treatment.
  • Assay procedure Prepare 2 ⁇ LS9/compound solution: T 0 : add 199 ⁇ L 2 mg/mL liver S9 solution + 100 ⁇ L of 8 mM NADPH solution + 100 ⁇ L of 20 mM UDPGA solution + 1200 ⁇ L of ACN, vortex at 1000 rpm for 5 min, then add 1 ⁇ L of 4 mM cpd solution. T 240 : add 199 ⁇ L 2 mg/mL liver S9 solution + 100 ⁇ L of 8 mM NADPH solution + 100 ⁇ L of 20 mM UDPGA solution, prewarm the T 240 sample at 37°C for 5 min and 1 ⁇ L of 4 mM cpd solution was added.
  • T 240-w/o add 199 ⁇ L 2 mg/mL liver S9 solution + 200 ⁇ L of buffer, prewarm the T 240-w/o sample at 37°C for 5 min and 1 ⁇ L of 4 mM cpd solution was added.
  • T 240 min incubation add 1200 ⁇ L of ACN and then vortex at 1000 rpm for 5 min.
  • Protein precipitation centrifuge quenched samples at 14000 rpm for 5 min.
  • Sample preparation evaporate an aliquot of 1200 ⁇ L of the supernatant under N 2 stream until dry.
  • the imide ring of the linker connecting an antibody and the payload exists as an equilibrium between an opened ring and a closed five-membered imide ring as shown in FIG.19.
  • Those ADC species with an opened ring and a closed five- membered imide ring show the same or comparable activities in the research described herein.
  • EXAMPLE 75 To assess whether an anti-HBV antibody conjugated with a TLR7 agonist as an antibody drug conjugate (ADC) could break B cell tolerance and elicit an antibody response to the HBV sAg protein in an AAV-HBV mouse model, male C57BL/6 mice were transduced with 1E11 viral genomes of AAV8-HBV virus intravenously (I.V.) and six weeks later measured HBV sAg levels in their serum to determine if they exhibited a CHB (chronic hepatitis B) phenotype. Mice that had HBV sAg levels ⁇ 1 ⁇ g/mL in the serum were included in the study.
  • ADC antibody drug conjugate
  • mice were then treated with an anti-HBV sAg mAb (mAb3) conjugated with an TLR7 agonist (LP6A) at either 44 ⁇ g or 0.44 ⁇ g per injection or given PBS five times, one week apart subcutaneously (SC).
  • mAb3 The sequence of mAb3 is shown in Table 15 below.
  • CHB mice treated with anti-HBV sAg-TLR7 ADC at both doses display rapid and sustained reduction in circulating HBV sAg levels until end of study (day 120).
  • serum was obtained and anti-HBV sAg IgG titers were measured via ELISA to determine endogenous antibody responses.
  • HBV sAg-TLR7 ADC elicited HBV sAg IgG titers above background while none of the PBS mice showed HBV sAg IgG titers which is characteristic of the AAV-HBV mouse model.
  • HBV proteins One of the hallmarks of chronic hepatitis B infection is tolerogenic immune response to the HBV proteins. This is most notable for immunity to HBV sAg in which there is a minimal T cell response and no HBV sAg IgG responses despite HBV sAg being abundantly expressed.

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Abstract

Provided herein are TLR7 agonists, linker-payloads, and antibody-drug-conjugates (ADCs) thereof. Also provided are methods of treating diseases, such as cancer and chronic hepatitis B infection, with the TLR7 agonists and antibody-drug-conjugates thereof.

Description

TLR7 AGONISTS AND ANTIBODY-DRUG-CONJUGATES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Appl. No. 63/429,096, filed November 30, 2022 and U.S. Provisional Appl. No.63/578,109, filed August 22, 2023, both of which are incorporated by reference as if fully set forth herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] This application contains a sequence listing. It has been submitted electronically as an XML file titled “2387951.xml.” The sequence listing is 30,417bytes in size and was created on November 27, 2023. It is hereby incorporated by reference in its entirety herein. FIELD [0003] Provided herein are TLR7 agonists and antibody-drug-conjugates (ADCs) thereof. BACKGROUND [0004] Toll-like receptors (TLRs) are a subset of pattern recognition receptors (PRRs) and play a key role in the innate immune response. TLRs are divided into two groups depending on subcellular localization, with endosomal TLRs being of pharmaceutical interest. Of these endosomal TLRs, TLR7 has been extensively studied as a target for small molecule agonists. See, Patinote, et al., Eur. J. Med. Chem., 2020, 193:112238; U.S. Patent No.9,944,649. TLR7 agonists have been reported to have antiviral and antibacterial activity, as well as activity as vaccine adjuvants and in the treatment of allergic diseases and asthma. Of interest herein, TLR7 agonists have been studied as cancer immunotherapeutics. One TLR7 agonist has been approved by the U.S. FDA, Aldara® (imiquimod) which is indicated for treatment of actinic keratosis, superficial basal cell carcinoma and external genital warts. Immune [0005] ADCs combine the power of antibody specificity with the ability to site specifically target a particular type of cell or tissue with a payload. Research in this area has drawn significant interest and has led to marketed pharmaceutical products, including ADCETRIS® (brentuximab vedotin) and KADCYLA™ (ado-trastuzumab emtansine). ADCs having TLR7 agonist payloads have been reported. See, e.g., U.S. Patent Nos.10,472,420, 10,780,180, 10,548,985, 10,722,591, 10,675,358; PCT Publication No. WO 2020/181050. However, no such ADCs have been approved for human use. [0006] Thus, there is a continuing need for TLR agonists and ADCs thereof for treatment of various diseases, including cancer and chronic hepatitis B. SUMMARY [0007] In one aspect, the present disclosure provides TLR7 agonists of Formula I for use in the compositions and methods provided herein:
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R3 is -CO2R23, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring. [0008] In another aspect, the present disclosure provides TLR7 agonist-linkers of Formula II for use in preparation of ADCs provided herein:
Figure imgf000004_0002
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined for Formula I; R9 is a divalent group formed by removal of a terminal hydrogen (i.e., a hydrogen distal from the phenyl group to which R9 is attached) from an R3 group, as defined in claim 1; and L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD. [0009] In certain embodiments, the ADCs provided herein are useful in methods of treatment, methods of imaging, or methods of diagnosis. [0010] Also provided herein are antibody-drug-conjugates (ADCs) comprising an antigen- binding domain (ABD) specific to an antigen and a Toll-like receptor 7 (TLR7) agonist, and use of the ADCs for treatment of a disease. In some embodiments, the antigen is an HBV surface antigen (HBS sAg) and the disease is chronic Hepatitis B. [0011] In one aspect, the present disclosure provides an antibody-drug-conjugate (ADC), comprising (a) an antigen-binding domain (ABD) having binding specificity to a hepatitis B virus surface antigen (HBV sAg) and (b) a Toll-like receptor 7 (TLR7) agonist. [0012] In some embodiments, the ADC further comprises a divalent linker that links the ABD to the TLR7 agonist. [0013] In some embodiments, the ADC is according to Formula IV:
Figure imgf000005_0001
or a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R9 is a divalent group formed by removal of a hydrogen from R3, R3 being a group attached to the phenyl group at the position of R9; R3 is -CO2H, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and k is an integer from one to thirty. [0014] In some embodiments, the ADC comprises ABD linked to a compound of Formula III:
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as described elsewhere for Formula I; L is any group or moiety that links to ABD; R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form the 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. [0015] In some embodiments, ABD-L1 is linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39. In some embodiments, the ABD is linked to a compound selected from LP1, LP6, LP7, LP8, LP10, and LP11. [0016] In some embodiments, the ADC is according to Formula V:
Figure imgf000007_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as described elsewhere for Formula I; R10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene- PEG-NH-; ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites; and k is an integer from one to thirty. [0017] In some embodiments, the ADC is according to Formula VI:
Figure imgf000008_0001
or a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1, R2, R16, R11, R12, R13, R14, R15, X, and x are as defined for Formula III; and k is an integer from one to thirty. [0018] In some embodiments, the ADC comprises ABD-L1 linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39. [0019] In some embodiments, k is 1, 2, 3, 4, or 5. In some embodiments, k is 2. [0020] In some embodiments, the ABD comprises a heavy chain and the C-terminus of the heavy chain is conjugated to L1. In some embodiments, the ABD comprises two heavy chains and the C-terminus of each of the two heavy chains is conjugated to L1. In some embodiments, L1 is linked to a cysteine residue of the ABD. [0021] In some embodiments, the ABD is an antibody against a HBV sAg or a fragment thereof. In some embodiments, the ABD is a human antibody or a humanized antibody. In some embodiments, the ABD is IgG1 or IgG2a. In some embodiments, the ABD comprises a scFv having binding specificity to a HBV sAg. In some embodiments, the ABD comprises VH chain and VL chain of an antibody against a HBV sAg. [0022] In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody against a HBV sAg. In some embodiments, the ABD comprises an Fc region, wherein the Fc region comprises a modification for enhanced binding to FcγR. [0023] In another aspect, the present disclosure provides a pharmaceutical composition comprising the ADC disclosed herein and one or more pharmaceutically acceptable carriers, excipients, or diluents. [0024] In yet another aspect, the present disclosure provides a method of treatment, comprising administering to a subject in need thereof an effective amount of the ADC or the pharmaceutical composition disclosed herein. [0025] In some embodiments, the subject has chronic Hepatitis B. In some embodiments, the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or the pharmaceutical composition. [0026] In some embodiments, the method further comprises, before the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject. In some embodiments, the method further comprises, after the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject to assess therapeutic efficacy of the ADC or the pharmaceutical composition. [0027] In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated twice, three times, or more. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated at least at 1-week intervals, at 2-week intervals, at 3-week intervals, or at 4-week intervals. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated at 1-week intervals, at 2-week intervals, at 3-week intervals, or at 4-week intervals. In some embodiments, the step of administrating the ADC or the pharmaceutical composition is repeated at 1-month intervals, at 2-month intervals, or 3-month intervals. [0028] In some embodiments, the ADC or pharmaceutical composition is administered by oral, intravenous, intraperitoneal, inhalation, intranasal, intramuscular, or subcutaneous administration. [0029] One aspect of the present disclosure provides the ADC or the pharmaceutical composition for use in treatment. In some embodiments, the ADC or the pharmaceutical composition is for use in treatment of chronic Hepatitis B in a subject in need thereof. [0030] Another aspect of the present disclosure provides the ADC or the pharmaceutical composition for manufacture of a medicament. In some embodiments, the medicament is for the treatment of chronic Hepatitis B in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS [0031] FIG.1 shows in vitro plasma stability of anti-HER2 Ab-LP1 ADC (Example 68). [0032] FIG.2 shows in vitro plasma stability of anti-HER2 Ab-LP6A ADC (Example 68). [0033] FIG.3 shows in vitro plasma stability of anti-HER2 Ab-LP11A ADC (Example 68). [0034] FIG.4 shows in vitro plasma stability of anti-HER2 Ab-LP7A ADC (Example 68). [0035] FIG.5 shows the conjugation scheme for conjugating antibodies with linker payloads provided herein (Examples 63, 64 and 69). [0036] FIG.6 is a preparative SEC chromatogram of a conjugation mixture demonstrating a clean separation of ADC monomer from aggregates (“HMW”) and unconjugated linker payload (“Free drug”). [0037] FIG. 7 is an analytical SEC chromatogram of an SEC purified antibody-TLR7 conjugate showing a monomer purity of 99.7%. [0038] FIG.8 shows a LC-ESI-MS spectrum of a deglycosylated and reduced ADC sample. The calculated average DAR value was 1.84. The deconvoluted mass spectra exhibited light chain species (LC, LC1) and heavy chain species (HC, HC1, HC2, etc.). The average DAR can be calculated from the LC and HC drug-loading. [0039] FIG.9 shows HIC chromatograms of an antibody and its LP11A conjugate, revealing a mixture of three species: DAR2 species (51%), DAR4 species (28%) and unconjugated antibody (21%). The average DAR of this ADC is 2.1. [0040] FIG. 10 depicts results following a single treatment of anti-HER2 Ab-LP6A ADC (Table 3) in the N87 xenograft tumor model. Dosing was performed at Day 0. Regression of tumor was observed after treatment with 5 mg/kg (gray circle) of anti-HER2 Ab-LP6A ADC, while treatment with 1 mg/kg (gray square) anti-HER2 Ab-LP6A ADC resulted in tumor stasis, when compared to saline treated animals (open circle). Regression of N87 gastric tumors was not observed in the N87 xenograft mice treated with 5 mg/kg of isotype control Ab-LP6A ADC (Table 3) (black circle) or 0.5 mg/kg (gray triangle) anti-HER2 Ab-LP6A ADC or 0.1 mg/kg (gray diamond) anti-HER2 Ab-LP6A ADC when compared to saline treated animals (open circle). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0041] FIG.11 depicts results following treatment of human N87 xenograft tumors with a single dose of anti-HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC or anti-HER2 Ab-LP7A ADC (Table 3). Dosing was performed at Day 0. Regression of tumor was observed after treatment with 5 mg/kg of anti-HER2 Ab-LP6A ADC (gray circle), 5 mg/kg of anti-HER2 Ab- LP11A ADC (gray square) or 5 mg/kg of anti-HER2 Ab-LP7A ADC (gray triangle), when compared to saline treated animals (open circle). Regression of N87 gastric tumors was not observed in the N87 xenograft mice treated with 5 mg/kg of isotype control Ab-LP6A ADC (black circle) (Table 3), isotype control Ab-LP11A ADC (black square) (Table 3), or isotype control Ab-LP7A ADC (black triangle) (Table 3) when compared to saline treated animals (open circle). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0042] FIG. 12 depicts results following treatment of trastuzumab-resistant HER2medium human JIMT-1 xenograft tumors. Dosing was initiated at Day 0 and subsequently every 7 days for a total of 4 doses of anti-HER2 Ab-LP6A ADC (Table 3) or in combination with pertuzumab. Regression of tumor was observed after treatment with 5 mg/kg of anti-HER2 Ab-LP6A ADC in combination with 5 mg/kg pertuzumab (gray square), while treatment with 5 mg/kg of anti- HER2 Ab-LP6A ADC alone (gray circle) resulted in tumor stasis for 45 days, when compared to 5 mg/kg unconjugated mAb2 alone (open circle) treated animals. Regression of JIMT-1 breast tumors was not observed following treatment with 5 mg/kg of isotype control Ab-LP6A ADC (black circle) (Table 3), isotype control Ab-LP6A ADC combined with 5 mg/kg pertuzumab (black square), or 5 mg/kg unconjugated mAb2 combined with 5 mg/kg pertuzumab (open square), when compared to 5 mg/kg unconjugated mAb2 alone (open circle) treated animals. Data represent mean tumor volumes (mean+/-SEM) over time (post- dose). [0043] FIG.13 depicts results following treatment of MC38 tumors engineered to express human CD20. Beginning at Day 0, tumor bearing mice were treated with 3 total doses of anti- CD20 Ab-LP6A ADC (Table 3) with each dose separated by seven days. Tumor regression was observed in four of five mice after treatment with 5 mg/kg of anti-CD20 Ab-LP6A ADC (gray square) when compared to saline treated animals (open circle). Regression of MC38hCD20 tumors was not observed in the MC38hCD20 syngeneic mice treated with 5 mg/kg of anti-mIgG2a Ctrl Ab-LP1 ADC (black circle) (Table 3) or 5 mg/kg unconjugated anti- CD20 Ab (open square), when compared to 5 mg/kg saline treated animals (open circle). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0044] FIG.14 depicts HBV sAg levels measured in the chronic hepatitis B (CHB) disease mouse model after treatment with an anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS three times, two weeks apart subcutaneously (SC). (Example 51; Experiment 1). [0045] FIG.15 depicts HBV sAg levels measured in the chronic hepatitis B (CHB) disease mouse model after treatment with an anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS three times, two weeks apart subcutaneously (SC). (Example 51; Experiment 2). [0046] FIG.16 depicts results following parental MC38 tumor cell rechallenge in mice having initially cleared MC38.hTAAPos engrafted tumors (the same as used in Fig.13). On day 60 after MC38.hTAAPos tumor cell inoculation, tumor free mice (black square) were rechallenged with parental MC38 cells without overexpression of human TAA. Compared to control naïve mice (open circle), mice previously treated with anti-CD20 -LP6A conjugate are protected against tumor rechallenge. Data represent mean tumor volumes (mean+/-SEM) over time (post-rechallenge). [0047] FIG. 17 depicts results following treatment of mice having been inoculated with MC38.hTAAPos tumor cells with 3 doses every seven days of anti-CD20-LP11A conjugate in wild type mice (closed symbols with solid lines) and in humanized IFNAR mice (open symbols with dashed lines) that lack the ability to respond to murine type I IFN. Regression of tumor was observed after treatment with 5 mg/kg of anti-CD20-LP11A conjugate (closed triangle) when compared to saline treated animals (closed circle) and isotype control antibody conjugate (closed square). Regression of MC38.hTAAPos tumors was not observed in the humanized IFNAR mice treated with 5 mg/kg of anti-CD20-LP11A conjugate (open triangle); isotype control antibody conjugate (open square) or saline treated animals (open circle). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0048] FIG.18 depicts results following treatment of mice having been inoculated with MC38.hTAAPos tumor cells with 3 doses every seven days of anti-CD20-LP6A conjugate with or without 5 doses every four days of anti-CD20 x anti-hCD3 bispecific antibody in mice humanized for TAA and human CD3. Regression of tumor was observed after treatment with 2.5 mg/kg of anti-CD20-LP6A conjugate in combination with 2.5 mg/kg anti-CD20 x anti- hCD3 bispecific antibody (black triangle), while treatment with 2.5 mg/kg of anti-CD20 in combination with 2.5 mg/kg isotype control for the bispecific antibody (open triangle), 2.5 mg/kg of anti-CD20x anti-hCD3 bispecific antibody alone (black circle); 2.5 mg/kg of isotype control antibody-(NC-1) in combination with 2.5 mg/kg anti-CD20 x anti-hCD3 bispecific antibody (black square) resulted in tumor growth delay when compared to mice treated with 2.5 mg/kg isotype control for the bispecific antibody (open circle) or 2.5 mg/kg of isotype control antibody in combination with 2.5 mg/kg isotype control for the bispecific antibody (open square). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0049] FIG.19 depicts the ring opening of the imide bond of the antibody-drug conjugates from the conjugation of the cysteine thiol with the maleimide of the linker-payload. Ring- opening of the imide bond under physiological conditions affords two regio-isomers that one is the thiol attached to the alpha carbon and the other is the thiol attached to the beta carbon to the carboxylic acid group, respectively. [0050] FIG. 20 is a scheme showing one possible metabolic pathway for the compound referenced herein as Qo-LP11A. [0051] FIG.21 is a scheme showing another possible metabolic pathway for the compound referenced herein as Qo-LP11A. [0052] FIG. 22 shows hepatitis B virus surface antigen (HBV sAg) levels measured in a chronic hepatitis B (CHB) disease mouse model after treatment with an anti-sAg monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate-buffered saline (PBS) 5 times, 1 week apart subcutaneously. mAb3+LP6A was effective in reducing the HBV sAg levels as compared to the PBS control. [0053] FIG.23 shows anti-hepatitis B virus surface antigen (HBsAG) IgG titers measured in a chronic hepatitis B (CHB) disease mouse model at day 120 (D120) post-first treatment with anti-surface antigen (sAg) monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate- buffered saline (PBS) 5 times, 1 week apart subcutaneously. Mab3+LP6A-treated mice had higher titers as compared to PBS-treated control mice. DETAILED DESCRIPTION I. Definitions [0054] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. [0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. [0056] The singular forms "a," "an," and "the" include plural references, unless the context clearly dictates otherwise. [0057] As used herein "subject" is an animal, such as a mammal, including human, such as a patient. [0058] As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmacokinetic behavior of such compounds, compositions and mixtures. Biological activities can be observed in in vitro systems designed to test for such activities. [0059] As used herein, "antigen-binding domain" or “ABD” means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide- containing construct that is capable of specifically binding a particular antigen of interest. As used herein, “antigen-binding domain” includes antibodies and antigen-binding fragments of antibodies. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. [0060] The phrase “specifically binds,” or “binds specifically to,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10-8 M or less (e.g., a smaller KD denotes tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. Antibodies can, for example, be identified by real-time, label free bio-layer interferometry assay on an Octet® HTX biosensor, which bind specifically to a target antigen. Moreover, multi-specific antibodies that bind to one domain in the target antigen and one or more additional antigens or a bi-specific that binds to two different regions of the target antigen are nonetheless considered antibodies that “specifically bind”, as used herein. In addition to neutralizing antibodies, antibodies that bind specifically to the target antigen, but are non- neutralizing, also can be used within the scope of the present disclosure to generate antibody- drug conjugates. Such antibodies may function, for example, to deliver a payload to the cells expressing a target antigen. [0061] The term “high affinity” antibody refers to those mAbs having a binding affinity to a target antigen, expressed as KD, of at least 10-8 M; preferably 10-9 M; more preferably 10-10M, even more preferably 10-11 M, even more preferably 10-12 M, as measured by real-time, label free bio-layer interferometry assay, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA. [0062] The phrase or term “slow off rate,” “Koff,” or “kd” refers to an antibody that dissociates from a target antigen, with a rate constant of 1x10-3 s-1 or less, preferably 1x10-4 s-1 or less, as determined by real-time, label free bio-layer interferometry assay, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™. [0063] As used herein, "unrelated antigens" are proteins, peptides or polypeptides that have less than 95% amino acid identity to one another. [0064] The term "antibody," as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Three CDRs of VH are referred to as HCDR1, HCDR2, and HCDR3, and three CDRs of VL are referred to as LCDR1, LCDR2 and LCDR3. [0065] As used herein, the term “antigen-binding fragment" of an antibody means any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. [0066] As used herein, the term "human antibody" means antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. [0067] As used herein, the term “humanized antibody” means chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety. [0068] As used herein, the term "recombinant human antibody", means all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. [0069] The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed in WO 2016/100807 or US 2016/0176953 A1, each of which are incorporated herein by reference in their entirety. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. [0070] As used herein in the context of amino acid sequences, the phrase “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. [0071] As used herein, the term "surface plasmon resonance", refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.). [0072] As used herein, the term "KD", means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., antibody-antigen interaction). Unless indicated otherwise, the KD values disclosed herein refer to KD values determined by surface plasmon resonance assay at 25° C. [0073] As used herein, pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'- ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and inorganic salts, such as but not limited to, sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, mesylates, and fumarates. [0074] As used herein, the terms “treat,” “treating,” or “treatment” refer to the reduction or amelioration of the severity of at least one symptom or indication of the disease, e.g., cancer or hepatitis B infection, due to the administration of a therapeutic agent such as a disclosed antibody to a subject in need thereof. The terms include inhibition of progression of disease or of worsening of infection. The terms also include positive prognosis of disease, e.g., the subject may be free of infection, the subject may have reduced or no viral titers, the subject may have tumor shrinkage, upon administration of a therapeutic agent such as a disclosed antibody or antibody-drug conjugate. The therapeutic agent may be administered at a therapeutic dose to the subject. [0075] The terms “prevent,” “preventing,” or “prevention” refer to inhibition of manifestation of any symptoms or indications of a disease (e.g., cancer or hepatitis B infection) upon administration of a disclosed antibody or antibody-drug conjugate. The term includes prevention of the spread of infection in a subject exposed to the virus or at risk of having hepatitis B infection. [0076] The phrase “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). [0077] As used herein, amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the compound or pharmaceutical composition. [0078] As used herein, the IC50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response. [0079] Where moieties are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical moieties that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-. [0080] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain saturated hydrocarbon radical. The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkyl. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, including those groups having 10 or fewer carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having six or fewer carbon atoms. Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n- heptyl, n-octyl, and the like. [0081] The term "alkenyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain hydrocarbon radical having one or more carbon-carbon double bonds. The term "alkenylene" by itself or as part of another substituent means a divalent radical derived from an alkenyl. Typically, an alkenyl (or alkenylene) group will have from 1 to 24 carbon atoms, including those groups having 10 or fewer carbon atoms. A "lower alkenyl" or "lower alkenylene" is a shorter chain alkenyl or alkenylene group, generally having six or fewer carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., ethenyl), 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and the higher homologs and isomers. [0082] The term "alkynyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain hydrocarbon radical having one or more carbon-carbon triple bonds, which can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Examples of alkynyl groups include, but are not limited to, ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. [0083] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. [0084] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, containing at least one heteroatom in the chain selected from O, N, P, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may have an alkyl substituent to fulfill valency and/or may optionally be quaternized. The heteroatom(s) O, N, P, Si and S may be placed at any interior position of the heteroalkyl group (i.e., not at the point of attachment to the rest of the molecule). Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2- CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2- S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and –CH2-O- Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-O- CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-O-CH2-CH2-NH-CH2-, -CH2-CH2-S-CH2-CH2- and - CH2-S-CH2-CH2-NH-CH2-. For alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula –C(O)2R'- represents both –C(O)2R'- and –R'C(O)2-. [0085] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively, including bicyclic, tricyclic and bridged bicyclic groups. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. The terms "cycloalkylene" and "heterocycloalkylene" by themselves or as part of another substituent means a divalent radical derived from a cycloalkyl or heterocycloalkyl. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornanyl, bicyclo(2.2.2)octanyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, 1- or 2-azabicyclo(2.2.2)octanyl, and the like. [0086] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (in some embodiments from 1 to 3 rings) which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups that contain from one to four heteroatoms selected from N, O, and S in the ring(s), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. The terms "arylene" and "heteroarylene" by themselves or as part of another substituent means a divalent radical derived from an aryl or heteroaryl. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1- isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. The term "heteroarylium" refers to a heteroaryl group that is positively charged on one or more of the heteroatoms. [0087] Each of the above terms are meant to include both substituted and unsubstituted forms of the indicated radical. Non-limiting examples of substituent moieties for each type of radical are provided below. [0088] Substituent moieties for alkyl, heteroalkyl, alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups are, in some embodiments, selected from, deuterium, -OR', =O, =NR', =N-OR', -NR'R", -SR', halo, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", - NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", - S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and –NO2 in a number ranging from zero to the number of hydrogen atoms in such radical. In some embodiments, substituent moieties for cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups also include substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl. R', R", R"' and R"" each in some embodiments are independently are hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound provided herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituent moieties, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and –CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like). [0089] Substituent moieties for aryl and heteroaryl groups are, in some embodiments, selected from deuterium, halo, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl, -OR', -NR'R", -SR', - SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", - NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", - S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and –NO2, -R', -N3, -CH(Ph)2, fluoro(C1- C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of hydrogens on the aromatic ring system; and where R', R", R"' and R"" are, in some embodiments, independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound provided herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. [0090] Two of the substituent moieties on adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -Q'-C(O)-(CRR')q-Q''-, wherein Q' and Q'' are independently –NR-, -O-, -CRR'- or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently –CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula – (CRR')s-X'-(CR''R''')d-, where s and d are independently integers of from 0 to 3, and X' is –O-, -NR'-, -S-, -S(O)-, -S(O)2-, or –S(O)2NR'-. The substituent moieties R, R', R" and R'" are, in some embodiments, independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. [0091] The terms "halo," by itself or as part of another substituent, means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1- C4)alkyl" is meant to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like. [0092] The term "oxo" as used herein means an oxygen atom that is double bonded to a carbon atom. [0093] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). [0094] Certain ADCs provided herein possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present disclosure. The ADCs provided herein do not include those which are known in the art to be too unstable to synthesize and/or isolate. II. TLR7 Agonists [0095] In one aspect, provided herein are TLR7 agonists for use in the compositions and methods provided herein. In some embodiments, the TLR7 agonists are compounds of Formula I:
Figure imgf000023_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R3 is -CO2R23, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring. In some embodiments, the compound of the formula (I) is not a compound of the formula:
Figure imgf000023_0002
[0001] In some embodiments, in the TLR7 agonist according to Formula I, R5 and R6 are selected from (i), (ii), and (iii): (i) R5 and R6 each H; (ii) R5 is H and R6 is alkyl; (iii) R5 and R6, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8, together with the N to which they are attached, form a heterocyclic ring. [0096] In some embodiments, the TLR7 agonists are selected with the proviso that R4 is not substituted with hydroxyl. [0097] In some embodiments, the TLR7 agonists are selected with the proviso that the alkylene and heteroalkylene portions of R3 are not substituted with oxo. [0098] In some embodiments, the TLR7 agonists are selected with the proviso that the compound is not 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine, which corresponds to P3 in Table 1; or (4-((2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3-methoxyphenyl)methanol, which corresponds to P1 in Table 1. [0099] In some embodiments, R1 is H. In some embodiments, R1 is halo, -NHR4, -OR4, -NH- OR4 or -R4, and is a straight chain of 6 atoms in length. In some embodiments, R1 is halo. In some embodiments, R1 is -NHR4. In some embodiments, R1 is -OR4. In some embodiments, R1 is -NH-OR4. In some embodiments, R1 is -R4. In some embodiments, R1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt. In some embodiments, R1 is -NH- n-pentyl. In some embodiments, R1 is -NH-O-n-butyl. In some embodiments, R1 is -O-n- pentyl. In some embodiments, R1 is -n-hexyl. In some embodiments, R1 is -NH-CH2CH2-OEt. [0100] In some embodiments, R2 is halo. In some embodiments, R2 is alkoxy. In some embodiments, R2 is methoxy. In some embodiments, R2 is H. [0101] In some embodiments, R3 is -CO2R23, -CONHR23, , -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y. In some embodiments, R3 is -CONHR23. In some embodiments, R3 is - alkylene-Y. In some embodiments, R3 is -heteroalkylene-Y. In some embodiments, R3 is - heteroalkylene-arylene-Y. In some embodiments, R3 is -(hydroxy)heteroalkylene-Y. In some embodiments, R3 is -(amino)heteroalkylene-Y. In some embodiments, R3 is alkylene-PEG-Y. In some embodiments, R3 is -CONH2, -COOH, -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. In some embodiments, R3 is -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2- O-alkylene-Y. In some embodiments, R3 is -C(Me)2OH, -CO2H -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH -CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, -CH2OCH2CO-1-piperazinyl, -(R)- CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2, or -CH2-1-piperazinyl. In some embodiments, R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, - CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH - CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, - CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl. [0102] In some embodiments, R4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl. In some embodiments, R4 is n-butyl. In some embodiments, R4 is n-pentyl. In some embodiments, R4 is n-hexyl. In some embodiments, R4 is ethoxyethyl. [0103] In some embodiments, R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring. In some embodiments, R5 and R6 are each H. In some embodiments, R5 is H and R6 is alkyl. In some embodiments, R5 and R6, together with the N to which they are attached, form 1-piperazinyl. [0104] In some embodiments, Y is OH. In some embodiments, Y is a divalent glycine group of the formula -NHCH2C(O)-. In some embodiments, Y is -NR5R6. In some embodiments, Y is -COZ. In some embodiments, Y is -OH, -NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2 or -CO-1-piperazinyl. In some embodiments, Y is -OH, -NH2, 1-piperazinyl, -COOH or -CO-1- piperazinyl. [0105] In some embodiments, Z is -OH. In some embodiments, Z is alkoxy. In some embodiments, Z is -NR7R8. In some embodiments, Z is -OH, ethoxy, -N-n-Pr2 or 1-piperazinyl. In some embodiments, Z is -OH or 1-piperazinyl. [0106] In some embodiments, R7 and R8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1-piperazinyl. In some embodiments, R7 and R8, together with the N to which they are attached, form 1-piperazinyl. [0107] In some embodiments, the TLR7 agonist is selected from compounds P1-P39 and P41-P48 in Table 1 and pharmaceutically acceptable salts of any of these:
Figure imgf000026_0001
Figure imgf000027_0001
Table 1
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
[0108] In some embodiments, a TLR7 agonist can be a known TLR7 agonist, e.g., 852A, imiquimod, resiquimod, gardiquimod loxoribine, bropirimine, 3M-011, 3M-052, DSR-6434, DSR-29133, SC1, SZU-101, SM-360320, and SM-276001. Exemplary TLR7 agonists are described in, for example, Chi et al., Front. Pharmacol.8:34, 31 May 2017, which is hereby incorporated by reference in its entirety. III. Synthesis of the TLR7 Agonists [0109] The TLR7 agonists of the disclosure can be synthesized in any suitable fashion. Non- limiting examples of synthetic schemes for the synthesis of the TLR7 agonists of the disclosure are presented herein in Schemes 1-7. [0110] Scheme 1. Synthesis of Intermediate Aa starting from Compound 1
Figure imgf000031_0001
[0111] Scheme 2. Synthesis of Intermediates Starting from Compound 5
Figure imgf000031_0002
Figure imgf000032_0001
[0112] Scheme 3. Synthesis of payloads P1, P2, P20, P23, P27, P29, P32, P33, P37 and P39
Figure imgf000032_0002
Figure imgf000033_0001
[0114] Scheme 5. Synthesis of payloads P22, P25, P31, P35, P21, P24, P30 and P34.
Figure imgf000034_0001
Figure imgf000035_0001
O
Figure imgf000036_0001
IV. Linker-TLR7-Agonist (Linker-Payload) [0117] In one aspect, provided herein are TLR7 agonist-linkers for use in preparing ADCs. In some embodiments, the ADC comprises ABD linked to a linker-TLR7 agonist according to Formula II:
Figure imgf000037_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined elsewhere for Formula I; R9 is a divalent group formed by removal of a hydrogen from R3, R3 being a group attached to the phenyl group at the position of R9; and L is any group or moiety that links, connects, or bonds an antigen-binding domain (ABD), as defined elsewhere herein, with a compound of Formula I. In some embodiments, the compound of Formula (II) is not a compound of the formula:
Figure imgf000037_0002
. [0118] In some embodiments, R9 is -alkylene-Y1-, -heteroalkylene-Y1-, -heteroalkylene-arylene-Y1-, -(hydroxy)heteroalkylene-Y1, -(amino)heteroalkylene-Y1, or - alkylene-PEG-Y1. In some embodiments, R9 is -alkylene-Y1-. In some embodiments, R9 is - heteroalkylene-Y1-. In some embodiments, R9 is -heteroalkylene-arylene-Y1-. In some embodiments, R9 is -(hydroxy)heteroalkylene-Y1. In some embodiments, R9 is - (amino)heteroalkylene-Y1. In some embodiments, R9 is -alkylene-PEG-Y1. In another embodiment, R9 is -CH2-Y1-, -CH2-O-heteroalkylene-Y1-, or -CH2-O-alkylene-Y1-. In another embodiment, R9 is -C(Me)2O-, -CO-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, - CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CO-, - CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, - CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH-, or -CH2-1-piperazin-4-yl. [0119] In embodiment, Y1 is -O-. In some embodiments, Y1 is a divalent glycine group of the formula -NHCH2C(O)-. In some embodiments, Y1 is -NR5-. In some embodiments, Y1 is - COZ1, wherein Z1 is -O-, -NR7-, -O-alkylene-, or 1-piperazin-4-yl. In some embodiments, Y1 is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl. [0120] In some embodiments, Z1 is -O-. In some embodiments, Z1 is -NR7-. In some embodiments, R7 is H. In some embodiments, R7 is alkyl. [0121] In some embodiments, Z1 is 1-piperazin-4-yl. In some embodiments, Y1 is 1- piperazin-4-yl. In some embodiments, Y1 is -CO-1-piperazin-4-yl. [0122] In some embodiments, linkers L for use herein may be found, for example, in Antibody-Drug Conjugates and Immunotoxins, Phillips, G. L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates, Wang, J., Shen, W.-C., and Zaro, J. L., Eds.; Springer International Publishing, 2015. In some embodiments, the L group for the ADCs provided herein is sufficiently stable to exploit the circulating half-life of the antigen binding domain and, at the same time, capable of releasing its payload after antigen-mediated internalization of the ADC. Linker L can be cleavable or non-cleavable. Cleavable linkers for use as L herein include linkers that are cleaved by intracellular metabolism following internalization, e.g., cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers for use as L herein include linkers that release an attached payload via lysosomal degradation of the antigen binding domain following internalization. Suitable L linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction labile linkers, self- immolative linkers, and non-cleavable linkers. Suitable L linkers also include, but are not limited to, those that are or comprise peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, and para-aminobenzyl (PAB) units. [0123] The term “PEG” or “polyethylene glycol,” by itself or in combination with another term, means, unless otherwise stated, the group –(OCH2CH2O)n-, wherein n is an integer from about 1 to about 100, such as, about 1 to about 10, about 2 to about 8, about 4 to about 20, about 4 to about 12, and about 12 to about 30). Examples of PEG groups include, but are not limited to, the following [insert ChemDraw structures]. PEG groups can have any suitable molecular weight, such as from about 60 g/mol to about 6,000 g/mol, about 60 g/mol to about 600 g/mol, about 100 g/mol to about 500 g/mol, about 300 g/mol to about 1,200 g/mol, about 200 g/mol to about 800 g/mol, about 200 g/mol to about 1,000 g/mol, about 500 g/mol to about 1,000 g/mol, about 500 g/mol to about 2,500 g/mol, or about 800 g/mol to about 2,200 g/mol. [0124] Any linker molecule or linker technology known in the art can be used as L to create or construct an ADC provided herein. In some embodiments, the L linker is a cleavable linker. In other embodiments, the L linker is a non-cleavable linker. In some embodiments, L linkers that can be used in the ADCs provided herein include linkers that comprise or consist of e.g., MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine- alanine), dipeptide site in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide site in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof. Additional examples of L linkers that can be used in the ADCs provided herein are disclosed, e.g., in U.S. Pat. No. 7,754,681 and in Ducry, Bioconjugate Chem., 2010, 21:5-13, and the references cited therein. [0125] In some embodiments, the L linkers are stable in physiological conditions. In some embodiments, the L linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value. In some embodiments, an L linker comprises an enzyme-cleavable moiety. In one embodiment, enzyme-cleavable L linkers include, but are not limited to, peptide bonds, ester linkages, and hydrazones. In some embodiments, the L linker comprises a cathepsin-cleavable linker. [0126] In some embodiments, the L linker comprises a non-cleavable moiety. [0127] In some embodiments, the L linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L- or D-α-amino acids. In some embodiments, the L linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or combination thereof. In some embodiments, one or more side chains of the amino acids is linked to a side chain group, described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the L linker comprises lysine, valine, and citrulline. In some embodiments, the L linker comprises lysine, valine, and alanine. In some embodiments, the L linker comprises valine and alanine. [0128] In some embodiments, the L linker comprises a self-immolative group. The self- immolative group can be any such group known to those of skill in the art. In particular embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those of skill in the art will recognize that a self-immolative group is capable of carrying out a chemical reaction which releases the remaining atoms of an L linker from a payload. [0129] In other embodiments, the L group can be modified with one or more enhancement groups. In some embodiments, the enhancement group can be linked to the side chain of any amino acid in L. In one embodiment, amino acids for linking enhancement groups include lysine, asparagine, aspartate, glutamine, glutamate, and citrulline. The link to the enhancement group can be a direct bond to the amino acid side chain, or the link can be indirect via a spacer and/or reactive group. In one embodiment, spacers and reactive groups include any described herein. In some embodiments, the enhancement group can be any group that imparts a beneficial effect to the payload, linker payload, or ADC including, but not limited to, biological, biochemical, synthetic, solubilizing, imaging, detecting, and reactivity effects, and the like. In some embodiments, the enhancement group is a hydrophilic group. In some embodiments, the enhancement group is a cyclodextrin. In some embodiments, the enhancement group is an alkyl sulfonic acid, heteroalkyl sulfonic acid, alkenyl sulfonic acid, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, heteroalkenyl amine (e.g., quaternary amine), or heteroalkenyl sugar. In some embodiments, sugars include, without limitation, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like. In some embodiments, sugars include sugar acids such as glucuronic acid, further including conjugated forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like. The cyclodextrin can be any cyclodextrin known to those of skill. In some embodiments, the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or mixtures thereof. In some embodiments, the cyclodextrin is alpha cyclodextrin. In some embodiments, the cyclodextrin is beta cyclodextrin. In some embodiments, the cyclodextrin is gamma cyclodextrin. In some embodiments, the enhancement group is capable of improving solubility of the remainder of the ADC. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or non-substituted. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)1-5SO3H, –(CH2)n–NH-(CH2)1-5SO3H, –(CH2)n–C(O)NH-(CH2)1-5SO3H, –(CH2CH2O)m–C(O)NH-(CH2)1-5SO3H, –(CH2)n–N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, –(CH2)n– C(O)N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, or –(CH2CH2O)m–C(O)N((CH2)1-5C(O)NH(CH2)1- 5SO3H)2, wherein n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In one embodiment, the alkyl or alkenyl sulfonic acid is –(CH2)1-5SO3H. In some embodiments, the heteroalkyl or heteroalkenyl sulfonic acid is –(CH2)n–NH-(CH2)1-5SO3H, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)n–C(O)NH-(CH2)1-5SO3H, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2CH2O)m–C(O)NH-(CH2)1-5SO3H, wherein m is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is – (CH2)n–N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)n–C(O)N((CH2)1- 5C(O)NH(CH2)1-5SO3H)2, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2CH2O)m–C(O)N((CH2)1- 5C(O)NH(CH2)1-5SO3H)2, wherein m is 1, 2, 3, 4, or 5. [0130] In some embodiments, L comprises a maleimido (for conjugation with a thiol, e.g., cysteine, of an antigen binding domain), an N-hydroxysuccinimido ester (for conjugation with an amine, e.g., lysine, of an antigen binding domain) or cyclooctynyl group (for conjugation with an antigen binding domain using click chemistry). See, e.g., WO 2020/132658; Chio et al. Methods Mol. Biol.2020, 2078:83-87. [0131] In some embodiments, L contains a maleimido group. In such embodiments, the maleimido group in L reacts with a cysteine residue on an antigen binding domain to form a carbon-sulfur bond. [0132] In some embodiments, L contains an N-hydroxysuccinimido ester group. In such embodiments, the N-hydroxysuccinimido ester group reacts with a lysine residue on an antigen binding domain to form an amide bond. [0133] In some embodiments, L contains an alkyne which can react via click chemistry with an azide, e.g., to form a click chemistry product. In some embodiments, the alkyne group reacts with an azide on a modified antigen binding domain. In some embodiments, L contains a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc. 1961,357-396; Wang et al. J. Am. Chem. Soc. 2003, 125(11), 3192-3193; and Agard et al. J. Am. Chem. Soc.2004, 126(46), 15046-15047). In some embodiments, the reactive group is an alkyne that is capable of undergoing a 1,3- cycloaddition reaction with an azide. Alkynes that may be used in such embodiments include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts. Alkynes that may be used in such embodiments also include, but are not limited to, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted, e.g., fluorinated alkynes, aza-cycloalkynes and bicyclo[6.1.0]nonyne. In other embodiments, alkynes are useful for conjugating antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups. In some embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, e.g., heavy chain Gln295, with a compound bearing an amino group and an azide group, in the presence of the enzyme transglutaminase. [0134] In some embodiments, L is selected from 2-maleimido-1-ethyl, 2-maleimidoacetyl, and 3-maleimidopropanoyl. In certain embodiments, L is selected from:
Figure imgf000042_0001
Figure imgf000043_0001
,
Figure imgf000044_0001
. [0135] In some embodiments, L is a group selected from 2-maleimido-1-ethyl, 2- maleimidoacetyl, 3-maleimidopropanoyl, ,
Figure imgf000045_0001
[0136] In some embodiments, the linker-TLR7 agonist is selected from those in Table 2 and pharmaceutically acceptable salts of any of these:
Figure imgf000045_0002
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
[0137] In some embodiments, when the payload (i.e., TLR7 agonist) has an alcohol group (i.e., -OH), then the payload can be converted to a prodrug prior to attachment to a linking group and formation of the ADC. See, e.g., WO 2020/146541. In this embodiment, the payloads of Formula I can be converted to linker-TLR7 agonist of Formula III:
Figure imgf000050_0001
or a pharmaceutically acceptable salt thereof, wherein: L is a linker as defined elsewhere herein; R1, R2 and X are as defined elsewhere for Formula I; R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7- OR 8-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form the 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. [0138] In some embodiments, the linker-TLR7 agonist has Formula III, wherein R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form a 4-, 5-, or 6-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, - (amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. In some embodiments, R16 is the moiety of R3 Formula I that links the phenyl ring to the oxygen atom of the alcohol. [0139] In some embodiments, the TLR7 agonist used in preparing the linker-TLR7 agonist according to Formula III is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, or P39. V. Synthesis of the TLR7 Agonist-Linkers [0140] Scheme 8. Synthesis of Linker-payloads LP1, LP2, LP3 and LP4.
Figure imgf000051_0001
[0141] Scheme 9. Synthesis of Linker-payload LP5.
Figure imgf000052_0001
[0142] Scheme 10. Synthesis of Linker-payload LP6A.
Figure imgf000052_0002
[0143] Scheme 11. Synthesis of Linker-payloads LP7A and LP10A.
Figure imgf000053_0001
[0144] Scheme 12. Synthesis of Linker-payload LP8A.
Figure imgf000053_0002
Figure imgf000054_0001
LP8A [0145] Scheme 13. Synthesis of Linker-payloads LP9 and LP12.
Figure imgf000054_0002
[0146] Scheme 14. Synthesis of Linker-payload LP6A, LP6B, LP7A, LP7B, LP7C, LP7D LP10A, LP10B, LP11A, LP11B, LP11C, LP11D, LP12 and LP14
Figure imgf000054_0003
Figure imgf000055_0001
Figure imgf000056_0001
[0147] Scheme 14A. Alternate synthesis of Linker-payload LP11A
Figure imgf000056_0002
Figure imgf000056_0004
Figure imgf000056_0003
Figure imgf000057_0002
[0148] Scheme 15. Synthesis of Linker-payload LP8B2
Figure imgf000057_0001
(SEQ ID NO: 16)
Figure imgf000058_0001
Figure imgf000059_0001
VI. ADCs for Use in Compositions and Methods [0151] In one aspect, the present disclosure provides an antibody-drug-conjugate (ADC) comprising an antigen-binding domain (ABD) (e.g., an ABD having binding specificity for a target antigen such as HBV sAg or a tumor specific antigen) and a TLR7 agonist. In some embodiments, the ADC further comprises a divalent linker that links the ABD to the TLR7 agonist. The ABD can bind to the TLR7 agonist with or without a linker, at any location along the ABD as long as the ABD is able to bind its target. [0152] In some embodiments, the ADC is according to Formula IV:
Figure imgf000060_0001
or a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R9 is a divalent group formed by removal of a hydrogen from R3, R3 being a group attached to the phenyl group at the position of R9; R3 is -CO2H, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and k is an integer from one to thirty. [0153] In some embodiments, the ABC comprises one TLR7 agonist molecule conjugated to an ABD having binding specificity for a target antigen. In some embodiments, the ADC comprises more than one TLR7 agonist molecules per an ABD. In some embodiments, two, three, four, five or more TLR7 agonist molecules are conjugated to one ABD. When the ADC is according to Formula IV, k can be 1, 2, 3, 4, or 5. In some embodiments, k is 2. In some embodiments, k is 1. In some embodiments, k is 4. In some embodiments, k is 5 or more. [0154] In some embodiments, the ADC is selected with the proviso that the ADC does not comprise 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine or (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl)methyl)-3-methoxyphenyl)methanol. [0155] In some embodiments, R1 is halo, -NHR4, -OR4, -NH-OR4 or -R4, and has a straight chain of 6 atoms in length. In some embodiments, R1 is halo. In some embodiments, R1 is - NHR4. In some embodiments, R1 is -OR4. In some embodiments, R1 is -NH-OR4. In some embodiments, R1 is -R4. In some embodiments, R1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n- pentyl, -n-hexyl or -NH-CH2CH2-OEt. In some embodiments, R1 is -NH-n-pentyl. In some embodiments, R1 is -NH-O-n-butyl. In some embodiments, R1 is -O-n-pentyl. In some embodiments, R1 is -n-hexyl. In some embodiments, R1 is -NH-CH2CH2-OEt. [0156] In some embodiments, R2 is halo. In some embodiments, R2 is alkoxy. In some embodiments, R2 is methoxy. In some embodiments, R2 is H. [0157] In some embodiments, R3 is -CO2H, -CONHR23, -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y. In some embodiments, R3 is CONHR23. In some embodiments, R3 is - alkylene-Y. In some embodiments, R3 is -heteroalkylene-Y. In some embodiments, R3 is - heteroalkylene-arylene-Y. In some embodiments, R3 is -(hydroxy)heteroalkylene-Y. In some embodiments, R3 is -(amino)heteroalkylene-Y. In some embodiments, R3 is alkylene-PEG-Y. In some embodiments, R3 is -CONH2, -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene- Y. In some embodiments, R3 is -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. In some embodiments, R3 is -C(Me)2OH, -CO2H -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH -CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, -CH2OCH2CO-1-piperazinyl, -(R)- CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2, or -CH2-1-piperazinyl. In some embodiments, R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, - CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH - CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, - CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl. [0158] In some embodiments, R4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl. In some embodiments, R4 is n-butyl. In some embodiments, R4 is n-pentyl. In some embodiments, R4 is n-hexyl. In some embodiments, R4 is ethoxyethyl. [0159] In some embodiments, R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring. In some embodiments, R5 and R6 are each H. In some embodiments, R5 is H and R6 is alkyl. In some embodiments, R5 and R6, together with the N to which they are attached, form 1-piperazinyl. [0160] In some embodiments, Y is OH. In some embodiments, Y is a divalent glycine group of the formula -NHCH2C(O)-. In some embodiments, Y is -NR5R6. In some embodiments, Y is -COZ. In some embodiments, Y is -OH, -NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2 or -CO-1-piperazinyl. In some embodiments, Y is -OH, -NH2, 1-piperazinyl, -COOH or -CO-1- piperazinyl. [0161] In some embodiments, Z is -OH. In some embodiments, Z is alkoxy. In some embodiments, Z is -NR7R8. In some embodiments, Z is -OH, ethoxy, -N-n-Pr2 or 1-piperazinyl. In some embodiments, Z is -OH or 1-piperazinyl. [0162] In some embodiments, R7 and R8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1-piperazinyl. In some embodiments, R7 and R8, together with the N to which they are attached, form 1-piperazinyl. [0163] In some embodiments, ABD-L1 is linked to a compound selected from P1-P43 by removal of a hydrogen from the group at the position corresponding to R3 of the compound. [0164] In some embodiments, R9 is -alkylene-Y1-, -heteroalkylene-Y1-, -heteroalkylene-arylene-Y1-, -(hydroxy)heteroalkylene-Y1, -(amino)heteroalkylene-Y1, or - alkylene-PEG-Y1. In some embodiments, R9 is -alkylene-Y1-. In some embodiments, R9 is - heteroalkylene-Y1-. In some embodiments, R9 is -heteroalkylene-arylene-Y1-. In some embodiments, R9 is -(hydroxy)heteroalkylene-Y1. In some embodiments, R9 is - (amino)heteroalkylene-Y1. In some embodiments, R9 is -alkylene-PEG-Y1. In some embodiments, R9 is -CH2-Y1-, -CH2-O-heteroalkylene-Y1-, or -CH2-O-alkylene-Y1-. In some embodiments, R9 is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, - CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CO-, - CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, - CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH-, or -CH2-1-piperazin-4-yl. [0165] In some embodiments, Y1 is -O-. In some embodiments, Y1 is a divalent glycine group of the formula -NHCH2C(O)-. In some embodiments, Y1 is -NR5-. In some embodiments, Y1 is -COZ1, wherein Z1 is -O-, -NR7-, -O-alkylene-, or 1-piperazin-4-yl. In some embodiments, Y1 is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl. [0166] In some embodiments, Z1 is -O-. In some embodiments, Z1 is -NR7-. In some embodiments, R7 is H. In some embodiments, R7 is alkyl. [0167] In some embodiments, Z1 is 1-piperazin-4-yl. In some embodiments, Y1 is 1- piperazin-4-yl. In some embodiments, Y1 is -CO-1-piperazin-4-yl. A. L1 Divalent Groups [0168] In some embodiments, the ADC of the present disclosure comprises a TLR7 linked to an ABD indirectly via a linker. In some embodiments, the linker is a divalent linker (L1) that links the ABD to the TLR7 agonist according to Formula IV. In some embodiments, when the ADC is conjugated to TLR7 agonist indirectly via a linker, the divalent linker (L1) is created by the reaction between the linker (L) and the ADC for the conjugation. [0169] Linkers (L1) for use herein may be found, for example, in Antibody-Drug Conjugates and Immunotoxins, Phillips, G. L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates, Wang, J., Shen, W.-C., and Zaro, J. L., Eds.; Springer International Publishing, 2015. In certain embodiments, the L1 group for the ADCs provided herein is sufficiently stable to exploit the circulating half- life of the antigen binding domain and, at the same time, capable of releasing its payload after antigen-mediated internalization of the ADC. Linker L1 can be cleavable or non-cleavable. Cleavable linkers for use as L1 herein include linkers that are cleaved by intracellular metabolism following internalization, e.g., cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers for use as L1 herein include linkers that release an attached payload via lysosomal degradation of the antigen binding domain following internalization. Suitable L1 linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction labile linkers, self-immolative linkers, and non- cleavable linkers. Suitable L1 linkers also include, but are not limited to, those that are or comprise peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, and para-aminobenzyl (PAB) units. [0170] Any linker molecule or linker technology known in the art can be used as L1 to create or construct an ADC provided herein. In certain embodiments, the L1 linker is a cleavable linker. In other embodiments, the L1 linker is a non-cleavable linker. In certain embodiments, L1 linkers that can be used in the ADCs provided herein include linkers that comprise or consist of e.g., MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), dipeptide site in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide site in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof. Additional examples of L1 linkers that can be used in the ADCs provided herein are disclosed, e.g., in U.S. Pat. No. 7,754,681 and in Ducry, Bioconjugate Chem., 2010, 21:5-13, and the references cited therein. [0171] In certain embodiments, the L1 linkers are stable in physiological conditions. In certain embodiments, the L1 linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value. In some embodiments, an L1 linker comprises an enzyme-cleavable moiety. In some embodiments, enzyme-cleavable L1 linkers include, but are not limited to, peptide bonds, ester linkages, and hydrazones. In some embodiments, the L1 linker comprises a cathepsin-cleavable linker. [0172] In some embodiments, the L1 linker comprises a non-cleavable moiety. [0173] In some embodiments, the L1 linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L- or D-α-amino acids. In some embodiments, the L1 linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or combination thereof. In certain embodiments, one or more side chains of the amino acids is linked to a side chain group, described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the L1 linker comprises lysine, valine, and citrulline. In some embodiments, the L1 linker comprises lysine, valine, and alanine. In some embodiments, the L1 linker comprises valine and alanine. [0174] In some embodiments, the L1 linker comprises a self-immolative group. The self- immolative group can be any such group known to those of skill in the art. In particular embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those of skill in the art will recognize that a self-immolative group is capable of carrying out a chemical reaction which releases the remaining atoms of an L1 linker from a payload. [0175] In other embodiments, the L1 group can be modified with one or more enhancement groups. In certain embodiments, the enhancement group can be linked to the side chain of any amino acid in L1. In some embodiments, amino acids for linking enhancement groups include lysine, asparagine, aspartate, glutamine, glutamate, and citrulline. The link to the enhancement group can be a direct bond to the amino acid side chain, or the link can be indirect via a spacer and/or reactive group. In some embodiments, spacers and reactive groups include any described herein. In certain embodiments, the enhancement group can be any group that imparts a beneficial effect to the payload, linker payload, or ADC including, but not limited to, biological, biochemical, synthetic, solubilizing, imaging, detecting, and reactivity effects, and the like. In certain embodiments, the enhancement group is a hydrophilic group. In certain embodiments, the enhancement group is a cyclodextrin. In certain embodiments, the enhancement group is an alkyl, heteroalkyl, alkenyl, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, heteroalkenyl amine (e.g., quaternary amine), or heteroalkenyl sugar. In certain embodiments, sugars include, without limitation, monosaccharides, disaccharides, and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like. In certain embodiments, sugars include sugar acids such as glucuronic acid, further including conjugated forms such as glucuronides (i.e., via glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like. The cyclodextrin can be any cyclodextrin known to those of skill. In certain embodiments, the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or mixtures thereof. In certain embodiments, the cyclodextrin is alpha cyclodextrin. In certain embodiments, the cyclodextrin is beta cyclodextrin. In certain embodiments, the cyclodextrin is gamma cyclodextrin. In certain embodiments, the enhancement group is capable of improving solubility of the remainder of the ADC. In certain embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or non-substituted. In certain embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)1-5SO3H, –(CH2)n–NH-(CH2)1-5SO3H, –(CH2)n–C(O)NH-(CH2)1-5SO3H, – (CH2CH2O)m–C(O)NH-(CH2)1-5SO3H, –(CH2)n–N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, –(CH2)n– C(O)N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, or –(CH2CH2O)m–C(O)N((CH2)1-5C(O)NH(CH2)1- 5SO3H)2, wherein n is 1, 2, 3, 4, or 5, and m is 1, 2, 3, 4, or 5. In some embodiments, the alkyl or alkenyl sulfonic acid is –(CH2)1-5SO3H. In another embodiment, the heteroalkyl or heteroalkenyl sulfonic acid is –(CH2)n–NH-(CH2)1-5SO3H, wherein n is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)n–C(O)NH-(CH2)1-5SO3H, wherein n is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2CH2O)m–C(O)NH-(CH2)1-5SO3H, wherein m is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)n–N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, wherein n is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2)n– C(O)N((CH2)1-5C(O)NH(CH2)1-5SO3H)2, wherein n is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is –(CH2CH2O)m–C(O)N((CH2)1- 5C(O)NH(CH2)1-5SO3H)2, wherein m is 1, 2, 3, 4, or 5. [0176] In another embodiment, L1 contains a 3-thiosuccinimido group (resulting from conjugation of a maleimido group with a thiol, e.g., cysteine, of an antigen binding domain), an amido group (resulting from conjugation of a N-hydroxysuccinimido group with an amine, e.g., lysine, of an antigen binding domain) or a triazolocyclooctyl group (resulting from conjugation of a cyclooctynyl group using click chemistry with an antigen binding domain modified to contain an azido group). See, e.g., WO 2020/132658; Chio et al. Methods Mol. Biol.2020, 2078:83-87. [0177] In certain embodiments, L1 contains a 3-thiosuccinimido group. In such embodiments, the 3-thiosuccinimido group in L1 results from reaction of a cysteine residue on an antigen binding domain with a maleimido group of L to form a carbon-sulfur bond. [0178] In certain embodiments, L1 is derived from L containing a maleimido group. In such embodiments, the maleimido group in L reacts with a cysteine residue on an antigen binding domain to form a carbon-sulfur bond. [0179] In some embodiments, L1 is derived from L containing an N-hdroxysuccinimido ester group. In such embodiments, the N-hydroxysuccinimido ester group reacts with a lysine residue on an antigen binding domain to form an amide bond. [0180] In other embodiments, L1 is derived from L containing an alkyne which can react via click chemistry with an azide, e.g., to form a click chemistry product. In some embodiments, the alkyne group reacts with an azide on a modified antigen binding domain. In certain embodiments, L contains a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc.1961,357-396; Wang et al. J. Am. Chem. Soc.2003, 125(11), 3192-3193; and Agard et al. J. Am. Chem. Soc.2004, 126(46), 15046-15047). In some embodiments, the reactive group is an alkyne that is capable of undergoing a 1,3-cycloaddition reaction with an azide. Alkynes that may be used in such embodiments include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts. Alkynes that may be used in such embodiments also include, but are not limited to, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted, e.g., fluorinated alkynes, aza-cycloalkynes and bicyclo[6.1.0]nonyne. In other embodiments, alkynes are useful for conjugating antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, e.g., heavy chain Gln295, with a compound bearing an amino group and an azide group, in the presence of the enzyme transglutaminase. [0181] In some embodiments, L1 contains an amido group. In such embodiments, the amido group in L1 results from reaction of an N-hydroxysuccinimido ester group of L with a lysine residue on an antigen binding domain to form an amide bond. [0182] In other embodiments, L1 contains a cyclic group which results from reaction of an alkyne via click chemistry with an azide, e.g., to form a click chemistry product. In some embodiments, the alkyne group reacts with an azide on a modified antigen binding domain. In some embodiments, an antigen-binding domain contains an alkyne group that reacts with an azide on L. See, e.g., click chemistry, Huisgen Proc. Chem. Soc.1961,357-396; Wang et al. J. Am. Chem. Soc. 2003, 125(11), 3192-3193; and Agard et al. J. Am. Chem. Soc. 2004, 126(46), 15046-15047. In some embodiments, the alkyne group is an alkyne that is capable of undergoing a 1,3-cycloaddition reaction with an azide. Alkynes that may be used in such embodiments include strained alkynes, e.g., those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts. Alkynes that may be used in such embodiments also include, but are not limited to, dibenzoazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted, e.g., fluorinated alkynes, aza-cycloalkynes and bicycle[6.1.0]nonyne. In other embodiments, alkynes are useful for conjugating antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, e.g., heavy chain Gln295, with a compound bearing an amino group and an azide group, in the presence of the enzyme transglutaminase. [0183] In another embodiment, L1 is a group derived from 2-maleimido-1-ethyl, 2- maleimidoacetyl, 3-maleimidopropanoyl, ,
Figure imgf000068_0001
[0184] In certain embodiments, L1 is or contains a divalent group selected from:
Figure imgf000069_0001
. [0185] In another embodiment, L1 is or contains a group selected from ,
Figure imgf000069_0002
O
Figure imgf000070_0001
Figure imgf000071_0001
[0186] In some embodiments, L1 is non-cleavable under physiological conditions. In some embodiments, L1 is cleavable under physiological conditions. In some embodiments, L1 is an acid-labile linker, a hydrolysis-labile linker, an enzymatically cleavable linker, a reduction labile linkers or a self-immolative linker. In some embodiments, L1 is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit. In some embodiments, L1 comprises one or more amino acids. In some embodiments, L1 comprises a self-immolative group. In some embodiments, L1 comprises p-aminobenzyl (PAB) or p- aminobenzyloxycarbonyl (PABC). In some embodiments, L1 comprises a maleimido, an N- hydroxysuccinimido ester or cyclooctynyl group. In some embodiments, L1 is a group derived from 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, ,
Figure imgf000071_0002
, O O O O N H N N H N H O O O O N N O N NH O , O NH2 , O O O O O H O O O HN H N N H N N N N N N N H H H H O O O O O O O O O OH N NH , H 2 , O
Figure imgf000072_0001
Figure imgf000073_0001
[0187] In some embodiments, the ADC comprises ABD linked LP1-LP15. B. Antigen-binding Domains (ABD) [0188] In one embodiment, antigen-binding domains, i.e., ABD in Formula IV, for use in the ADCs provided herein include any molecule that specifically interacts with a particular antigen. [0189] In certain embodiments, the ABD is an antibody or antigen-binding fragment of an antibody. In certain embodiments, the ABD is an antibody. [0190] In some embodiments, the ABD is an antibody comprising an Fc region modified to enhance binding affinity to FcγR. In some embodiments, ABD is an antibody with one or more mutations selected from F243L, R292P, Y300L, V305I, and P396L. In some embodiments, ABD is an antibody with one or more mutation selected from S239D and I332E. In some embodiments, ABD is an antibody with one or more mutations selected from S239D, I332E, and A330L. In some embodiments, ABD is an antibody with one or more mutations selected from S298A, E333A and K334A. In some embodiments, ABD is an antibody with one or more mutations selected from L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, ABD is an antibody with one or more mutations selected from D270E, K326D, A330M, and K334E. In some embodiments, ABD is an antibody with L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in one heavy chain and D270E, K326D, A330M, and K334E in the opposing heavy chain. In some embodiments, ABD is an antibody with one or more mutations selected from G236A, S239D, and I332E. In some embodiments, ABD is an antibody with one or more mutations selected from M252Y, S254T, and T256E. In some embodiments, ABD is an antibody with one or more mutations selected from M428L and N434S. In some embodiments, ABD is an antibody with one or more mutations selected from S267E and L328F. In some embodiments, ABD is an antibody with one or more mutations selected from N325S and L328F. [0191] In some embodiment, ABD is an antibody that comprises a glutamine residue. Antibodies comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain (glutaminyl-modified antibodies) are within the skill of the practitioners in the art. In other embodiments, ABD is an N297Q mutant antibody. In further embodiments, Z is an antibody that has one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites. See, e.g., U.S. Patent No.9,676,871 and U.S. Patent Application Publication No.2003/0138785. [0192] In certain embodiments, the antibody is aglycosylated. In some embodiments, the antibody is glucosylated. [0193] In certain embodiments, ABD is an antibody that is a monoclonal antibody, human antibody, humanized antibody, camelised antibody, or chimeric antibody. In other embodiments, ABD is an antibody of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In some embodiments, ABD has a molecular weight of at least 500, 600, 700, 800, 900, 1000, 10000, 50000 or 100000 Daltons. [0194] In other embodiments, antigen-binding domains that can be used in the ADCs provided herein include antibodies, antigen-binding fragments of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., (see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein)), and aptamers or portions thereof. In some embodiments, ABD comprises a scFv having binding specificity to a target antigen. [0195] Methods for determining whether two molecules specifically bind one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antigen-binding domain, as used herein, includes polypeptides that bind a target antigen or a portion thereof with a KD of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured in a surface plasmon resonance assay. [0196] In certain embodiments, the framework regions (FRs) of the antibodies or antigen- binding fragment thereof for use in the ADCs provided herein may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. [0197] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody. [0198] The antigen-binding domains for use in the ADCs provided herein may comprise or consist of antigen-binding fragments of full antibody molecules. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. [0199] Non-limiting examples of antigen-binding fragments for use in the ADCs provided herein include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. In other embodiments, an antigen-binding fragment of an antibody includes other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains. [0200] In certain embodiments, an antigen-binding fragment of an antibody will comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain. [0201] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody for use in the ADCs provided herein include: (i) VH- CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH- CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2- CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In further embodiments, an antigen-binding fragment may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). [0202] In another embodiment, the antigen-binding domains used in the ADCs provided herein may comprise or consist of human antibodies and/or recombinant human antibodies, or antigen-binding fragments thereof. [0203] In another embodiment, the antigen-binding domains used in the ADCs provided herein may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. In some embodiments, such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. [0204] In another embodiment, the antigen-binding domains used in the ADCs provided herein also include bispecific antigen-binding molecules, such as bispecific antibodies. Methods for making bispecific antibodies are known in the art and may be used to construct bispecific antigen-binding molecules for use herein. Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). See also, e.g., US2018/0134794, which discloses bispecific antigen-binding molecules. Briefly, bispecific antigen binding molecules may comprise a first antigen-binding domain (also referred to herein as "D1"), and a second antigen-binding domain (also referred to herein as "D2"). The simultaneous binding of the two separate epitopes by the bispecific antigen-binding molecule results in effective ligand blocking with minimal activation of target signaling. In certain embodiments, D1 and D2 domains of a bispecific antibody are non-competitive with one another. Non-competition between D1 and D2 means that, the respective monospecific antigen binding proteins from which D1 and D2 were derived do not compete with one another for binding to the target. Exemplary antigen-binding protein competition assays are known in the art. In certain embodiments, D1 and D2 bind to different (e.g., non-overlapping, or partially overlapping) epitopes on the target. Bispecific antigen-binding molecules may be constructed using the antigen-binding domains of two separate monospecific antibodies. For example, a collection of monoclonal monospecific antibodies may be produced using standard methods known in the art. The individual antibodies thus produced may be tested pairwise against one another for cross-competition to the target protein. If two different antibodies are able to bind to the target at the same time (i.e., do not compete with one another), then the antigen-binding domain from the first antibody and the antigen-binding domain from the second, non- competitive antibody can be engineered into a single bispecific antibody. A bispecific antigen- binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. Any antigen binding construct which has the ability to simultaneously bind two separate, non- identical epitopes of the target molecule is regarded as a bispecific antigen-binding molecule. Bispecific antigen-binding molecules, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology) as will be known to a person of skill in the art. In another embodiment, bispecific antibodies are also provided wherein one arm of the bispecific antibody binds to an epitope on a first target protein, and the other arm of the bispecific antibody binds to a second epitope on a second target protein. Other exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. (Epub: Dec.4, 2012)). [0205] In another embodiment, the antigen binding domains for use in the ADCs provided herein also include antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art. In some embodiments, variants include variants of any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art having one or more conservative substitutions. For example, the antigen binding domains include antibodies or antigen binding fragments thereof having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art. In another embodiment, the antigen binding domains include antibodies or antigen binding fragments thereof also include variants having substantial sequence identity to any of the HCVR, LCVR, and/or CDR amino acid sequences known in the art. In certain embodiments, residue positions which are not identical differ by conservative amino acid substitutions. [0206] Sequence identity between two different amino acid sequences is typically measured using sequence analysis software. Sequence analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another algorithm when comparing a sequence provided herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402. [0207] The antigen-binding domains for use in the ADCs provided herein encompass proteins having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind the target proteins. Such variant antigen-binding domains comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence but exhibit biological activity that is essentially equivalent to that of the described antibodies. [0208] Two antigen-binding domains are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Some antigen- binding domains will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied. [0209] In some embodiments, two antigen-binding domains are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. [0210] In some embodiments, two antigen-binding domains are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching. [0211] In some embodiments, two antigen-binding domains are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known. [0212] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antigen-binding domain or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) in a well- controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding domain. [0213] Bioequivalent variants of antigen-binding domains for use in the ADCs provided herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen- binding domains may include variants comprising amino acid changes which modify the glycosylation characteristics of the antigen-binding domain, e.g., mutations which eliminate or remove glycosylation. [0214] In certain embodiments, the antigen-binding domains for use in the ADCs provided herein bind to a human target protein but not to target protein from other species. In other embodiments, the antigen-binding domains for use in the ADCs provided herein bind to a human target protein and to a target protein from one or more non-human species. For example, the antigen-binding domains for use in the ADCs provided herein may bind to a human target protein and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee target protein. In some embodiments, the antigen-binding domains specifically bind human target protein and cynomolgus monkey (e.g., Macaca fascicularis) target protein. In other embodiments, antigen-binding domains for use herein bind human target protein but do not bind, or bind only weakly, to cynomolgus monkey target protein. 1. ABD Sequences [0215] In some embodiments, the ABD comprises the heavy chain and light chain of an antibody. [0216] In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. 2. Linkage Sites [0217] The ABD can be linked to the TLR7 agonist directly or indirectly via a linker, through an attachment at a particular amino acid within the ABD. Exemplary amino acid attachments that can be used in the context of this embodiment of the disclosure include, e.g., lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358- 361; WO 2005/089808; US 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non-natural amino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidic amino acids (see, e.g., WO 2012/05982). Linkers can also be conjugated to an ABD via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130, and Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997). [0218] In some examples, ABD is bonded to the linker through a lysine residue. In some embodiments, the antibody or antigen binding molecule is bonded to the linker through a cysteine residue, lysine residue, or glutamine residue. In certain embodiments, the ABD is bonded to the linker through a cysteine residue. In certain embodiments, a linker maleimide moiety bonds to an antibody cysteine residue. In certain embodiments, the ABD is bonded to the linker through a lysine residue. In certain embodiments, a linker N-hydroxysuccinimide moiety bonds to an antibody lysine residue to form an amide linkage. [0219] In certain embodiments, the ABD is bonded to the linker through a glutamine residue (see, e.g., Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997 and Dennler et al., Bioconjugate Chem. 2014, 25:569-578). Antibodies comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues. In certain embodiments, antibodies or antigen binding molecules are engineered by mutations, for example insertions or deletions to facilitate reaction via transglutaminase. In certain embodiments, antibodies or antigen binding molecules are engineered to remove one or more glycosylation sites. In certain embodiments, antibodies or antigen binding molecules are engineered to add one or more glutamine residues. In certain embodiments, glutamine residues are added within a TGase recognition tag, as described herein. Techniques for engineering glutamine residues into an antibody polypeptide chain (glutaminyl-modified antibodies or antigen binding molecules) are within the skill of the practitioners in the art. In certain embodiments, the antibody is aglycosylated. [0220] In certain embodiments, ABD comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, ABD comprises two heavy chain polypeptides, each with one Gln295 or Q295 residue. In further embodiments, ABD comprises one or more glutamine residues at a site other than a heavy chain 295. Included herein are antibodies of this section bearing N297Q mutation(s) described herein. In certain embodiments, a glutamine residue is added at the heavy chain C-terminus. [0221] In certain embodiments, the glutamine is polypeptide engineered with a glutamine- containing tag (e.g., glutamine-containing peptide tags, Q-tags or TGase recognition tag). The term “TGase recognition tag” or “Q-Tag” refers to a sequence of amino acids comprising a glutamine residue that when incorporated into (e.g., appended to) a polypeptide sequence, under suitable conditions, is recognized by a transglutaminase (“TGase”) and leads to cross- linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reactive group. The recognition tag may be a peptide sequence that is not naturally present in the polypeptide. In certain embodiments, the TGase recognition tag comprises at least one glutamine. In certain embodiments, the TGase recognition tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., conventional amino acid Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, He, Met, Pro, Thr, Lys, or Trp or nonconventional amino acid). In certain embodiments, the TGase recognition tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 2), LLQG (SEQ ID NO: 1), LSLSQG (SEQ ID NO: 17), GGGLLQGG (SEQ ID NO: 18), GLLQG (SEQ ID NO: 19), LLQ, GSPLAQSHGG (SEQ ID NO: 20), GLLQGGG (SEQ ID NO: 21), GLLQGG (SEQ ID NO: 22), GLLQ (SEQ ID NO: 23), LLQLLQGA (SEQ ID NO: 3), LLQGA (SEQ ID NO: 5), LLQYQGA (SEQ ID NO: 12), LLQGSG (SEQ ID NO: 6), LLQYQG (SEQ ID NO: 4), LLQLLQG (SEQ ID NO: 3), SLLQG (SEQ ID NO: 7), LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), and LLQGR (SEQ ID NO: 11). See for example, WO2012059882, the entire contents of which are incorporated herein. [0222] In some embodiments, ABD includes an antibody heavy chain and further includes a TGase recognition tag at the C-terminus of the antibody heavy chain. In some embodiments, ABD includes an antibody heavy chain and further includes a TGase recognition tag at the C- terminus of the antibody heavy chain, wherein the TGase recognition tag is the pentapeptide sequence LLQGA (SEQ ID NO: 5). In some embodiments, ABD includes two antibody heavy chains and further includes a TGase recognition tag at the C-terminus of each antibody heavy chain. In some embodiments, ABD includes two antibody heavy chains and further includes a TGase recognition tag at the C-terminus of each antibody heavy chain, wherein the TGase recognition tag is the pentapeptide sequence LLQGA (SEQ ID NO: 5). [0223] ABD can be also modified at one or more glutamine residues via transglutaminase (see, e.g., Jeger et al., Angew Chem Int Ed Engl., 2010, 49:9995-9997 and Dennler et al., Bioconjugate Chem. 2014, 25:569-578). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be coupled to a primary amine compound to provide a moiety capable of reacting with a reactive group on a linker-payload. In certain embodiments, the primary amine compound provides a diene or dienophile. In certain embodiments, the primary amine compound provides a diene or dienophile, and the linker- payload provides a complementary dienophile or diene, respectively, for conjugation via a Diels-Alder reaction. In certain embodiments, the primary amine compound provides an azido group. In certain embodiments, the primary amine compound provides an azido group, and the linker-payload provides a complementary alkyne, for conjugation via a click reaction. [0224] In some embodiments, the ABD comprises a heavy chain and the heavy chain is linked to ABD directly or indirectly via a linker. In some embodiments, the ABD comprises a light chain and the light chain is linked to ABD directly or indirectly via a linker. [0225] In some embodiments, the ABD comprises a heavy chain and the C-terminus of the heavy chain is linked to ABD directly or indirectly via a linker. In some embodiments, the ABD comprises a light chain and the C-terminus of the light chain is linked to ABD directly or indirectly via a linker. [0226] In some embodiments, the ABD comprises two heavy chains and each of the two heavy chains is linked to ABD directly or indirectly via a linker. In some embodiments, the ABD comprises two light chains and each of the two light chains is linked to ABD directly or indirectly via a linker. [0227] In some embodiments, the ABD comprises two heavy chains and C-terminus of each of the two heavy chains is linked to ABD directly or indirectly via a linker. In some embodiments, the ABD comprises two light chains and C-terminus of each of the two light chains is linked to ABD directly or indirectly via a linker. 3. Epitope Mapping and Related Technologies [0228] The epitope to which the antigen-binding domains bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a target protein. Alternatively, the relevant epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of the target protein. In some embodiments, the epitope is located on or near the binding domain of the target protein. In other embodiments, the epitope is located outside of the binding domain of the target protein. [0229] Various techniques known to persons of ordinary skill in the art can be used to determine the epitope with which the antigen-binding domains used in the ADCs provided herein interact. Exemplary techniques that can be used to determine an epitope or binding domain of a particular antigen-binding domain include, e.g., point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen-binding domain to the deuterium-labeled protein. Next, the protein/antigen-binding domain complex is transferred to water to allow hydrogen- deuterium exchange to occur at all residues except for the residues protected by the antigen- binding domain (which remain deuterium-labeled). After dissociation of the antigen-binding domain, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antigen-binding domain interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystal structure analysis can also be used to identify the amino acids within a polypeptide with which an antigen-binding domain interacts. 4. Synthesis of ABDs [0230] In one embodiment, the antibodies for use in the ADCs provided herein are fully human antibodies. Methods for generating monoclonal antibodies, including fully human monoclonal antibodies are known in the art. Any such known methods can be used in the context of the present disclosure to make human antibodies that specifically bind to a human protein target. [0231] Using VELOCIMMUNE™ technology, for example, or any other similar known method for generating fully human monoclonal antibodies, high affinity chimeric antibodies to a human protein target are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, mouse constant regions are replaced with a desired human constant region, for example wild-type or modified IgG1 or IgG4, to generate a fully human antibody. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region. In certain instances, fully human antibodies are isolated directly from antigen-positive B cells. [0232] Monoclonal antibodies can be generated by any techniques with which those having ordinary skill in the art will be familiar. Such methods include, but are not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B-cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein. For example, fully human monoclonal antibodies can be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. Methods for obtaining fully human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun.6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol.8:455-58; Jakobovits et al., 1995 Ann. N. Y. Acad. Sci.764:525-35. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes may be mini-gene constructs, or transloci on yeast artificial chromosomes, which undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue. Fully human monoclonal antibodies may be obtained by immunizing the transgenic mice, which may then produce human antibodies specific for a target antigen. Lymphoid cells of the immunized transgenic mice can be used to produce human antibody-secreting hybridomas according to the methods described herein. Polyclonal sera containing fully human antibodies may also be obtained from the blood of the immunized animals. [0233] Another method for generating human antibodies of the present disclosure includes immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Patent No. 4,464,456. Such an immortalized B-cell line (or lymphoblastoid cell line) producing a monoclonal antibody that specifically binds to a target antigen can be identified by immunodetection methods as provided herein, for example, an ELISA, and then isolated by standard cloning techniques. The stability of the lymphoblastoid cell line producing an antibody against a target antigen can be improved by fusing the transformed cell line with a murine myeloma to produce a mouse-human hybrid cell line according to methods known in the art (see, e.g., Glasky et al., Hybridoma 8:377-89 (1989)). Still another method to generate human monoclonal antibodies is in vitro immunization, which includes priming human splenic B-cells with a target antigen, followed by fusion of primed B-cells with a heterohybrid fusion partner. See, e.g., Boerner et al., 1991 J. Immunol.147:86-95. [0234] In certain embodiments, a B-cell that is producing an antibody against a target antigen is selected and the light chain and heavy chain variable regions are cloned from the B-cell according to molecular biology techniques known in the art (WO 92/02551; U.S. Patent 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B-cells from an immunized animal may be isolated from the spleen, lymph node, or peripheral blood sample by selecting a cell that is producing an antibody that specifically binds to a target antigen. B-cells may also be isolated from humans, for example, from a peripheral blood sample. [0235] Methods for detecting single B-cells that are producing an antibody with the desired specificity are well known in the art, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibody, and the like. Methods for selection of specific antibody-producing B-cells include, for example, preparing a single cell suspension of B-cells in soft agar that contains a target antigen. Binding of the specific antibody produced by the B-cell to the antigen results in the formation of a complex, which may be visible as an immune-precipitate. [0236] The methods for obtaining antibodies of the present disclosure can also adopt various phage display technologies known in the art. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Human or murine immunoglobulin variable region gene combinatorial libraries may be created in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind specifically to a target antigen or variant or fragment thereof. See, e.g., U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments may be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with the sequence encoding a phage coat protein. A fusion protein may be a fusion of the coat protein with the light chain variable region domain and/or with the heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments may also be displayed on a phage particle (see, e.g., U.S. Patent No.5,698,426). [0237] Antibody fragments fused to another protein, such as a minor coat protein, can be also used to enrich phage with antigen. Then, using a random combinatorial library of rearranged heavy (VH) and light (VL) chains from mice immune to the target antigen (e.g., HBV sAg, tumor specific antigen), diverse libraries of antibody fragments are displayed on the surface of the phage. These libraries can be screened for complementary variable domains, and the domains purified by, for example, affinity column. See Clackson et al., Nature, V.352 pp.624-628 (1991). [0238] Heavy and light chain immunoglobulin cDNA expression libraries may also be prepared in lambda phage, for example, using λlmmunoZapTM(H) and λImmunoZapTM(L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B-cell population, and used to create heavy and light chain immunoglobulin cDNA expression libraries in the λImmunoZap(H) and λImmunoZap(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid that allows high level expression of monoclonal antibody fragments from E. coli. [0239] In some embodiments, in a hybridoma the variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers. These primers may be synthesized by one of ordinary skill in the art or may be purchased from commercially available sources. (See, e.g., Stratagene (La Jolla, California), which sells primers for mouse and human variable regions including, among others, primers for VHa, VHb, VHc, VHd, CH1, VL and CL regions.) These primers may be used to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTMH or ImmunoZAPTML (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced using these methods (see Bird et al., Science 242:423-426, 1988). [0240] Once cells producing antibodies according to the disclosure have been obtained using any of the above-described immunization and other techniques, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein. The antibodies produced therefrom may be sequenced and the CDRs identified and the DNA coding for the CDRs may be manipulated as described previously to generate other antibodies according to the disclosure. [0241] The binding agents of the present disclosure preferably modulate activity of the target antigen in the cell-based assay described herein and/or the in vivo assay described herein and/or bind to one or more of the domains described herein and/or cross-block the binding of one of the antibodies described in this application and/or are cross-blocked from binding the target antigen by one of the antibodies described in this application. Accordingly, such binding agents can be identified using the assays described herein. [0242] In certain embodiments, antibodies are generated by first identifying antibodies that bind to one or more of the domains provided herein and/or neutralize in the cell-based and/or in vivo assays described herein and/or cross-block the antibodies described in this application and/or are cross-blocked from binding a target antigen by one of the antibodies described in this application. The CDR regions from these antibodies are then used to insert into appropriate biocompatible frameworks to generate binding agents against the target antigen. The non-CDR portion of the binding agent may be composed of amino acids or may be a non- protein molecule. The assays described herein allow the characterization of binding agents. Preferably the binding agents of the present disclosure are antibodies as defined herein. [0243] Other antibodies according to the disclosure may be obtained by conventional immunization and cell fusion procedures as described herein and known in the art. [0244] Molecular evolution of the complementarity determining regions (CDRs) in the center of the antibody binding site also has been used to isolate antibodies with increased affinity, for example, antibodies having increased affinity for c-erbB-2, as described by Schier et al., 1996, J. Mol. Biol. 263:551. Accordingly, such techniques are useful in preparing antibodies to a target antigen. Antigen binding proteins directed against a target antigen can be used, for example, in assays to detect the presence of a target antigen, either in vitro or in vivo. The antigen binding proteins also may be employed in purifying a target antigenby immunoaffinity chromatography. [0245] Although human, partially human, or humanized antibodies will be suitable for many applications, particularly those involving administration of the antibody to a human subject, other types of antigen binding proteins will be suitable for certain applications. Non-human antibodies of the present disclosure can be, for example, derived from any antibody-producing animal, such as mouse, rat, rabbit, goat, donkey, or non-human primate (such as monkey (e.g., cynomolgus or rhesus monkey) or ape (e.g., chimpanzee)). An antibody from a particular species can be made by, for example, immunizing an animal of that species with the desired immunogen (e.g., HBV sAg, tumor specific antigen or using an artificial system for generating antibodies of that species (e.g., a bacterial or phage display-based system for generating antibodies of a particular species), or by converting an antibody from one species into an antibody from another species by replacing, e.g., the constant region of the antibody with a constant region from the other species, or by replacing one or more amino acid residues of the antibody so that it more closely resembles the sequence of an antibody from the other species. In some embodiments, the antibody is a chimeric antibody comprising amino acid sequences derived from antibodies from two or more different species. [0246] Antigen binding proteins may be prepared, and screened for desired properties, by any of a number of conventional techniques. Certain of the techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of an antigen binding protein of interest (e.g., an anti-HBV sAg antibody, a tumor specific antigen), and manipulating the nucleic acid through recombinant DNA technology. The nucleic acid may be fused to another nucleic acid of interest, or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues, for example. Furthermore, the antigen binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems, using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). [0247] Any expression system known in the art can be used to make the recombinant polypeptides of the present disclosure. Expression systems are detailed comprehensively above. In general, host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired polypeptide. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). [0248] It will be appreciated that an antibody of the present disclosure may have at least one amino acid substitution, providing that the antibody retains binding specificity. Therefore, modifications to the antibody structures are encompassed within the scope of the present disclosure. These may include amino acid substitutions, which may be conservative or non- conservative that do not destroy the target binding capability of an antibody. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. A conservative amino acid substitution may also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. [0249] Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid mimetics for a member from another class with different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule. [0250] Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations. [0251] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. [0252] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues. [0253] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. [0254] In certain embodiments, variants of antibodies include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines. [0255] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. According to certain embodiments, preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991), which are each incorporated herein by reference. [0256] In certain embodiments, antibodies of the present disclosure may be chemically bonded with polymers, lipids, or other moieties. [0257] The binding agents may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to display one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region. Such structures can be a naturally occurring polypeptide or polypeptide “fold” (a structural motif), or can have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally occurring polypeptide or fold. These scaffolds can be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus. [0258] Typically, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and tendamistat domains may be used (See e.g., Nygren and Uhlen, 1997, Curr. Opin. in Struct. Biol., 7, 463-469). [0259] Humanized antibodies can be produced using techniques known to those skilled in the art (Zhang, W., et al., Molecular Immunology.42(12):1445-1451, 2005; Hwang W. et al., Methods. 36(1):35-42, 2005; Dall’Acqua WF, et al., Methods 36(1):43-60, 2005; and Clark, M., Immunology Today.21(8):397-402, 2000). [0260] Additionally, one skilled in the art will recognize that suitable binding agents include portions of these antibodies, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and/or HCDR3. The non-CDR portion of the antibody may be a non-protein molecule, wherein the binding agent cross-blocks the binding of an antibody disclosed herein to a target antigen. The non-CDR portion of the antibody may be a non-protein molecule in which the antibody exhibits a similar binding pattern to a target antigen in a competition binding assay as that exhibited by at least one of antibodies disclosed herein. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to a target antigen and/or neutralizes a target antigen. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant antibody, and the recombinant antibody exhibits a similar binding pattern to a target antigen in the target epitope competition binding assay (described hereinbelow) as that exhibited by at least one of the antibodies disclosed herein, and/or neutralizes the target antigen. [0261] Where an antibody comprises one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as described above, it may be obtained by expression from a host cell containing DNA coding for these sequences. A DNA coding for each CDR sequence may be determined on the basis of the amino acid sequence of the CDR and synthesized together with any desired antibody variable region framework and constant region DNA sequences using oligonucleotide synthesis techniques, site-directed mutagenesis and polymerase chain reaction (PCR) techniques as appropriate. DNA coding for variable region frameworks and constant regions is widely available to those skilled in the art from genetic sequences databases such as GenBank®. In some embodiments, the heavy chain and the light chain of the antibody are expressed from a single DNA construct. In some embodiments, the heavy chain and the light chain of the antibody are expressed from two or more separate DNA constructs. [0262] Once synthesized, the DNA encoding an antibody of the present disclosure or fragment thereof may be propagated and expressed according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection using any number of known expression vectors. Thus, in certain embodiments expression of an antibody fragment may be preferred in a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol.178:497-515). In certain other embodiments, expression of the antibody or a fragment thereof may be preferred in a eukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells) or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma (such as a mouse NSO line), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells. [0263] One or more replicable expression vectors containing DNA encoding an antibody variable and/or constant region may be prepared and used to transform an appropriate cell line, for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacteria, such as E. coli, in which production of the antibody will occur. In order to obtain efficient transcription and translation, the DNA sequence in each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operatively linked to the variable domain sequence. Particular methods for producing antibodies in this way are generally well-known and routinely used. For example, basic molecular biology procedures are described by Maniatis et al. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory, New York, (2001)). DNA sequencing can be performed as described in Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International plc sequencing handbook, and site directed mutagenesis can be carried out according to methods known in the art (Kramer et al., Nucleic Acids Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92 (1985); Kunkel et al., Methods in Enzymol.154:367-82 (1987); the Anglian Biotechnology Ltd. handbook). Additionally, numerous publications describe techniques suitable for the preparation of antibodies by manipulation of DNA, creation of expression vectors, and transformation and culture of appropriate cells (Mountain A and Adair, J R in Biotechnology and Genetic Engineering Reviews (ed. Tombs, M P, 10, Chapter 1, 1992, Intercept, Andover, UK); “Current Protocols in Molecular Biology”, 1999, F.M. Ausubel (ed.), Wiley Interscience, New York). [0264] Where it is desired to improve the affinity of antibodies according to the disclosure containing one or more of the above-mentioned CDRs can be obtained by a number of affinity maturation protocols including maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutation strains of E. coli. (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7-88, 1996) and sexual PCR (Crameri, et al., Nature, 391, 288-291, 1998). All of these methods of affinity maturation are discussed by Vaughan et al. (Nature Biotech., 16, 535-539, 1998). [0265] It will be understood by one skilled in the art that some proteins, such as antibodies, may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the protein as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperizine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, R.J. Journal of Chromatography 705:129-134, 1995). 5. Bioequivalents [0266] The antigen-binding domains for use in the ADCs provided herein encompass proteins having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind the target proteins. Such variant antigen-binding domains comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. [0267] Two antigen-binding domains are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple doses. Some antigen- binding domains will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied. [0268] In one embodiment, two antigen-binding domains are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. [0269] In one embodiment, two antigen-binding domains are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching. [0270] In one embodiment, two antigen-binding domains are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known. [0271] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antigen-binding domain or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) in a well- controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding domain. [0272] Bioequivalent variants of antigen-binding domains for use in the ADCs provided herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen- binding domains may include variants comprising amino acid changes which modify the glycosylation characteristics of the antigen-binding domain, e.g., mutations which eliminate or remove glycosylation. 6. Species Selectivity and Species Cross-Reactivity [0273] In certain embodiments, the antigen-binding domains for use in the ADCs provided herein bind to a human target protein but not to target protein from other species. In other embodiments, the antigen-binding domains for use in the ADCs provided herein bind to a human target protein and to a target protein from one or more non-human species. For example, the antigen-binding domains for use in the ADCs provided herein may bind to a human target protein and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzee target protein. In one embodiment, the antigen-binding domains specifically bind human target protein and cynomolgus monkey (e.g., Macaca fascicularis) target protein. In other embodiments, antigen-binding domains for use herein bind human target protein but do not bind, or bind only weakly, to cynomolgus monkey target protein. 7. Exemplary Antibodies and Antigen Targets [0274] The antigen binding domain (ABD) for use in the ADCs provided herein can have binding specificity for any antigen (target protein) deemed suitable to those of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., receptor) or a surface protein. ABD against a Hepatitis B virus (HBV) antigen [0275] Chronic Hepatitis B infection is typically associated with increase of circulating HBV DNA and HBV surface antigen (HBV sAg) in the serum. Sustained loss of circulating HBV sAg and HBV DNA are the hallmarks of a successful control of infection, or a “functional cure.” Current therapy with nucleoside analogs reduces HBV load in plasma but rarely is accompanied by HBV sAg loss. Thus, the nucleoside therapy needs to be given lifelong to prevent viral rebound. Some embodiments of the present disclosure relate to an ADC targeting an HBV antigen. The ADC can be used for treatment of Hepatitis B. In some embodiments, the ADC comprises an ABD specific to HBV sAg, where HBV sAg could relate to a non-infectious HBV sAg particle, infectious HBV virion, or HBV sAg expressing cells. [0276] In some embodiments, the ABD comprises the heavy chain and light chain of an antibody specific to HBV sAg. In some embodiments, the ABD comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of an antibody specific to HBV sAg . In some embodiments, the ABD is an antibody specific to HBV sAg . In some embodiments, the HBV antigen is HBV sAg available from Prospect Bio with Cat No. HBS-872. ABD against a tumor antigen [0277] Some embodiments of the present disclosure relate to an ADC targeting a tumor antigen. The ADC can be used for treatment of cancer. In some embodiments, the ADC comprises an ABD specific to a tumor antigen. [0278] In some embodiments, the antigen is expressed on a tumor. In some embodiments, the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed, or modified on a particular type of tumor. In one embodiment, the antigen is expressed on solid tumors. Exemplary antigens include, but are not limited to, lipoproteins; alpha1-antitrypsin; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CD152, or an antibody which binds to one or more tumor-associated antigens or cell-surface receptors disclosed in US Publication No. 2008/0171040 or US Publication No. 2008/0305044; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); T- cell receptors; surface membrane proteins; integrins, such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, BCMA, SLAMF7, GPNMB, UPK3A, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, endoglin, Epcam, EphA2, ErbB2/Her2, ErbB3/Her3, ErbB4/Her4, ETV6-AML, Fra-1, FOLR1, GAGE proteins, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/EBNA1, HLA/k-ras, HLA/MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins, MART-1, mesothelin, ML-IAP, Muc1, Muc16, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART- 3, Steap-1, Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP- 2, tyrosinase, and uroplakin-3, and fragments of any of the above-listed polypeptides; cell- surface expressed antigens; MUC16; c-MET; molecules such as class A scavenger receptors including scavenger receptor A (SR-A), and other membrane proteins such as B7 family- related member including V-set and Ig domain-containing 4 (VSIG4), Colony stimulating factor 1 receptor (CSF1R), asialoglycoprotein receptor (ASGPR), and Amyloid beta precursor-like protein 2 (APLP-2). In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antigen is HER2. In some embodiments, the antigen is human HER2. In some embodiments, the antigen is STEAP2. In some embodiments the antigen is human STEAP2. In some embodiments, the MAGE proteins are selected from MAGE-1, -2, -3, -4, - 6, and -12. In some embodiments, the GAGE proteins are selected from GAGE-1 and GAGE- 2. Antibody Scaffold [0279] In certain embodiments, the antibody comprises a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In the present disclosure, this position is referred to as glutamine 295, or as Gln295, or as Q295. Those of skill in the art will recognize that this is a conserved glutamine residue in the wild type sequence of many antibodies. In other embodiments, the antibody can be engineered to comprise a glutamine residue. In certain embodiments, the antibody comprises one or more N297Q mutations. Techniques for modifying an antibody sequence to include a glutamine residue are within the skill of those in the art (see, e.g., Ausubel et al. Current Protoc. Mol. Biol. (John Wiley & Sons)). [0280] In embodiments where the antibody contains a Q295 residue, an N297Q mutation, or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites, a payload of Formula I where R3 is -alkylene-Y, - alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene- Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y; where Y is -NR5R6; and R5 and R6 each H may be directly conjugated to the antibody to form an ADC of Formula V:
Figure imgf000100_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined elsewhere for Formula I; R10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or - alkylene-PEG-NH-; ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites; and k is an integer from one to thirty. [0281] In another embodiment, R10 is -alkylene-NH-, -heteroalkylene-NH- or -heteroalkylene-arylene-NH-. In another embodiment, R10 is -alkylene-NH-. In another embodiment, R10 is -heteroalkylene-NH-. In another embodiment, R10 is -heteroalkylene-arylene-NH-. In another embodiment, R10 is -CH2-NH-, -CH2-O- heteroalkylene-NH-, or -CH2-O-alkylene-NH-. In another embodiment, R10 is - CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4- NH-1-phenyl), -CH2OCH(NH-)CH2OH or -CH2NH-. In another embodiment, R10 is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4- NH-1-phenyl) or -CH2NH-. [0282] In another embodiment, the ADC provided herein has the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, or ABD- P34, wherein ABD is attached to the payload (i.e., TLR7 agonist) on the amino group of R3. [0283] In another embodiment, the ADCs provided herein for use in the compositions and methods provided herein are prepared from linker-TLR7 agonist of Formula III and have Formula VI:
Figure imgf000101_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2, R11, R12, R13, R14, R15, R16, L1, ABD and x are as defined elsewhere for Formula III; and k is an integer from one to thirty. [0284] In another embodiment, the ADC provided herein has the formula ABD-L1-P1, ABD- L1-P2, ABD-L1-P6, ABD-L1-P8, ABD-L1-P17, ABD-L1-P18, ABD-L1-P19, ABD-L1-P20, ABD-L1- P23, ABD-L1-P27, ABD-L1-P29, ABD-L1-P32, ABD-L1-P33, ABD-L1-P37, or ABD-L1-P39, where ABD-L1 is attached to the payload (i.e., TLR7 agonist). VII. Synthesis of the ADCs [0285] Also provided herein is a method of synthesizing the ADCs comprising an ABD, linker and TLR7 agonist. Each component of the ADCs (i.e., ABD, linker, and TLR7 agonist) can be individually synthesized and then linked or conjugated to generate the ADCs. [0286] In some embodiments, the method involves the step of partial reduction of an antigen-binding domain with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced cysteine residues with maleimide functionalized linker- payload (i.e., TLR7 agonist). In some embodiments, an antigen-binding domain is partially reduced via addition of 1.5 - 3.0- fold molar excess of TCEP in PBS pH 7.4 and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 h at 37 °C. The reduced antigen-binding domain may be buffer exchanged into PBS with 1% w/v polysorbate 20. Linker-payloads can be added at a linker-payload / antigen-binding domain molar ratio of 5 - 10 and reacted for an additional 2 h at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO). The mixture may be purified, e.g., via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase) to afford the ADCs provided herein. [0287] Alternatively, in the case of ADCs of Formula V, in some embodiments, the reaction of ABD and the payload (i.e., TLR7 agonist) is mediated by a transglutaminase enzyme. In another embodiment, the transglutaminase enzyme is a murine transglutaminase enzyme. In another embodiment, when the Gln of ABD is Q295 of an N297 antibody, then prior to reaction of ABD with the payload, ABD is reacted with a PNGase, such as without limitation PNGaseF, to deglycosylate N297. [0288] Chemical site-selective protein modification has become increasingly popular for antibody-based bio-conjugates. Amongst all bioorthogonal reactions developed to date, the [4+2] cycloaddition of 1,2,4,5-tetrazines (s-tetrazines, Tz) with various dienophiles, referred as inverse electron demand Diels–Alder (IEDDA) reaction, is the one that satisfies most of the bioorthogonal criteria (e.g., fast, selective, biocompatible and catalyst-free) necessary for the conjugations. In this work, the tetrazine-linkers were designed to have two functions: (1) tetrazine-linker as a handle that has an additional chemical moiety (such as an amine) to be attached with an antibody while the tetrazine-moiety can react with a linker-payload to generate an ADC; (2) tetrazine-linker as the linker of the linker-payload that can be attached with an antibody-handle (Titas Deb, et al., Chem. Rev. 2021, 121, 12, 6850–6914; Astrid- Caroline Knall and Christian Slugovc. Chem. Soc. Rev., 2013, 42, 5131). [0289] In one embodiment, the ADCs provided herein are selected from those in Table 3:
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
VIII. Pharmaceutical Compositions [0290] In one aspect, the present disclosure provides a pharmaceutical composition comprising an ADC described herein a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a target antigen (e.g., an HBV sAg). In some embodiments, the pharmaceutical composition comprises an immunocomplex (IC) of the ADC and the HBV sAg. [0291] The TLR7 agonists or ADCs can be formulated into suitable pharmaceutical preparations. Typically, the TLR7 agonists or ADCs described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999). [0292] In the compositions, effective concentrations of one or more TLR7 agonists or ADCs or pharmaceutically acceptable salts is (are) mixed with a suitable pharmaceutical carrier. In certain embodiments, the concentrations of the TLR7 agonists or ADCs in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder disclosed herein. [0293] Typically, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a TLR7 agonists or ADC is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved or ameliorated. Pharmaceutical carriers suitable for administration of the TLR7 agonists or ADCs provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. [0294] In some embodiments, the TLR7 agonist or ADC is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems described herein and well known to those of skill in the art, and then extrapolated therefrom for dosages for humans. In some embodiments, the ADC is administered in a method to achieve a therapeutically effective concentration of the payload. In some embodiments, a companion diagnostic (see, e.g., Olsen D and Jorgensen J T, Front. Oncol., 2014 May 16, 4:105, doi: 10.3389/fonC.2014.00105) is used to determine the therapeutic concentration and safety profile of the TLR7 agonist or ADC in specific subjects or subject populations. [0295] The concentration of TLR7 agonist or ADC in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation and excretion rates of the TLR7 agonist or ADC, the physicochemical characteristics of the TLR7 agonist or ADC, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to ameliorate one or more of the symptoms of a disease or disorder disclosed herein. [0296] The compositions may be administered at once or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. [0297] The compositions may include other active compounds to obtain desired combinations of properties. The TLR7 agonists or ADCs provided herein, or pharmaceutically acceptable salts thereof as described herein, may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the general art to be of value in treating one or more of the diseases or medical conditions referred to herein. It is to be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein. [0298] The pharmaceutical composition can be in any form appropriate for human or veterinary medicine, including a liquid, an oil, an emulsion, a gel, a colloid, an aerosol or a solid. [0299] The pharmaceutical composition can be formulated for administration by any route of administration appropriate for human or veterinary medicine, including enteral and parenteral routes of administration. [0300] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration. IX. Dosing [0301] The compounds and pharmaceutical compositions provided herein may be dosed in certain therapeutically or prophylactically effective amounts, certain time intervals, certain dosage forms, and certain dosage administration methods as described below. [0302] The methods provided herein encompass treating a patient regardless of subject's age, although some diseases or disorders are more common in certain age groups. [0303] The TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, can be administered repeatedly if necessary, for example, until the subject experiences stable disease or regression, or until the subject experiences disease progression or unacceptable toxicity. [0304] The TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day), intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term "daily" is intended to mean that a therapeutic compound, such as the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administered once or more than once each day, for example, for a period of time. The term "continuous" is intended to mean that a therapeutic compound, such as the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administered daily for an uninterrupted period of at least 10 days to 52 weeks. The term "intermittent" or "intermittently" as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. The term "cycling" as used herein is intended to mean that a therapeutic compound, such as the TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is administered daily or continuously but with a rest period. In some such embodiments, administration is once a day for two to six days, then a rest period with no administration for five to seven days. X. Methods of Treatment [0305] In some embodiments, a method of treating a subject with a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is provided. In some embodiments, a method of treating a subject with a pharmaceutical composition comprising a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, is provided. The pharmaceutical composition comprises any of the TLR7 agonists or ADCs disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [0306] The TLR7 agonists or ADCs provided herein are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by expression, signaling or activity of the target protein of the antigen-binding domain. [0307] In certain embodiments, the ADCs provided herein are used to treat primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the TLR7 agonists or ADCs provided herein are used to treat one or more of the following cancers: acute myelogenous leukemia, adult T-cell leukemia, astrocytomas, bladder cancer, breast cancer, PRLR positive (PRLR+) breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastomata, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), Kaposi's sarcoma, kidney cancer, leiomyosarcomas, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC)), lymphomas, malignant gliomas, malignant mesothelioma, melanoma, mesothelioma, malignant mesothelioma, MFH/fibrosarcoma, multiple myeloma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic carcinoma, prostate cancer, castrate-resistant prostate cancer, renal cell carcinoma, residual cancer wherein “residual cancer” means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy, rhabdomyosarcoma, stomach cancer, synovial sarcoma, thyroid cancer, uterine cancer and Wilms' tumor. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. [0308] In some embodiments, the subject has chronic hepatitis B. In some embodiments, the ADCs provided herein are used to treat chronic hepatitis B. [0309] In some embodiments, the subject is diagnosed to have chronic hepatitis B. In some embodiments, the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or the pharmaceutical composition. In some embodiments, the treatment method provided herein further comprises the step of measuring circulating HBV DNA or HBV sAg in serum of the subject before administration of the ADC or the pharmaceutical composition. In some embodiments, the treatment method provided herein further comprises the step of measuring circulating HBV DNA or HBV sAg in serum of the subject to assess therapeutic efficacy of the ADC or the pharmaceutical composition after administration. [0310] In the context of the methods of treatment provided herein, the TLR7 agonists or ADCs may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein). VII. COMBINATION THERAPY WITH A SECOND ACTIVE AGENT [0311] Provided herein are compositions comprising any of the TLR7 agonists or ADCs provided herein in combination with one or more additional therapeutically active components, and methods of treatment comprising administering such combinations to a subject. [0312] The TLR7 agonists or ADCs provided herein may be co-formulated with and/or administered in combination with one or more additional therapeutically active component(s) selected from a MET antagonist (e.g., an anti-MET antibody (e.g., onartuzumab, emibetuzumab, and H4H14639D) or small molecule inhibitor of MET), an EGFR antagonist (e.g., an anti-EGFR antibody (e.g., cetuximab or panitumumab) or small molecule inhibitor of EGFR (e.g., gefitinib or erlotinib)), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 (e.g., trastuzumab or T-DM1 {KADCYLA®}), anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII (e.g., an anti-EGFRvIII antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX- 4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody or small molecule kinase inhibitor such as, e.g., imatinib mesylate or sunitinib malate), a PDGF ligand inhibitor (e.g., anti-PDGF-A, -B, -C, or -D antibody, aptamer, siRNA, etc.), a VEGF antagonist (e.g., a VEGF-Trap such as aflibercept, see, e.g., US 7,087,411 (also referred to herein as a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 2011/0027286 such as H1H685P), a FOLH1 antagonist (e.g., an anti-FOLH1 antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin (e.g., anti-UPK3A) antibody), a MUC16 antagonist (e.g., an anti-MUC16 antibody), a Tn antigen antagonist (e.g., an anti-Tn antibody), a CLEC12A antagonist (e.g., an anti- CLEC12A antibody), a TNFRSF17 antagonist (e.g., an anti- TNFRSF17 antibody), a LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), a CD20 x CD3 bispecific antibody, a PD-1 blocking agent (e.g., an anti-PD-1 antibody such as pembrolizumab or nivolumab), etc. Other agents that may be beneficially administered in combination with antibodies provided herein include, e.g., tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors. [0313] Illustratively, a PD-1 inhibitor such as an anti-PD-1 antibody can be combined with a TLR7 agonist or ADC as described herein. [0314] In some embodiments, provided herein are pharmaceutical compositions comprising any of the TLR7 agonists or ADCs provided herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxotere™; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. [0315] The TLR7 agonists or ADCs provided herein may also be administered and/or co- formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and/or NSAIDs. [0316] The additional therapeutically active component(s), e.g., any of the agents listed above or derivatives thereof, may be administered just prior to, concurrent with, or shortly after the administration of a TLR7 agonist or ADC provided herein. In some embodiments, provided are pharmaceutical compositions in which a TLR7 agonist or ADC provided herein is co- formulated with one or more of the additional therapeutically active component(s) as described herein. [0317] As used herein, the term "in combination" includes the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). However, the use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject with a disease or disorder. A first therapy (e.g., an ADC provided herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent) to the subject. Triple therapy is also contemplated herein. [0318] Administration of the compound provided herein, or a derivative thereof and one or more second active agents to a subject can occur simultaneously or sequentially by the same or different routes of administration. The suitability of a particular route of administration employed for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without decomposing prior to entering the blood stream) and the disease or disorder being treated. XI. Additional Embodiments and Clauses [0319] The disclosure is further described by the following non-limiting embodiments. Embodiment 1. A compound of Formula I:
Figure imgf000111_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R3 is -CO2R23, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; with the proviso that the compound is not a compound of the formula:
Figure imgf000112_0001
Embodiment 2. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein: R1 is halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is halo, or alkoxy; R3 is -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R23 is H, alkyl or aryl; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; X is CH or N; Y is -OH, Gly, -NR5R6 or -COZ; Z is -OH or -NR7R8; R5 and R6 are selected from (i), (ii), and (iii): (i) R5 and R6 each H; (ii) R5 is H and R6 is alkyl; (iii) R5 and R6, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8, together with the N to which they are attached, form a heterocyclic ring. Embodiment 3. The compound of embodiment 1 or 2 selected with the proviso that R4 is not substituted with hydroxyl. Embodiment 4. The compound of any one of embodiments 1-3 selected with the proviso that the alkylene and heteroalkylene portions of R3 are not substituted with oxo. Embodiment 5. The compound of any of embodiments 1-4 selected with the proviso that the compound is not 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine or (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl)methyl)-3-methoxyphenyl)methanol. Embodiment 6. The compound of any one of embodiments 1-5, wherein R1 is halo, -NHR4, - OR4, -NH-OR4 or -R4, and is a straight chain of 6 atoms in length. Embodiment 7. The compound of any one of embodiments 1-6, wherein R1 is halo. Embodiment 8. The compound of any one of embodiments 1-6, wherein R1 is -NHR4. Embodiment 9. The compound of any one of embodiments 1-6, wherein R1 is -OR4. Embodiment 10. The compound of any one of embodiments 1-6, wherein R1 is -NH-OR4. Embodiment 11. The compound of any one of embodiments 1-6, wherein R1 is -R4. Embodiment 12. The compound of any one of embodiments 1-5, wherein R1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt. Embodiment 13. The compound of any one of embodiments 1-5, wherein R1 is -NH-n-pentyl. Embodiment 14. The compound of any one of embodiments 1-5, wherein R1 is -NH-O-n- butyl. Embodiment 15. The compound of any one of embodiments 1-5, wherein R1 is -O-n-pentyl. Embodiment 16. The compound of any one of embodiments 1-5, wherein R1 is -n-hexyl. Embodiment 17. The compound of any one of embodiments 1-5, wherein R1 is -NH-CH2CH2- OEt. Embodiment 18. The compound of any one of embodiments 1-17, wherein R2 is alkoxy. Embodiment 19. The compound of any one of embodiments 1-17, wherein R2 is methoxy. Embodiment 20. The compound of any one of embodiments 1-17, wherein R2 is H. Embodiment 21. The compound of any one of embodiments 1-17, wherein R2 is halo. Embodiment 22. The compound of any one of embodiments 1-21, wherein R3 is -CONHR23, -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, - (amino)heteroalkylene-Y, or -alkylene-PEG-Y. Embodiment 23. The compound of any one of embodiments 1-22, wherein R3 is CONHR23. Embodiment 24. The compound of any one of embodiments 1-22, wherein R3 is -alkylene-Y. Embodiment 25. The compound of any one of embodiments 1-22, wherein R3 is - heteroalkylene-Y. Embodiment 26. The compound of any one of embodiments 1-22, wherein R3 is -heteroalkylene-arylene-Y. Embodiment 27. The compound of any one of embodiments 1-22, wherein R3 is -(hydroxy)heteroalkylene-Y. Embodiment 28. The compound of any one of embodiments 1-22, wherein R3 is -(amino)heteroalkylene-Y. Embodiment 29. The compound of any one of embodiments 1-22, wherein R3 is -alkylene-PEG-Y. Embodiment 30. The compound of any one of embodiments 1-22, wherein R3 is -CONH2, - CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. Embodiment 31. The compound of any one of embodiments 1-22, wherein R3 is -CH2-Y, - CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. Embodiment 32. The compound of any one of embodiments 1-22, wherein R3 is -C(Me)2OH, -CO2H -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH -CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, -CH2OCH2CO-1-piperazinyl, -(R)- CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2, or -CH2-1-piperazinyl. Embodiment 33. The compound of any one of embodiments 1-22, wherein R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, - CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH - CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, - CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl. Embodiment 34. The compound of any one of embodiments 1-33, wherein R4 is n-butyl, n- pentyl, n-hexyl or ethoxyethyl. Embodiment 35. The compound of any one of embodiments 1-33, wherein R4 is n-butyl. Embodiment 36. The compound of any one of embodiments 1-33, wherein R4 is n-pentyl. Embodiment 37. The compound of any one of embodiments 1-33, wherein R4 is n-hexyl. Embodiment 38. The compound of any one of embodiments 1-33, wherein R4 is ethoxyethyl. Embodiment 39. The compound of any one of embodiments 1-38, wherein R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring. Embodiment 40. The compound of any one of embodiments 1-38, wherein R5 and R6 are each H. Embodiment 41. The compound of any one of embodiments 1-38, wherein R5 is H and R6 is alkyl. Embodiment 42. The compound of any one of embodiments 1-38, wherein R5 and R6, together with the N to which they are attached, form 1-piperazinyl. Embodiment 43. The compound of any one of embodiments 1-42, wherein Y is OH. Embodiment 44. The compound of any one of embodiments 1-42, wherein Y is Gly. Embodiment 45. The compound of any one of embodiments 1-42, wherein Y is -NR5R6. Embodiment 46. The compound of any one of embodiments 1-42, wherein Y is -COZ. Embodiment 47. The compound of any one of embodiments 1-42, wherein Y is -OH, Gly, - NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2 or -CO-1-piperazinyl. Embodiment 48. The compound of any one of embodiments 1-47, wherein Z is -OH. Embodiment 49. The compound of any one of embodiments 1-47, wherein Z is alkoxy. Embodiment 50. The compound of any one of embodiments 1-47, wherein Z is -NR7R8. Embodiment 51. The compound of any one of embodiments 1-47, wherein Z is -OH, ethoxy, -N-n-Pr2 or 1-piperazinyl. Embodiment 52. The compound of any one of embodiments 1-47, wherein Z is -OH or 1- piperazinyl. Embodiment 53. The compound of any one of embodiments 1-52, wherein R7 and R8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1- piperazinyl. Embodiment 54. The compound of any one of embodiments 1-52, wherein R7 and R8, together with the N to which they are attached, form 1-piperazinyl. Embodiment 55. A compound selected from:
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0003
and pharmaceutically acceptable salts of any of these compounds. Embodiment 56. A compound of Formula II:
Figure imgf000121_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined for Formula I in embodiment 1; R9 is a divalent group formed by removal of a terminal hydrogen (i.e., a hydrogen distal from the phenyl group to which R9 is attached) from an R3 group, as defined in embodiment 1; and L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; with the proviso that the compound is not a compound of the formula:
Figure imgf000121_0002
Embodiment 57. The compound of embodiment 56, wherein R9 is - alkylene-Y1-, - heteroalkylene-Y1-, -heteroalkylene-arylene-Y1-, -(hydroxy)heteroalkylene-Y1, - (amino)heteroalkylene-Y1, or -alkylene-PEG-Y1. Embodiment 58. The compound of embodiment 56 or 57, wherein R9 is -alkylene-Y1-. Embodiment 59. The compound of embodiment 56 or 57, wherein R9 is -heteroalkylene-Y1-. Embodiment 60. The compound of embodiment 56 or 57, wherein R9 is -heteroalkylene- arylene-Y1-. Embodiment 61. The compound of embodiment 56 or 57, wherein R9 is - (hydroxy)heteroalkylene-Y1. Embodiment 62. The compound of embodiment 56 or 57, wherein R9 is - (amino)heteroalkylene-Y1. Embodiment 63. The compound of embodiment 56 or 57, wherein R9 is -alkylene-PEG-Y1. Embodiment 64. The compound of embodiment 56 or 57, wherein R9 is -CH2-Y1-, -CH2-O-heteroalkylene-Y1-, or -CH2-O-alkylene-Y1-. Embodiment 65. The compound of embodiment 56 or 57, wherein R9 is -C(Me)2O-, C(O)-, - CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, - CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CO-, - CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, - CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH-, or -CH2-1-piperazin-4-yl. Embodiment 66. The compound of any one of embodiments 55-65, wherein Y1 is -O-. Embodiment 67. The compound of any one of embodiments 55-65, wherein Y1 is Gly. Embodiment 68. The compound of any one of embodiments 55-65, wherein Y1 is -NR5-. Embodiment 69. The compound of any one of embodiments 55-65, wherein Y1 is -COZ1. Embodiment 70. The compound of any one of embodiments 55-65, wherein Y1 is -O-, Glycine, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl. Embodiment 71. The compound of embodiment 69, wherein Z1 is -O-. Embodiment 72. The compound of embodiment 69, wherein Z1 is -NR7-. Embodiment 73. The compound of embodiment 69, wherein Z1 is -O- or 1-piperazin-4-yl. Embodiment 74. The compound of embodiment 68, wherein R5 is H. Embodiment 75. The compound of embodiment 68, wherein R5 is alkyl. Embodiment 76. The compound of any one of embodiments 55-75, wherein L is non- cleavable under physiological conditions. Embodiment 77. The compound of any one of embodiments 55-75, wherein L is cleavable under physiological conditions. Embodiment 78. The compound of embodiment 77, wherein L is an acid-labile linker, a hydrolysis-labile linker, an enzymatically cleavable linker, a reduction labile linkers or a self- immolative linker. Embodiment 79. The compound of any one of embodiments 55-78, wherein L is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit. Embodiment 80. The compound of any one of embodiments 55-79, wherein L comprises one or more amino acids. Embodiment 81. The compound of any one of embodiments 55-80, wherein L comprises a self-immolative group. Embodiment 82. The compound of any one of embodiments 55-81, wherein L comprises p- aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC). Embodiment 83. The compound of any one of embodiments 55-82, wherein L comprises a maleimido, an N-hydroxysuccinimido ester or cyclooctynyl group. Embodiment 84. The compound of any one of embodiments 55-83, wherein L is a group selected from 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl,
Figure imgf000123_0001
, OH ,
Figure imgf000124_0001
Figure imgf000125_0001
. Embodiment 85. A compound selected from:
Figure imgf000125_0002
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
and pharmaceutically acceptable salts of any of these compounds. Embodiment 86. A compound of Formula III:
Figure imgf000129_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined for Formula I in embodiment 1; L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form the 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene, heteroalkylene-arylene, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. Embodiment 87. The compound of embodiment 86, wherein R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6- membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form a 4-, 5-, or 6-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene, heteroalkylene-arylene, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. Embodiment 88. The compound of embodiment 86 or embodiment 87, wherein the TLR7 agonist used in preparing the compound is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, P39, P41, P42, or P43. Embodiment 89. An antibody-drug-conjugate (ADC), comprising the compound of any one of embodiments 1-88 or compounds of the formulae:
Figure imgf000130_0001
Embodiment 90. The ADC of embodiment 89 having Formula IV:
Figure imgf000130_0002
or a pharmaceutically acceptable salt thereof, wherein: R1, R2, R9 and X are as defined for Formula I in embodiment 1 and Formula II in embodiment 56; L1 is a divalent linker; ABD is an antigen-binding domain; and k is an integer from one to thirty. Embodiment 91. The ADC of embodiment 89 or 90 that is ABD-LP1, ABD-LP6A, ABD-LP7A, ABD-LP8A, ABD-LP10A or ABD-LP11A. Embodiment 92. The ADC of embodiment 89 having Formula V:
Figure imgf000131_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined for Formula I in embodiment 1; R10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG- NH-; and ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites; and k is an integer from one to thirty. Embodiment 93. The ADC of embodiment 92, wherein R10 is -alkylene-NH-, -heteroalkylene- NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene- NH-, or -alkylene-PEG-NH-. Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein R10 is -alkylene- NH-. Embodiment 95. The ADC of embodiment 92 or embodiment 93, wherein R10 is - heteroalkylene-NH-. Embodiment 96. The ADC of embodiment 92 or embodiment 93, wherein R10 is - heteroalkylene-arylene-NH-. Embodiment 97. The ADC of embodiment 92 or embodiment 93, wherein R10 is -- (hydroxy)heteroalkylene-NH-. Embodiment 98. The ADC of embodiment 92 or embodiment 93, wherein R10 is - (amino)heteroalkylene-NH-. Embodiment 99. The ADC of embodiment 92 or embodiment 93, wherein R10 is -alkylene- PEG-NH-. Embodiment 100. The ADC of embodiment 92 or embodiment 93, wherein R10 is -CH2-NH-, -CH2-O-heteroalkylene-NH-, or -CH2-O-alkylene-NH-. Embodiment 101. The ADC of embodiment 92 or embodiment 93, wherein R10 is - CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4- NH-1-phenyl), -CH2OCH(NH-)CH2OH or -CH2NH-. Embodiment 102. The ADC of embodiment 92 or embodiment 93, wherein R10 is - CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4- NH-1-phenyl) or -CH2NH-. Embodiment 103. The ADC of any one of embodiments 89-102 having the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, ABD-P34 or ABD-P41, wherein ABD is attached to the payload (i.e., TLR7 agonist) on the amino group of R3. Embodiment 104. The ADC of embodiment 89 having Formula VI:
Figure imgf000132_0001
or a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1, R2, R16, R11, R12, R13, R14, R15, X, and x are as defined for Formula III in embodiment 86; and k is an integer from one to thirty. Embodiment 105. The ADC of embodiment 104 having the formula ABD-L1-P1, ABD-L1-P2, ABD-L1-P6, ABD-L1-P8, ABD-L1-P17, ABD-L1-P18, ABD-L1-P19, ABD-L1-P20, ABD-L1-P23, ABD-L1-P27, ABD-L1-P29, ABD-L1-P32, ABD-L1-P33, ABD-L1-P37, ABD-L1-P39, or ABD-L1- P42, where ABD-L1 is attached to the payload (i.e., TLR7 agonist) on the alcohol group of R3. Embodiment 106. The ADC of any one of embodiments 89-105, wherein ABD has binding specificity for a transmembrane molecule (e.g., receptor) expressed on a tumor. Embodiment 107. A pharmaceutical composition, comprising a compound of any one of embodiments 1-88 or an ADC of any one of embodiments 89-106, and a pharmaceutically acceptable carrier. Embodiment 108. A method of treating or diagnosing disease, comprising administering to a subject a compound of any one of embodiments 1-88 or an ADC of any one of embodiments 89-106 or a pharmaceutical composition of embodiment 107. Embodiment 109. The method of embodiment 108, wherein the method treats a disease. Embodiment 110. The method of embodiment 108 or 109, wherein the disease is cancer. [0320] The disclosure is further described by the following non-limiting clauses. Clause 1. An antibody-drug-conjugate (ADC), comprising a. an antigen-binding domain (ABD) having binding specificity to a hepatitis B virus surface antigen (HBV sAg); and b. a Toll-like receptor 7 (TLR7) agonist. Clause 2. The ADC of clause 1, further comprising a divalent linker that links the ABD to the TLR7 agonist. Clause 3. The ADC of clause 2, wherein the ADC is according to Formula IV:
Figure imgf000133_0001
or a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R9 is a divalent group formed by removal of a hydrogen from R3, R3 being a group attached to the phenyl group at the position of R9; R3 is -COOH, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, - heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and k is an integer from one to thirty. Clause 4. The ADC of clause 3, selected with the proviso that the ADC does not comprise 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4- diamine or (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3- methoxyphenyl)methanol. Clause 5. The ADC of any one of clauses 1-3, wherein R1 is halo, -NHR4, -OR4, -NH-OR4 or -R4, and has a straight chain of 6 atoms in length. Clause 6. The ADC of clause 5, wherein R1 is halo. Clause 7. The ADC of clause 5, wherein R1 is -NHR4. Clause 8. The ADC of clause 5, wherein R1 is -OR4. Clause 9. The ADC of clause 5, wherein R1 is -NH-OR4. Clause 10. The ADC of clause 5, wherein R1 is -R4. Clause 11. The ADC of clause 5, wherein R1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt. Clause 12. The ADC of clause 5, wherein R1 is -NH-n-pentyl. Clause 13. The ADC of clause 5, wherein R1 is -NH-O-n-butyl. Clause 14. The ADC of clause 5, wherein R1 is -O-n-pentyl. Clause 15. The ADC of clause 5, wherein R1 is -n-hexyl. Clause 16. The ADC of clause 5, wherein R1 is -NH-CH2CH2-OEt. Clause 17. The ADC of any one of clauses 3-16, wherein R2 is alkoxy. Clause 18. The ADC of any one of clauses 3-16, wherein R2 is methoxy. Clause 19. The ADC of any one of clauses 3-16, wherein R2 is H. Clause 20. The ADC of any one of clauses 3-16, wherein R2 is halo. Clause 21. The ADC of any one of clauses 3-20, wherein R3 is -CONHR23, -alkylene-Y, - heteroalkylene-Y or -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, - (amino)heteroalkylene-Y, or -alkylene-PEG-Y. Clause 22. The ADC of clause 21, wherein R3 is CONHR23. Clause 23. The ADC of clause 21, wherein R3 is -alkylene-Y. Clause 24. The ADC of clause 21, wherein R3 is -heteroalkylene-Y. Clause 25. The ADC of clause 21, wherein R3 is -heteroalkylene-arylene-Y. Clause 26. The ADC of clause 21, wherein R3 is -(hydroxy)heteroalkylene-Y. Clause 27. The ADC of clause 21, wherein R3 is -(amino)heteroalkylene-Y. Clause 28. The ADC of clause 21, wherein R3 is -alkylene-PEG-Y. Clause 29. The ADC of any one of clauses 3-20, wherein R3 is -CONH2, -CH2-Y, -CH2-O- heteroalkylene-Y, or -CH2-O-alkylene-Y. Clause 30. The ADC of any one of clauses 3-20, wherein R3 is -CH2-Y, -CH2-O- heteroalkylene-Y, or -CH2-O-alkylene-Y. Clause 31. The ADC of any one of clauses 3-20, wherein R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH,-CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, - CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2,-CONH2 or -CH2-1-piperazinyl. Clause 32. The ADC of any one of clauses 3-20, wherein R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, - CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH - CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, - CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl. Clause 33. The ADC of any one of clauses 3-32, wherein R4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl. Clause 34. The ADC of clause 33, wherein R4 is n-butyl. Clause 35. The ADC of clause 33, wherein R4 is n-pentyl. Clause 36. The ADC of clause 33, wherein R4 is n-hexyl. Clause 37. The ADC of clause 33, wherein R4 is ethoxyethyl. Clause 38. The ADC of any one of clauses 3-37, wherein R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring. Clause 39. The ADC of clause 38, wherein R5 and R6 are each H. Clause 40. The ADC of clause 38, wherein R5 is H and R6 is alkyl. Clause 41. The ADC clause 38, wherein R5 and R6, together with the N to which they are attached, form 1-piperazinyl. Clause 42. The ADC of any one of clauses 3-41, wherein Y is OH. Clause 43. The ADC of any one of clauses 3-41, wherein Y is Glycine. Clause 44. The ADC of any one of clauses 3-41, wherein Y is -NR5R6. Clause 45. The ADC of any one of clauses 3-41, wherein Y is -COZ. Clause 46. The ADC of any one of clauses 3-41, wherein Y is -OH, Glycine, -NH2, 1- piperazinyl, -COOH, -COOEt, -CONPr2 or -CO-1-piperazinyl. Clause 47. The ADC of any one of clauses 3-41, wherein Y is -OH, -NH2, 1-piperazinyl, - COOH or -CO-1-piperazinyl. Clause 48. The ADC of clause 45, wherein Z is -OH. Clause 49. The ADC of clause 45, wherein Z is alkoxy. Clause 50. The ADC of clause 45, wherein Z is -NR7R8. Clause 51. The ADC of clause 45, wherein Z is -OH, ethoxy, -N-n-Pr2 or 1-piperazinyl. Clause 52. The ADC of clause 45, wherein Z is -OH or 1-piperazinyl. Clause 53. The ADC of any one of clauses 3-52, wherein R7 and R8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1- piperazinyl. Clause 54. The ADC of any one of clauses 3-53, wherein R7 and R8, together with the N to which they are attached, form 1-piperazinyl. Clause 55. The ADC of clause 3, wherein ABD-L1 is linked to a compound selected from P1-P43 and pharmaceutical salts thereof by removal of a hydrogen from the group at the position corresponding to R3 of the compound, wherein the structures of P1-P43 are as follows:
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Clause 56. The ADC of any one of clauses 3-54, wherein R9 is -alkylene-Y1-, - heteroalkylene-Y1-, -heteroalkylene-arylene-Y1-, -(hydroxy)heteroalkylene-Y1, - (amino)heteroalkylene-Y1, or -alkylene-PEG-Y1-. Clause 57. The ADC of clause 56, wherein R9 is -alkylene-Y1-. Clause 58. The ADC of clause 56, wherein R9 is -heteroalkylene-Y1-. Clause 59. The ADC of clause 56, wherein R9 is -heteroalkylene-arylene-Y1-. Clause 60. The ADC of clause 56, wherein R9 is -(hydroxy)heteroalkylene-Y1. Clause 61. The ADC of clause 56, wherein R9 is -(amino)heteroalkylene-Y1. Clause 62. The ADC of clause 56, wherein R9 is -alkylene-PEG-Y1-. Clause 63. The ADC of clause 56, wherein R9 is -CH2-Y1-, -CH2-O-heteroalkylene-Y1-, or - CH2-O-alkylene-Y1-. Clause 64. The ADC of clause 56, wherein R9 is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, - CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CO-, - CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, - CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH-, or -CH2-1-piperazin-4-yl. Clause 65. The ADC of any one of clauses 56-64, wherein Y1 is -O-. Clause 66. The ADC of any one of clauses 56-64, wherein Y1 is Gly. Clause 67. The ADC of any one of clauses 56-64, wherein Y1 is -NR5-. Clause 68. The ADC of any one of clauses 56-64, wherein Y1 is -COZ1, wherein Z1 is -O-, -NR7-, -O-alkylene-, or 1-piperazin-4-yl. Clause 69. The ADC of any one of clauses 56-64, wherein Y1 is -O-, -NH-, 1-piperazin-4- yl, -COO- or -CO-1-piperazin-4-yl. Clause 70. The ADC of clause 68, wherein Z1 is -O-. Clause 71. The ADC of clause 68, wherein Z1 is -NR7-. Clause 72. The ADC of clause 71, wherein R7 is H. Clause 73. The ADC of clause 71, wherein R7 is alkyl. Clause 74. The ADC of clause 68, wherein Z1 is 1-piperazin-4-yl. Clause 75. The ADC of clause 69, wherein Y1 is 1-piperazin-4-yl. Clause 76. The ADC of clause 69, wherein Y1 is -CO-1-piperazin-4-yl. Clause 77. The ADC of any one of clauses 3-76, wherein L1 is non-cleavable under physiological conditions. Clause 78. The ADC of any one of clauses 3-76, wherein L1 is cleavable under physiological conditions. Clause 79. The ADC of clause 78, wherein L1 is an acid-labile linker, a hydrolysis-labile linker, an enzymatically cleavable linker, a reduction labile linkers or a self-immolative linker. Clause 80. The ADC of any one of clauses 3-79, wherein L1 is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit. Clause 81. The ADC of any one of clauses 3-80, wherein L1 comprises one or more amino acids. Clause 82. The ADC of clause 79, wherein L1 comprises a self-immolative group. Clause 83. The ADC of clause 80, wherein L1 comprises p-aminobenzyl (PAB) or p- aminobenzyloxycarbonyl (PABC). Clause 84. The ADC of any one of clauses 3-76, wherein L1 comprises a maleimido, an N- hydroxysuccinimido ester or cyclooctynyl group. Clause 85. The ADC of any one of clauses 3-76, wherein L1 is a group derived from 2- maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, ,
Figure imgf000143_0001
, O O O O N H N N H N O O H O O N N O N NH O , O NH2 , ,
Figure imgf000144_0001
Figure imgf000145_0001
. Clause 86. The ADC of clause 3, comprising ABD linked to a compound selected from LP1, LP6-LP12, and a pharmaceutically acceptable salt of these compounds, wherein the structures of LP1 and LP6-LP12 are as follows:
Figure imgf000145_0002
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Clause 87. The ADC of clause 3, comprising ABD linked to a compound of Formula III:
Figure imgf000149_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined for Formula I in clause 3; L is any group or moiety that links to ABD; R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form the 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene, heteroalkylene-arylene-, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. Clause 88. The ADC of clause 87, wherein R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form a 4-, 5-, or 6-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene, heteroalkylene-arylene, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six. Clause 89. The ADC of clause 3, wherein ABD-L1 is linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39. Clause 90. The ADC of clause 89, wherein the ABD is linked to a compound selected from LP1, LP6, LP7, LP8, LP10, and LP11. Clause 91. The ADC of clause 3, wherein the ADC is according to Formula V:
Figure imgf000150_0001
or a pharmaceutically acceptable salt thereof, wherein: R1, R2, and X are as defined for Formula In in clause 3; R10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or - alkylene-PEG-NH-; ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites; and k is an integer from one to thirty. Clause 92. The ADC of clause 91, wherein R10 is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or - alkylene-PEG-NH-. Clause 93. The ADC of clause 92, wherein R10 is -alkylene-NH-. Clause 94. The ADC of clause 92, wherein R10 is -heteroalkylene-NH-. Clause 95. The ADC of clause 92, wherein R10 is -heteroalkylene-arylene-NH-. Clause 96. The ADC of clause 92, wherein R10 is -(hydroxy)heteroalkylene-NH-. Clause 97. The ADC of clause 92, wherein R10 is -(amino)heteroalkylene-NH-. Clause 98. The ADC of clause 92, wherein R10 is -alkylene-PEG-NH-. Clause 99. The ADC of clause 91 or 92, wherein R10 is -CH2-NH-, -CH2-O-heteroalkylene- NH-, or -CH2-O-alkylene-NH-. Clause 100. The ADC of clause 91, wherein R10 is -CH2OCH2CH2NH-, - CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, - CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl)-, -CH2OCH(NH-)CH2OH or -CH2NH-. Clause 101. The ADC of clause 91, wherein R10 is -CH2OCH2CH2NH-, - CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, - CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl)- or -CH2NH-. Clause 102. The ADC of clause 3, comprising ABD linked to a compound selected from P4, P5, P7, P9, P11, P12, P19, P21, P24, P30, and P34 via an amino group of R3. Clause 103. The ADC of clause 2, wherein the ADC is according to Formula VI:
Figure imgf000152_0001
or a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form the 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene, heteroalkylene-arylene-, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; X is CH or N; x is zero, one, two, three, four, five, or six; and k is an integer from one to thirty. Clause 104. The ADC of clause 103, comprising ABD-L1 linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39. Clause 105. The ADC of clause 103 or 104, wherein k is 1, 2, 3, 4, or 5. Clause 106. The ADC of clause 103, wherein k is 2. Clause 107. The ADC of clause 103, wherein ABD comprises a heavy chain and C-terminus of the heavy chain is conjugated to L1. Clause 108. The ADC of clause 103, wherein ABD comprises two heavy chains and C- terminus of each of the two heavy chains is conjugated to L1. Clause 109. The ADC of any one of clauses 3-108, wherein L1 is linked to a cysteine residue of the ABD. Clause 110. The ADC of any one of clauses 1-109, wherein the ABD is an antibody against an HBV sAg or a fragment thereof. Clause 111. The ADC of any one of clauses 1-110, wherein the ABD is a human antibody or a humanized antibody. Clause 112. The ADC of any one of clauses 1-111, wherein the ABD is IgG1 or IgG2a. Clause 113. The ADC of any one of clauses 1-110, wherein the ABD comprises a scFv having binding specificity to an HBV sAg. Clause 114. The ADC of any one of clauses 1-113, wherein the ABD comprises VH chain and VL chain of an antibody against an HBV sAg. Clause 115. The ADC of any one of clauses 1-114, wherein the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of an antibody against an HBV sAg. Clause 116. The ADC of any one of clauses 1-102, wherein the ABD comprises an Fc region. Clause 117. The ADC of clause 116, wherein the Fc region comprises a modification for enhanced binding to FcγR. Clause 118. The ADC of any one of clauses 1-114, wherein said ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 25, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 29. Clause 119. The ADC of clause 118, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 30, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32. Clause 120. The ADC of clause 118 or 119, wherein said HCVR comprises the amino acid sequence of SEQ ID NO: 25. Clause 121. The ADC of claim 120, wherein said HCVR is a component of a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. Clause 122. The ADC of clause 118 or 119, wherein said LCVR comprises the amino acid sequence of SEQ ID NO: 29. Clause 123. The ADC of claim 120, wherein said LCVR is a component of a light chain comprising the amino acid sequence of SEQ ID NO: 34. Clause 124. The ADC of any one of clauses 118-123, wherein said ABD is a component of an antibody or antigen-binding fragment thereof. Clause 125. The ADC of clause 1, wherein the TLR7 agonist with a divalent linker is any one of LP1-5, LP6A-6B, LP7A-7E, LP8A-8B, LP9, LP10A-10B, LP11A-11D, and LP12-15 Clause 126. A pharmaceutical composition comprising the ADC of any one of clauses 1- 117 and one or more pharmaceutically acceptable carriers, excipients, or diluents. Clause 127. A method of treatment, comprising administering to a subject in need thereof an effective amount of the ADC of any one of clauses 1-117 or the pharmaceutical composition of clause 118. Clause 128. The method of clause 127, wherein the subject has Hepatitis B. Clause 129. The method of clause 127 wherein said Hepatitis B is chronic Hepatitis B. Clause 130. The method of clause 127 or 128, wherein the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or the pharmaceutical composition. Clause 131. The method of any one of clauses 127-130, further comprising, before the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject. Clause 132. The method of any one of clauses 127-131, further comprising, after the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject to assess therapeutic efficacy of the ADC or the pharmaceutical composition. Clause 133. The method of any one of clauses 127-132, wherein the step of administrating the ADC or the pharmaceutical composition is repeated. Clause 134. The method of clause 133, wherein the step of administrating the ADC or the pharmaceutical composition is repeated twice, three times, or more. Clause 135. The method of clause 133 or 134, wherein the step of administrating the ADC or the pharmaceutical composition is repeated at least at 1-week intervals, at 2-week intervals, at 3-week intervals, or at 4-week intervals. Clause 136. The method of clause 133 or 134, wherein the step of administrating the ADC or the pharmaceutical composition is repeated at 1-week intervals, at 2-week intervals, at 3- week intervals, or at 4-week intervals. Clause 137. The method of clause 133 or clause 134, wherein the step of administrating the ADC or the pharmaceutical composition is repeated at 1-month intervals, at 2-month intervals, or 3-month intervals. Clause 138. The method of any one of clauses 127-137, wherein the ADC or pharmaceutical composition is administered by oral, intravenous, intraperitoneal, inhalation, intranasal, intramuscular, or subcutaneous administration. Clause 139. The ADC of any one of clauses 1-117 or the pharmaceutical composition of clause 126 for use in treatment. Clause 140. The ADC of any one of clauses 1-117 or the pharmaceutical composition of clause 126 for use in treatment of chronic Hepatitis B in a subject in need thereof. Clause 141. Use of the ADC of any one of clauses 1-117 or the pharmaceutical composition of clause 126 for manufacture of a medicament. Clause 142. Use of the ADC of any one of clauses 1-117 or the pharmaceutical composition of clause 126 for manufacture of a medicament for the treatment of chronic Hepatitis B in a subject in need thereof.
XII. Examples [0321] The examples below are meant to illustrate certain embodiments provided herein, and not to limit the scope of this disclosure. EXAMPLE 1 [0322] Synthesis of Intermediate Aa (see Scheme 1) [0323] Methyl 4-{[2-chloro-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoate (3)
Figure imgf000157_0001
[0324] To a mixture of compound 1 (0.50 g, 2.7 mmol) in acetonitrile (10 mL) were added bromide 2 (0.76 g, 2.9 mmol) and potassium carbonate (0.74 g, 5.3 mmol), and the suspension was stirred at room temperature for 6 hours, which was monitored by LCMS (ESI m/z: 366.1 (M + H)+). To the resulting mixture were added potassium carbonate (0.37 g, 2.7 mmol) and 1-pentanamine (0.70 g, 7.9 mmol), and the reaction mixture was stirred at 85 oC for 5 hours, which was monitored by LCMS. After cooled to room temperature, the mixture was filtered, and the filtrate was concentrated in vacuo. The residue was diluted with water and extracted with ethyl acetate (x 3). The combined organic solution was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound 3 (0.23 g, 21% yield) as a light-yellow solid. ESI m/z: 417.2 (M + H)+. [0325] Methyl 4-{[2-azido-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoate (4)
Figure imgf000157_0002
[0326] To a solution of compound 3 (0.20 g, 0.48 mmol) in NMP (6 mL) were added sodium azide (0.23 g, 3.6 mmol) and zinc chloride (0.33 g, 2.4 mmol), and the reaction mixture was stirred at 150 oC for 6 hours, which was monitored by LCMS. After cooled to room temperature, the mixture was diluted with ethyl acetate (40 mL) and sat. aq. sodium bicarbonate (40 mL). The suspension was filtered, and the filtrate was extracted with ethyl acetate. The combined organic solution was washed with water and brine, dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound 4 (60 mg, 30% yield) as a light yellow solid. ESI m/z: 424.2 (M + H)+. [0327] Methyl 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoate (Aa) (method 1)
Figure imgf000158_0001
[0328] To a solution of compound 4 (0.12 g, 0.28 mmol) in anhydrous acetic acid (10 mL) was added zinc dust (0.56 g, 8.5 mmol) under the protection of nitrogen flow at 0 oC. The reaction mixture was then stirred at 85 oC for 4 hours. After cooled to room temperature, the mixture was filtered through the Celite, and the filtrate was concentrated in vacuo. The residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give intermediate Aa (65 mg, 58% yield) as a light-yellow solid. ESI m/z: 398.3 (M + H)+. EXAMPLE 2 [0329] General procedure I to synthesize compound 7
Figure imgf000158_0002
[0330] To a mixture of compound 5a,b (1.0 equiv.) in acetonitrile (0.2 M) were added compound 6a-c (1.1-1.2 equiv.) and potassium carbonate (2.0 equiv.), and the suspension was stirred at room temperature for 16 hours, which was monitored by LCMS. The resulting mixture was filtered, and the filtrate was concentrated in vacuo. The black residue was purified by silica gel flash chromatography to give compound 7a-d (34-94% yield) as a light-yellow solid. [0331] Methyl 4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxybenzoate (7a)
Figure imgf000159_0003
[0332] Following the general procedure I starting from compound 5a (0.50 g, 3.0 mmol) and 6a (0.85 g, 3.3 mmol), compound 7a (0.85 g, 83% yield) was obtained as a light yellow solid after purification by silica gel flash chromatography (50-100% ethyl acetate in petroleum ether over 20 minutes). ESI m/z: 347.1 (M + H)+, 369.1 (M + Na)+.1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 1.3 Hz, 1H), 7.52 (dd, J = 7.9, 1.4 Hz, 1H), 7.31 (d, J = 3.1 Hz, 1H), 6.57 (d, J = 7.9 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 5.58 (s, 2H), 4.86 (s, 2H), 3.95 (s, 3H), 3.90 (s, 3H) ppm. [0333] Methyl 4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzoate (7b)
Figure imgf000159_0001
[0334] Following the general procedure I starting from compound 5a (0.55 g, 3.3 mmol) and 6b (0.90 g, 3.9 mmol), compound 7b (1.1 g, 94% yield) was obtained as a light yellow solid after purification by silica gel flash chromatography (5-10% methanol in DCM over 20 minutes). ESI m/z: 317.1 (M + H)+, 339.1 (M + Na)+.1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 3.2 Hz, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.42 (d, J = 3.2 Hz, 1H), 5.62 (s, 2H), 4.87 (s, 2H), 3.90 (s, 3H) ppm. [0335] tert-Butyl N-{[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5- yl}methyl)phenyl]methyl}carbamate (7c)
Figure imgf000159_0002
[0336] Following the general procedure I starting from compound 5a (0.20 g, 1.2 mmol) and 6c (0.43 g, 1.4 mmol), compound 7c (0.30 g, 65% yield) was obtained as a light yellow solid after purification by silica gel flash chromatography (20-25% ethyl acetate in petroleum ether over 20 minutes). ESI m/z: 388.2 (M + H)+.1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 3.2 Hz, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 6.37 (d, J = 3.1 Hz, 1H), 5.55 (s, 2H), 4.82 (br s, 3H), 4.29 (d, J = 5.5 Hz, 2H), 1.45 (s, 9H) ppm. [0337] Methyl 4-[(2-amino-6-chloro-7H-purin-7-yl)methyl]-3-methoxybenzoate (7d) and methyl 4-[(2-amino-6-chloro-9H-purin-9-yl)methyl]-3-methoxybenzoate (7d’)
Figure imgf000160_0001
[0338] Following the general procedure I starting from compound 5b (0.50 g, 2.9 mmol) and 6a (0.84 g, 3.2 mmol), compound 7d (0.35 g, 34% yield) as a white solid and its isomer 7d’ (0.15 g, 15% yield) were separately obtained after purification by silica gel flash chromatography (0-5% methanol in DCM). [0339] 7d: ESI m/z: 348.1 (M + H)+, 717.3 (2M + Na)+; LCMS RetTime: 1.64 min; 1H NMR (400 MHz, DMSOd6) δ 8.48 (s, 1H), 7.54 (d, J = 1.5 Hz, 1H), 7.50 (dd, J = 8.0, 1.5 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 6.70 (s, 2H), 5.56 (s, 2H), 3.93 (s, 3H), 3.84 (s, 3H) ppm. [0340] 7d’: ESI m/z: 348.1 (M + H)+; LCMS RetTime: 1.67 min; 1H NMR (400 MHz, DMSOd6) δ 8.17 (s, 1H), 7.53 (d, J = 1.3 Hz, 1H), 7.50 (dd, J = 7.8, 1.4 Hz, 1H), 6.94 (s, 2H), 6.88 (d, J = 7.8 Hz, 1H), 5.29 (s, 2H), 3.93 (s, 3H), 3.85 (s, 3H) ppm. [0341] The structures of 7d and 7d’ were determined by NOE data of 9b and 9b’. EXAMPLE 3 [0342] General procedure II to synthesize intermediates Aa, Ba, Bb, Bc and Fa
Figure imgf000160_0002
[0343] To a suspension of compound 7a-d (1.0 equiv.) in acetonitrile (40-50 mM) were added potassium carbonate (4.0 equiv.) and 1-pentanamine 8a (5.0 equiv.) or O- butylhydroxylamine 8b (HCl salt, 2.0 equiv.), and the reaction mixture was stirred at 85 oC for 15 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give intermediate Aa, Ba, Bb, Bc or Fa (28-98% yield) as a solid. [0344] Methyl 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoate (Aa) (method 2)
Figure imgf000161_0001
[0345] Following the general procedure II starting from 7a (0.60 g, 1.7 mmol) with 1- pentanamine 8a (0.75 g, 8.6 mmol), intermediate Aa (1.5 g, 83% yield) was obtained as a yellow solid after purification by silica gel flash chromatography (0-10% methanol in DCM). ESI m/z: 398.3 (M + H)+. [0346] Methyl 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoate (Ba)
Figure imgf000161_0002
[0347] Following the general procedure II starting from 7a (0.45 g, 1.3 mmol) with O- butylhydroxylamine 8b (HCl salt, 0.33 g, 2.6 mmol), intermediate Ba (0.51 g, 98% yield) was obtained as a gray solid after purification by silica gel flash chromatography (0-10% methanol in DCM). ESI m/z: 400.3 (M + H)+, 821.5 (2M + Na)+; 1H NMR (400 MHz, CDCl3) δ 7.53-7.51 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 2.9 Hz, 1H), 6.07 (d, J = 2.9 Hz, 1H), 5.46 (s, 2H), 4.50 (br s, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.87 (t, J = 6.7 Hz, 2H), 1.55-1.45 (m, 2H), 1.37-1.18 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H) ppm. [0348] Methyl 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}benzoate (Bb)
Figure imgf000162_0001
[0349] Following the general procedure II starting from 7b (0.40 g, 1.3 mmol) with O- butylhydroxylamine 8b (HCl salt, 0.32 g, 2.6 mmol), intermediate Bb (0.39 g, 83% yield) was obtained as an orange solid after purification by silica gel flash chromatography (0-10% methanol in DCM). ESI m/z: 370.3 (M + H)+, 761.4 (2M + Na)+; 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 6.74 (d, J = 2.9 Hz, 1H), 6.07 (d, J = 2.9 Hz, 1H), 5.46 (s, 2H), 3.94-3.77 (m, 5H), 1.50-1.40 (m, 2H), 1.30-1.20 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H) ppm. [0350] tert-Butyl N-[(4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}phenyl)methyl]carbamate (Bc)
Figure imgf000162_0002
[0351] Following the general procedure II starting from 7c (0.14 g, 0.36 mmol) with O- butylhydroxylamine 8b (HCl salt, 91 mg, 0.72 mmol), intermediate Bc (45 mg, 28% yield) was obtained as a white solid after purification by silica gel flash chromatography (0-10% methanol in DCM). ESI m/z: 441.1 (M + H)+, 881.5 (2M + Na)+. [0352] Methyl 4-{[2-amino-6-(pentylamino)-7H-purin-7-yl]methyl}-3-methoxybenzoate (Fa)
Figure imgf000162_0003
[0353] Following the general procedure II starting from 7d (0.28 g, 0.81 mmol) with 1- pentanamine 8a (0.21 g, 2.4 mmol), intermediate Fa (0.29 g, 90% yield) was obtained as a white solid after purification by silica gel flash chromatography (0-5% methanol in DCM). ESI m/z: 399.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.97 (s, 1H), 7.53 (d, J = 1.2 Hz, 1H), 7.49 (dd, J = 7.9, 1.3 Hz, 1H), 6.71 (d, J = 7.9 Hz, 1H), 6.00 (t, J = 5.5 Hz, 1H), 5.66 (s, 2H), 5.58 (s, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 3.35-3.22 (m, 2H), 1.47-1.30 (m, 2H), 1.25-1.07 (m, 2H), 1.08-0.91 (m, 2H), 0.75 (t, J = 7.3 Hz, 3H) ppm. EXAMPLE 4 [0354] General procedure III to synthesize intermediates Ca, Cb and Cc
Figure imgf000163_0001
[0355] To a solution of compound 7a-c (1.0 equiv.) in 1-butanol (0.10-0.15 M) were added DIPEA (4 equiv.) and 2-ethoxyethan-1-amine 8c (2.0 equiv.), and the reaction mixture was protected with argon and stirred at 120 oC for 4-5 hours in a sealed tube, which was monitored by LCMS. After cooled, the reaction mixture was concentrated in vacuo, and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give intermediate Ca-c (51-61% yield) as a yellow solid. [0356] Methyl 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5- yl}methyl)-3-methoxybenzoate (Ca)
Figure imgf000163_0002
[0357] Following the general procedure III starting from 7a (0.17 g, 0.49 mmol) with amine 8c (87 mg, 0.98 mmol), intermediate Ca (0.12 g, 61% yield) was obtained as a yellow solid after purification by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)). ESI m/z: 400.3 (M + H)+. [0358] Methyl 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5- yl}methyl)benzoate (Cb)
Figure imgf000163_0003
[0359] Following the general procedure III starting from 7b (0.16 g, 0.49 mmol) with amine 8c (87 mg, 0.98 mmol), intermediate Cb (0.12 g, 61% yield) was obtained as a yellow solid after purification by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)). ESI m/z: 370.3 (M + H)+. [0360] tert-Butyl N-{[4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5- yl}methyl)phenyl]methyl}carbamate (Cc)
Figure imgf000164_0001
[0361] Following the general procedure III starting from 7c (0.18 g, 0.45 mmol) with amine 8c (80 mg, 0.90 mmol), intermediate Cc (90 mg, 51% yield) was obtained as a yellow solid after purification by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)). ESI m/z: 441.5 (M + H)+. EXAMPLE 5 [0362] Methyl 4-{[2-amino-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoate (Da) [0363]
Figure imgf000164_0002
[0364] To a solution of compound 7a (0.50 g, 1.4 mmol) in 1-pentanol (5 mL) was added HCl in dioxane (4 M, 1 mL), and the reaction mixture was sealed and stirred at 120 oC for 5 hours, which was monitored by LCMS. After cooled, the mixture was concentrated in vacuo and the residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give a mixture of compound Da and its hydrolysate P35-1 (0.51 g), which was used for the next step without further purification. ESI m/z: 399.2 (MDa + H)+, 385.3 (MP35-1 + H)+. EXAMPLE 6 [0365] General procedure IV to synthesize intermediates 9a and 9b
Figure imgf000165_0001
[0366] To a solution of compound 7a or 7d (1.0 equiv.) in dioxane and water (v/v = 4, 0.10- 0.12 M) were added potassium carbonate (5.0 equiv.), boric acid 8e (3.0 equiv.) and tetrakis(triphenylphosphine)palladium (0.20 equiv.). The reaction mixture was protected with nitrogen and stirred at 80 oC for 15 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was directly separated by silica gel column chromatography (0-5% methanol in DCM) to give compound 9a or 9b (53-90% yield) as a white solid. [0367] Methyl 4-[(2-amino-4-(hex-1-en-1-yl)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]-3- methoxybenzoate (9a)
Figure imgf000165_0002
[0368] Following the general procedure IV starting from 7a (0.40 g, 1.2 mmol), compound 9a (0.40 g, 90% yield) was obtained as a yellow solid. ESI m/z: 395.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.70 (d, J = 3.0 Hz, 1H), 7.57 (s, 1H), 7.44 (d, J = 7.8 Hz, 1H), 6.88-6.69 (m, 1H), 6.35 (m, 2H), 6.21 (d, J = 3.0 Hz, 1H), 5.83 (s, 2H), 5.52 (s, 2H), 3.98 (s, 3H), 3.83 (s, 3H), 2.05-1.95 (m, 2H), 1.20-1.01 (m, 4H), 0.77 (t, J = 7.0 Hz, 3H) ppm. [0369] Methyl 4-{[2-amino-6-(hex-1-en-1-yl)-7H-purin-7-yl]methyl}-3-methoxybenzoate (9b)
Figure imgf000165_0003
[0370] Following the general procedure IV starting from 7d (0.27 g, 0.78 mmol), compound 9b (0.18 g, 53% yield) was obtained as a yellow solid. ESI m/z: 396.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 8.39 (s, 1H), 7.57 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 6.92-6.77 (m, 1H), 6.60 (d, J = 7.8 Hz, 1H), 6.27 (d, J = 15.1 Hz, 1H), 6.14 (s, 2H), 5.58 (s, 2H), 3.97 (s, 3H), 3.84 (s, 3H), 2.08-1.95 (m, 2H), 1.21-1.01 (m, 4H), 0.77 (t, J = 7.0 Hz, 3H) ppm. [0371] Methyl 4-{[2-amino-6-(hex-1-en-1-yl)-9H-purin-9-yl]methyl}-3-methoxybenzoate (9b’)
Figure imgf000166_0001
[0372] Following the general procedure IV starting from 7d’ (0.10 g, 0.29 mmol), compound 9b’ (60 mg, 53% yield) was obtained as a yellow solid. ESI m/z: 396.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 8.09 (s, 1H), 7.53-7.49 (m, 3H), 6.80 (d, J = 7.4 Hz, 1H), 6.66 (d, J = 16.3 Hz, 1H), 6.40 (s, 2H), 5.29 (s, 2H), 3.94 (s, 3H), 3.85 (s, 3H), 2.40-2.28 (m, 2H), 1.37-1.31 (m, 4H), 0.92 (t, J = 7.0 Hz, 3H) ppm. [0373] Comparing the NOE data of 9b and 9b’: there is signal between the benzyl CH2 (5.58 ppm, s, 2H) and CH=CH (6.27 ppm, d, J = 15.1 Hz, 1H) in NOESY spectra of compound 9b, while no signal between benzyl CH2 (5.29 ppm, s, 2H) and CH=CH (6.66 ppm, d, J = 16.3 Hz, 1H) in NOESY spectra of compound 9b’. That result demonstrated that compounds 7d and 9b are desired intermediates. EXAMPLE 7 [0374] Methyl 4-({2-amino-4-hexyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxybenzoate (Ea)
Figure imgf000166_0002
[0375] To a solution of compound 9a (0.38 g, 0.96 mmol) in ethyl acetate (30 mL) was added palladium on carbon (containing 10% palladium, 40 mg) under nitrogen protection. The reaction mixture was stirred under hydrogen at room temperature for 15 hours, which was monitored by LCMS. The resulting mixture was filtered through the Celite and the filtrate was concentrated in vacuo to give compound Ea (0.38 g, 95% yield) as a white solid. ESI m/z: 397.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.66 (d, J = 2.9 Hz, 1H), 7.56 (s, 1H), 7.45 (d, J = 7.9 Hz, 1H), 6.25 (d, J = 7.9 Hz, 1H), 6.21 (d, J = 2.9 Hz, 1H), 5.81 (s, 2H), 5.48 (s, 2H), 3.96 (s, 3H), 3.83 (s, 3H), 2.49-2.41 (m, 2H), 1.36-1.25 (m, 2H), 1.21-0.93 (m, 6H), 0.78 (t, J = 7.2 Hz, 3H) ppm. EXAMPLE 8 [0376] Methyl 4-[(2-amino-6-hexyl-7H-purin-7-yl)methyl]-3-methoxybenzoate (Fb)
Figure imgf000167_0001
[0377] To a solution of compound 9b (0.20 g, 0.51 mmol) in ethanol (15 mL) was added palladium hydroxide (20 mg, 10wt%) under nitrogen protection, and the reaction mixture was stirred under hydrogen balloon (1.1 atm) at 50 oC for 15 hours, which was monitored by LCMS. After cooled, the resulting mixture was filtered through the Celite and the filtrate was concentrated in vacuo to give compound Fb (0.21 g, 95% yield) as a white solid, which was used for the next step without further purification. ESI m/z: 398.3 (M + H)+. EXAMPLE 9 [0378] General procedure V to reduce methyl benzoates to benzyl alcohols
Figure imgf000167_0002
[0379] To a stirred suspension of lithium aluminum tetrahydride (LAH, 2.0 equiv.) in anhydrous THF (0.04-0.20 M) was added dropwise a solution of the ester (1.0 equiv.) in anhydrous THF (0.02-0.20 M) for 5 minutes at 0 oC under nitrogen protection. The reaction mixture was stirred at 0 oC for 15 minutes and was then stirred at room temperature for half an hour until the ester was totally reduced according to LCMS. The resulting mixture was cooled to 0 oC and carefully quenched with sat. aq. sodium bicarbonate (5% vol.) and water (5% vol.). The mixture was filtered, and the filtrate was extracted with ethyl acetate (x 3). The combined organic solution was washed with brine and concentrated in vacuo. The crude product was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to give the payload (43% yield, TFA salt) as a white solid. [0380] Payload P1 [0381] (4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methanol (P1)
Figure imgf000168_0001
[0382] Following the general procedure V starting from intermediate Aa (65 mg, 0.16 mmol), payload P1 (35 mg, 43% yield, TFA salt) was obtained as a white solid. ESI m/z: 389.3 (M + H)+.1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 3.2 Hz, 1H), 7.22 (br s, 2H), 7.02 (s, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 6.18 (d, J = 3.2 Hz, 1H), 5.50 (s, 2H), 5.21 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 5.6 Hz, 2H), 3.83 (s, 3H), 3.41-3.49 (m, 2H), 1.52-1.40 (m, 2H), 1.30- 1.19 (m, 2H), 1.19-1.01 (m, 2H), 0.82 (t, J = 7.2 Hz, 3H) ppm. (COOH of TFA was not revealed.) 19F NMR (376 MHz, DMSOd6) δ -73.4 ppm. [0383] Payload P20 [0384] (4-{[2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methanol (P20)
Figure imgf000168_0002
[0385] Following the general procedure V starting from intermediate Ba (0.15 g, 0.38 mmol), payload P1 (0.13 g, 72% yield, TFA salt) was obtained as a white solid. ESI m/z: 372.5 (M + H)+.1H NMR (400 MHz, CDCl3) δ 12.06 (s, 1H), 10.73 (s, 1H), 7.90 (s, 2H), 7.11 (d, J = 2.9 Hz, 1H), 6.98 (s, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.64 (d, J = 7.8 Hz, 1H), 6.08 (d, J = 2.9 Hz, 1H), 5.35 (s, 2H), 5.17 (s, 1H), 4.46 (s, 2H), 3.95 (t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 1.59-1.48 (m, 2H), 1.36-1.23 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H) ppm. [0386] Payload P23 [0387] [4-({2-Amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxyphenyl]methanol (P23)
Figure imgf000169_0001
[0388] Following the general procedure V starting from intermediate Ca (30 mg, 75 µmol), payload P23 (15 mg, 41% yield, TFA salt) was obtained as a white solid. ESI m/z: 372.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.30 (s, 1H), 7.50-7.41 (m, 3H), 7.39 (d, J = 3.0 Hz, 1H), 7.02 (s, 1H), 6.83 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.19 (d, J = 3.0 Hz, 1H), 5.48 (s, 2H), 5.23 (s, 1H), 4.47 (s, 2H), 3.83 (s, 3H), 3.66 (q, J = 7.0 Hz, 2H), 3.48 (t, J = 5.9 Hz, 2H), 3.42-3.38 (m, 2H), 1.06 (t, J = 7.0 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. [0389] Payload P27 [0390] (4-{[2-Amino-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methanol (P27)
Figure imgf000169_0002
[0391] Following the general procedure V starting from intermediate Da (60 mg, 0.15 mmol), payload P27 (45 mg, 62% yield, TFA salt) was obtained as a white solid. ESI m/z: 371.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 13.17 (s, 1H), 7.97 (s, 2H), 7.68 (d, J = 2.9 Hz, 1H), 7.00 (s, 1H), 6.79 (d, J = 7.2 Hz, 1H), 6.50 (d, J = 7.7 Hz, 1H), 6.34 (d, J = 2.9 Hz, 1H), 5.43 (s, 2H), 5.20 (s, 1H), 4.46 (s, 2H), 4.42 (t, J = 6.3 Hz, 2H), 3.83 (s, 3H), 1.66-1.54 (m, 2H), 1.29- 1.09 (m, 4H), 0.81 (t, J = 7.1 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. [0392] Payload P29 [0393] [4-({2-Amino-4-hexyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxyphenyl]methanol (P29)
Figure imgf000170_0001
[0394] Following the general procedure V starting from intermediate Ea (0.38 g, 0.96 mmol), payload P29 (0.30 g, 65% yield, TFA salt) was obtained as a white solid. ESI m/z: 369.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 8.11-7.75 (m, 3H), 7.07 (s, 1H), 6.80 (d, J = 6.6 Hz, 1H), 6.45 (s, 1H), 6.30 (d, J = 6.6 Hz, 1H), 5.52 (s, 2H), 5.26 (s, 1H), 4.48 (s, 2H), 3.87 (s, 3H), 2.77-2.61 (m, 2H), 1.49-1.35 (m, 2H), 1.27-1.01 (m, 6H), 0.83 (t, J = 6.5 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. [0395] Payload P32 [0396] (4-{[2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}phenyl)methanol (P32)
Figure imgf000170_0002
[0397] Following the general procedure V starting from intermediate Bb (82 mg, 0.22 mmol), payload P32 (70 mg, 69% yield, TFA salt) was obtained as a white solid. ESI m/z: 342.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.12 (s, 1H), 10.76 (s, 1H), 7.93 (s, 2H), 7.28 (d, J = 3.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 6.08 (d, J = 3.0 Hz, 1H), 5.38 (s, 2H), 5.15 (br s, 1H), 4.45 (s, 2H), 4.03 (t, J = 6.6 Hz, 2H), 1.66-1.53 (m, 2H), 1.43-1.26 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. [0398] Payload P33 [0399] [4-({2-Amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5- yl}methyl)phenyl]methanol (P33)
Figure imgf000170_0003
[0400] Following the general procedure V starting from intermediate Cb (28 mg, 75 µmol), payload P33 (15 mg, 44% yield, TFA salt) was obtained as a white solid. ESI m/z: 342.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.29 (s, 1H), 7.63 (d, J = 3.0 Hz, 1H), 7.49 (t, J = 5.5 Hz, 1H), 7.42 (s, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 6.24 (d, J = 3.0 Hz, 1H), 5.59 (s, 2H), 5.21-5.14 (m, 1H), 4.44 (s, 2H), 3.63 (q, J = 6.8 Hz, 2H), 3.43 (t, J = 7.0 Hz, 2H), 3.39 (t, J = 7.0 Hz, 2H), 1.07 (t, J = 7.0 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. [0401] Payload P37 [0402] (4-{[2-Amino-6-(pentylamino)-7H-purin-7-yl]methyl}-3-methoxyphenyl)methanol (P37)
Figure imgf000171_0001
[0403] Following the general procedure V starting from intermediate Fa (0.28 g, 0.70 mmol), payload P37 (0.13 g, 38% yield, TFA salt) was obtained as a white solid. ESI m/z: 371.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 13.27 (s, 1H), 8.14 (s, 1H), 7.70 (t, J = 5.4 Hz, 1H), 7.56 (s, 2H), 7.02 (s, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 5.58 (s, 2H), 5.27 (s, 1H), 4.48 (s, 2H), 3.81 (s, 3H), 3.60-3.40 (m, 2H), 1.59-1.45 (m, 2H), 1.34-1.10 (m, 4H), 0.84 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. [0404] Payload P39 [0405] {4-[(2-Amino-6-hexyl-7H-purin-7-yl)methyl]-3-methoxyphenyl}methanol (P39)
Figure imgf000171_0002
[0406] Following the general procedure V starting from intermediate Fb (0.21 g, 0.53 mmol), payload P37 (0.13 g, 51% yield, TFA salt) was obtained as a white solid. ESI m/z: 370.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 8.40 (s, 1H), 7.05 (s, 1H), 6.80 (d, J = 7.8 Hz, 1H), 6.43 (d, J = 7.8 Hz, 1H), 6.32 (br s, 2H), 5.45 (s, 2H), 5.26 (s, 1H), 4.48 (s, 2H), 3.86 (s, 3H), 2.59- 2.48 (m, 2H), 1.45-1.28 (m, 2H), 1.24-1.04 (m, 6H), 0.82 (t, J = 7.2 Hz, 3H) ppm. (COOH of TFA was not revealed) 19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 10 [0407] Payload P2 [0408] 2-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)propan-2-ol (P2)
Figure imgf000172_0001
[0409] To a cooled (-5 to 5 oC) solution of intermediate Aa (50 mg, 0.13 mmol) in anhydrous THF (5.0 mL) was added methylmagnesium bromide (3.0 M in THF, 0.43 mL, 1.3 mmol) dropwise under nitrogen protection. The reaction mixture was stirred at 0 oC for half an hour and then at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was quenched with methanol and the volatiles were removed in vacuo. The residue was diluted with water and extracted with ethyl acetate (x 3). The combined organic solution was washed with brine and concentrated in vacuo. The crude product was purified by reversed phase flash chromatography (5-100% acetonitrile in aq. TFA (0.01%)) to give payload P2 (10 mg, 15% yield, TFA salt) as a white solid. ESI m/z: 398.3 (M + H)+. 1H NMR (400 MHz, MeODd4) δ 7.35 (d, J = 3.0 Hz, 1H), 7.23 (d, J = 1.4 Hz, 1H), 6.99 (dd, J = 8.0, 1.5 Hz, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.19 (d, J = 3.0 Hz, 1H), 5.49 (s, 2H), 3.92 (s, 3H), 3.55-3.48 (m, 2H), 1.50-1.44 (m, 8H), 1.33-1.29 (m, 4H), 0.87 (t, J = 6.3 Hz, 3H) ppm.19F NMR (376 MHz, MeODd4) δ -73.5 ppm. EXAMPLE 11 [0410] General procedure VI to synthesize piperazine analogues P3, P26, P28, P36 and P38
Figure imgf000173_0001
[0411] To a suspension of alcoholic payload P1, P27, P29, P37 or P39 (1.0 equiv.) in DCM (50 mM) was added a solution of thionyl chloride (1.2 equiv.) in DCM (0.1 M) dropwise at 0 oC. The reaction mixture was stirred at room temperature for 5 hours. The volatiles were removed in vacuo to give crude corresponding chloride P3-1, P26-1, P28-1, P36-1 or P38-1 as a yellow semi-solid, which was dissolved into DMF (25 mg/mL). To the solution were added potassium carbonate (2.0 equiv.) and N-Bocpiperazine (1.0 equiv.), and the reaction mixture was stirred at room temperature for 15 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-95% methanol in aq. TFA (0.01%)) to give Boc-payload P3-2, P26-2, P28-2, P36-2 or P38-2 as a white solid, which was dissolved in DCM (25 mg/mL). To the solution was added dropwise a solution of hydrochloride in dioxane (4 M, v / vDCM = 2 / 3, pH < 1). The reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-95% acetonitrile in aq. TFA (0.01%)) to give piperazine payload P3, P26, P28, P36 or P38 (31-72% yield) as a white solid. [0412] Payload P3 [0413] 5-({2-Methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P3)
Figure imgf000173_0002
[0414] Following the general procedure VI starting from P1, payload P3 (0.23 g, 31% yield, TFA salt) was obtained as a white solid. ESI m/z: 438.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.81 (s, 1H), 9.21 (br s, 1H), 7.55 (s, 2H), 7.41 (d, J = 3.0 Hz, 2H), 7.15 (s, 1H), 6.93 (d, J = 7.7 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.56 (s, 2H), 3.97 (s, 2H), 3.85 (s, 3H), 3.55-3.35 (m, 2H), 3.27 (br s, 4H), 2.99 (br s, 4H), 1.55-1.34 (m, 2H), 1.33-1.16 (m, 2H), 1.15-1.04 (m, 2H), 0.82 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -74.0 ppm. [0415] Payload P26 [0416] 5-({2-Methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-4-(pentyloxy)-5H- pyrrolo[3,2-d]pyrimidin-2-amine (P26)
Figure imgf000174_0001
[0417] Following the general procedure VI starting from P27, payload P26 (15 mg, 31% yield, TFA salt) was obtained as a white solid. ESI m/z: 439.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 9.10 (s, 1H), 8.21 (s, 2H), 7.70 (d, J = 2.9 Hz, 1H), 7.13 (s, 1H), 6.90 (d, J = 7.6 Hz, 1H), 6.52 (d, J = 7.7 Hz, 1H), 6.36 (d, J = 2.9 Hz, 1H), 5.46 (s, 2H), 4.41 (t, J = 6.3 Hz, 2H) 3.90-3.78 (m, 5H), 3.24 (br s, 4H), 2.95 (br s, 4H), 1.66-1.47 (m, 2H), 1.35-1.01 (m, 4H), 0.80 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -74.0 ppm. [0418] Payload P28 [0419] 4-Hexyl-5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-5H-pyrrolo[3,2- d]pyrimidin-2-amine (P28)
Figure imgf000174_0002
[0420] Following the general procedure VI starting from P29, payload P28 (46 mg, 53% yield, TFA salt) was obtained as a white solid. ESI m/z: 437.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 9.12 (s, 1H), 8.01 (br s, 2H), 8.00 (d, J = 3.0 Hz, 1H), 7.21 (s, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.48 (d, J = 2.9 Hz, 1H), 6.37 (d, J = 7.7 Hz, 1H), 5.56 (s, 2H), 4.02 (s, 2H), 3.89 (s, 3H), 3.27 (br s, 4H), 3.00 (br s, 4H), 2.78-2.64 (m, 2H), 1.50-1.38 (m, 2H), 1.20-1.08 (m, 6H), 0.83 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -74.2 ppm. [0421] Payload P36 [0422] 7-({2-Methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-N6-pentyl-7H-purine-2,6- diamine (P36)
Figure imgf000175_0001
[0423] Following the general procedure VI starting from P37, payload P36 (78 mg, 66% yield, TFA salt) was obtained as a white solid. ESI m/z: 439.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 9.02 (br s, 1H), 8.15 (s, 1H), 7.79 (t, J = 5.6 Hz, 1H), 7.72 (s, 2H), 7.10 (s, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 5.61 (s, 2H), 3.82 (s, 3H), 3.81 (s, 2H), 3.53- 3.45 (m, 2H), 3.20 (br s, 4H), 2.81 (br s, 4H), 2.55 (s, 1H), 1.58-1.42 (m, 2H), 1.37-1.02 (m, 4H), 0.84 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.9 ppm. [0424] Payload P38 [0425] 6-Hexyl-7-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-7H-purin-2-amine (P38)
Figure imgf000175_0002
[0426] Following the general procedure VI starting from P39, payload P38 (79 mg, 72% yield, TFA salt) was obtained as a white solid. ESI m/z: 438.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 8.72 (s, 2H), 8.49 (s, 1H), 7.10 (s, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.53 (br s, 1H), 6.51 (d, J = 7.6 Hz, 1H), 5.48 (s, 2H), 3.87 (s, 3H), 3.69 (br s, 2H), 3.14 (br s, 4H), 2.67 (br s, 4H), 2.60-2.53 (m, 2H), 1.40-1.28 (m, 2H), 1.25-1.00 (m, 6H), 0.83 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.8 ppm. EXAMPLE 12 [0427] General procedure VII to synthesize amides P22, P25, P31 and P35
Figure imgf000176_0001
[0428] To a solution of ester Ba, Ca, Bb or Cb (1.0 equiv.) in isopropanol or methanol (50- 100 mM) was added aq. sodium hydroxide or lithium hydroxide (1 M, 0.5 v of alcoholic solvent). The reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The residual solution was neutralized by diluted aq. hydrochloride (1 M) to pH 6 and the resulting mixture was concentrated. The residue was purified by reversed phase flash chromatography (0-95% acetonitrile in aq. TFA (0.01%)) to give acid P22-1, P25-1, P31-1 or P35-1 as a yellow solid, which was dissolved in DMF (50-80 mM). To the solution were added ammonium chloride (2.0 equiv.), HATU (1.2 equiv.) and DIPEA (3.0 equiv.), and the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give amide P22, P25, P31 and P35 (4-51% yield, TFA salt) as a white solid. [0429] Payload P22 [0430] 4-{[2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzamide (P22)
Figure imgf000176_0002
[0431] Following the general procedure VII starting from Ba, payload P22 (82 mg, 28% yield, TFA salt) was obtained as a white solid. ESI m/z: 385.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.09 (s, 1H), 10.75 (s, 1H), 7.95 (s, 1H), 7.94 (br s, 2H), 7.50 (d, J = 1.2 Hz, 1H), 7.38 (dd, J = 7.6 Hz and 1.2 Hz, 1H), 7.36 (s, 1H), 7.19 (d, J = 2.9 Hz, 1H), 6.53 (d, J = 7.6 Hz, 1H), 6.13 (t, J = 2.9 Hz, 1H), 5.41 (s, 2H), 3.98-3.81 (m, 5H), 1.49-1.36 (m, 2H), 1.30-1.15 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. [0432] Payload P25 [0433] 4-({2-Amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxybenzamide (P25)
Figure imgf000177_0001
[0434] Following the general procedure VII starting from Ca, payload P25 (60 mg, 31% yield, TFA salt) was obtained as a white solid. ESI m/z: 385.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.35 (s, 1H), 7.99 (s, 1H), 7.52 (d, J = 1.3 Hz, 1H), 7.47 (d, J = 3.0 Hz, 3H), 7.43- 7.36 (m, 3H), 6.67 (d, J = 7.9 Hz, 1H), 6.24 (d, J = 3.0 Hz, 1H), 5.57 (s, 2H), 3.90 (s, 3H), 3.63 (q, J = 5.7 Hz, 2H), 3.44 (t, J = 5.9 Hz, 2H), 3.33-3.28 (m, 2H), 1.01 (t, J = 7.0 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.4 ppm. [0435] Payload P31 [0436] 4-{[2-Amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}benzamide (P31)
Figure imgf000177_0002
[0437] Following the general procedure VII starting from Bb, payload P31 (5.0 mg, 4% yield, TFA salt) was obtained as a white solid. ESI m/z: 355.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.13 (s, 1H), 10.76 (s, 1H), 7.93 (s, 3H), 7.81 (d, J = 8.3 Hz, 2H), 7.35 (s, 1H), 7.31 (d, J = 2.9 Hz, 1H), 7.19 (d, J = 8.2 Hz), 6.12 (d, J = 2.9 Hz, 1H), 5.45 (s, 2H), 3.97 (t, J = 6.5 Hz, 2H), 1.55-1.44 (m, 2H), 1.35-1.23 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. [0438] Payload P35 [0439] 4-({2-Amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5- yl}methyl)benzamide (P35)
Figure imgf000178_0001
[0440] Following the general procedure VII starting from Cb, payload P35 (50 mg, 51% yield, TFA salt) was obtained as a white solid. ESI m/z: 355.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.26 (s, 1H), 7.93 (s, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 3.0 Hz, 1H), 7.50- 7.35 (m, 4H), 7.08 (d, J = 8.1 Hz, 2H), 6.28 (d, J = 2.9 Hz, 1H), 5.68 (s, 2H), 3.61 (d, J = 5.9 Hz, 2H), 3.41-3.37 (m, 2H), 3.35-3.28 (m, 2H), 1.02 (t, J = 7.0 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. [0441] 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxybenzoic acid (P47)
Figure imgf000178_0002
[0442] Following the general procedure VII starting from Aa but without amidation, crude acid P47 was collected. The crude acid was further purified by prep-HPLC to give pure P47 (5 mg, 60% yield) as a light yellow solid. ESI m/z: 384.2 (M + H)+. EXAMPLE 13 [0443] Payload P21 [0444] 5-{[4-(Aminomethyl)-2-methoxyphenyl]methyl}-N4-butoxy-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P21)
Figure imgf000179_0001
[0445] To a stirred suspension of lithium aluminum tetrahydride (39 mg, 1.0 mmol) in anhydrous THF (6 mL) was added dropwise a solution of compound P22 (65 mg, 0.17 mmol) in anhydrous THF (4 mL) over 10 minutes at 0 oC under nitrogen protection. The reaction mixture was stirred at 65 oC for 4 hours which was monitored by LCMS. The resulting mixture was cooled to 0 oC and carefully quenched with sodium sulfate decahydrate. The mixture was then filtered and the filtrate was concentrated in vacuo. The crude product was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give P21 (31 mg, 38% yield, TFA salt) as a light yellow solid. ESI m/z: 371.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.53 (s, 1H), 10.78 (s, 1H), 8.14 (br, 4H), 7.18 (d, J = 1.2 Hz, 1H), 7.12 (d, J = 2.9 Hz, 1H), 6.92 (d, J = 7.2 Hz, 1H), 6.68 (d, J = 7.2 Hz, 1H), 6.08 (d, J = 2.9 Hz, 1H), 5.39 (s, 2H), 4.00 (br s, 2H), 3.93 (t, J = 6.5 Hz, 2H), 3.85 (s, 3H), 1.59-1.48 (m, 2H), 1.38- 1.22 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.7 ppm. EXAMPLE 14 [0446] Payload P24 [0447] 5-{[4-(Aminomethyl)-2-methoxyphenyl]methyl}-N4-(2-ethoxyethyl)-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P24)
Figure imgf000179_0002
[0448] To a solution of compound P25 (29 mg, 75 µmol) in anhydrous THF (4 mL) was added dropwise a solution of borane in THF (1 M, 0.23 mL) at 65 oC under argon protection. The reaction mixture was refluxed at 65 oC for 4 hours under argon, which was monitored by LCMS. After cooled to 0 oC, the resulting mixture was carefully quenched with sat. aq. sodium bicarbonate (0.24 mL) and water (0.24 mL). The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give P24 (5 mg, 14% yield, TFA salt) as a light yellow solid. ESI m/z: 371.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.44 (s, 1H), 8.19 (s, 2H), 7.54 (t, J = 5.5 Hz, 1H), 7.48 (s, 2H), 7.39 (d, J = 3.0 Hz, 1H), 7.20 (s, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.52 (s, 2H), 4.01 (s, 2H), 3.84 (s, 3H), 3.72-3.60 (m, 2H), 3.50 (t, J = 5.9 Hz, 2H), 3.42 (q, J = 7.0 Hz, 2H), 1.07 (t, J = 7.0 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. EXAMPLE 15 [0449] Payload P30 [0450] 5-{[4-(Aminomethyl)phenyl]methyl}-N4-butoxy-5H-pyrrolo[3,2-d]pyrimidine-2,4- diamine (P30)
Figure imgf000180_0001
[0451] To a suspension of compound Bc (45 mg, 0.10 mmol) in DCM (1 mL) was added a solution of hydrochloride (4 M in dioxane, 0.5 mL), and the reaction mixture was stirred at room temperature for an hour until Boc was totally removed, which was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by reversed phase flash chromatography (5-95% acetonitrile in aq. TFA (0.01)) to give P30 (45 mg, 97% yield, TFA salt) as a white solid. ESI m/z: 341.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.56 (s, 1H), 10.82 (s, 1H), 8.19 (br s, 4H), 7.40 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 3.2 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.08 (d, J = 3.2 Hz, 1H), 5.42 (s, 2H), 4.10-3.90 (m, 4H), 1.71-1.52 (m, 2H), 1.42-1.28 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.7 ppm. EXAMPLE 16 [0452] Payload P34 [0453] 5-{[4-(Aminomethyl)phenyl]methyl}-N4-(2-ethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine- 2,4-diamine (P34)
Figure imgf000180_0002
[0454] Following the similar procedure as Payload P30 except substituting Cc for Bc, payload P34 (0.10 g, 60% yield, TFA salt) was obtained as a white solid. ESI m/z: 341.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.60 (s, 1H), 8.18 (s, 2H), 7.64 (d, J = 3.0 Hz, 1H), 7.61 (t, J = 5.6 Hz, 1H), 7.51 (s, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.14 (d, J = 8.2 Hz, 2H), 6.24 (d, J = 3.0 Hz, 1H), 5.63 (s, 2H), 3.99 (br s, 2H), 3.64 (q, J = 5.7 Hz, 2H), 3.47-3.41 (m, 4H), 1.09 (t, J = 7.0 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 17 [0455] Intermediate P3-1 was synthesized as described in the procedure of payload P3. EXAMPLE 18 [0456] 5-{[4-(Bromomethyl)-2-methoxyphenyl]methyl}-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P4-1)
Figure imgf000181_0001
[0457] To a solution of P1 (0.30 g, 0.81 mmol) in DCM was added phosphorus tribromide (0.38 mL), and the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give P4-1 (0.32 g, 91% yield) as a yellow solid. ESI m/z: 432.1 (M + H)+. EXAMPLE 19 [0458] General procedure VIII to synthesize ethers by alcohols reacting with benzyl halide
Figure imgf000181_0002
[0459] To a solution of alcohol P#-2 (2.0 equiv.) in anhydrous THF or DMF (40 mM) was added sodium hydride (60% in mineral, 2.5 equiv.) in one portion at -15 to 0 oC under nitrogen protection. The resulting suspension was stirred at -15 to 0 oC under nitrogen protection for 10 minutes. To the stirred mixture were then added tetrabutylammonium iodide (TBAI) (0.05 equiv.) and benzyl halide (1.0 equiv.), and the reaction mixture was stirred at -15 to 0 oC for 30 minutes, which was monitored by LCMS. The resulting mixture was quenched with methanol. The mixture (with Boc protection or propylidene protection) was directly used for the next step without further purification. Or the mixture was neutralized with TFA (to pH 6-7) and then concentrated in vacuo. The residual mixture was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to provide desired ester. EXAMPLE 20 [0460] General procedure IX to synthesize P4, P5, P10, P17, P18, P19, P41 and P42 by acidification using TFA
Figure imgf000182_0001
[0461] The quenched reaction mixture containing P#-3 was diluted with DCM (for P4, P5, P10, P41 and P42) or THF (for P17, P18 and P19). To the solution was added TFA (25% vol.), and the reaction mixture was stirred at room temperature for half an hour, which was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to give payload P4, P5, P10, P17, P18, P19, P41 or P42 (20%yield) as a white solid. EXAMPLE 21 [0462] Payload P4 [0463] 5-({4-[(2-Aminoethoxy)methyl]-2-methoxyphenyl}methyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P4)
Figure imgf000183_0001
[0464] Following the general procedures VIII and IX subsequently starting from benzyl bromide P4-1 reacting with N-Boc-aminoethanol P4-2, payload P4 (13 mg, 15% yield, di-TFA salt) was obtained as a white solid. ESI m/z: 413.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.43 (br s, 1H), 7.87 (br s, 3H), 7.50-7.35 (m, 4H), 7.06 (s, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 6.21 (d, J = 2.8 Hz, 1H), 5.54 (s, 2H), 4.50 (s, 2H), 3.48 (s, 3H), 3.70-3.40 (m, 4H), 3.02 (br s, 2H), 1.52-1.42 (m, 2H), 1.27-1.18 (m, 2H), 1.15-1.06 (m, 2H), 0.81 (t, J = 7.2Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 22 [0465] Payload P5 [0466] 5-({4-[(4-Aminobutoxy)methyl]-2-methoxyphenyl}methyl)-N4-pentyl-5H-pyrrolo[3,2- d]pyrimidine-2,4-diamine (P5)
Figure imgf000183_0002
[0467] Following the general procedures VIII and IX subsequently starting from benzyl bromide P4-1 reacting with N-Boc-aminobutanol P5-2, payload P5 (2 mg, 2% yield, formic acid salt) was obtained as a light yellow solid after purification by prep-HPLC (5-95% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 441.5 (M + H)+.1H NMR (400 MHz, MeODd4) δ 7.26 (d, J = 3.0 Hz, 1H), 6.97 (s, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.62 (d, J = 7.6 Hz, 1H), 6.12 (d, J = 3.0 Hz, 1H), 5.42 (s, 2H), 4.51 (br s, 1H), 4.41 (s, 2H), 3.82 (s, 3H), 3.51-3.38 (m, 4H), 2.90-2.81 (m, 2H), 1.70-1.55 (m, 4H), 1.42-1.33 (m, 2H), 1.22-1.13 (m, 2H), 1.10-0.99 (m, 2H), 0.77 (t, J = 7.4 Hz, 3H) ppm. EXAMPLE 23 [0468] Payload P6 [0469] 2-{2-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]ethoxy}ethan-1-ol (P6)
Figure imgf000184_0001
[0470] Following the general procedure VIII starting from benzyl bromide P4-1 reacting with diethylene glycol P6-2, payload P6 (12 mg, 23% yield, TFA salt) was obtained as an off-white solid. ESI m/z: 458.1 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.44 (s, 1H), 7.44 (s, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.34 (t, J = 5.2 Hz, 1H), 7.03 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 5.53 (s, 2H), 4.46 (s, 2H), 3.84 (s, 3H), 3.60-3.34 (11H, covered or partially covered by water peak), 1.52-1.34 (m, 2H), 1.28-1.20 (m, 2H), 1.19-1.00 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.7 ppm. EXAMPLE 24 [0471] Payload P8 [0472] 1-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-ol (P8)
Figure imgf000184_0002
[0473] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with tetraethylene glycol P8-2 (CAS: 112-60-7), payload P8 (36 mg, 6.4% yield, TFA salt) was obtained as an off-white solid. ESI m/z: 546.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.59 (s, 1H), 7.49 (s, 2H), 7.42 (d, J = 3.2 Hz, 1H), 7.34 (t, J = 5.2 Hz, 1H), 7.02 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.55 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.2 Hz, 1H), 5.54 (s, 2H), 4.46 (s, 2H), 3.84 (s, 3H), 3.80 (br s, 1H), 3.58-3.36 (m, 18H), 1.51-1.40 (m, 2H), 1.26-1.18 (m, 2H), 1.18- 1.01 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H) ppm. [0474] Payload P44 [0475] 1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-oic acid (P44)
Figure imgf000185_0001
[0476] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with Hydroxy-PEG3-CH2CO2 tBu (CAS: 518044-31-0), payload P44 (4.6 mg, 13% yield) was obtained as a white solid (tBu group was lost during the reaction). ESI m/z: 560.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.27 (d, J = 3.2 Hz, 1H), 7.00 (s, 1H), 6.80 (d, J = 7.6 Hz, 1H),6.46 (d, J = 7.6 Hz, 1H), 6.28 (s, 1H), 6.02 (d, J = 2.4 Hz, 1H), 5.43 (s, 2H), 4.44 (s, 2H), 3.84 (s, 3H), 3.54-3.50 (m, 17H), 1.42-1.38 (m, 2H), 1.23-1.18 (m, 2H), 1.11-1.05 (m, 2H), 0.80 (t, J = 7.6 Hz, 3H) ppm. [0477] Payload P45 [0478] 2-(2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]ethoxy}ethoxy)acetic acid (P45)
Figure imgf000185_0002
[0479] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with Hydroxy-PEG2-CH2CO2 tBu (CAS: 149299-82-1), payload P45 (3.9 mg, 15% yield) was obtained as a white solid (tBu group was lost during the reaction). ESI m/z: 516.3 (M + H)+. [0480] Payload P46 [0481] 2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]ethoxy}acetic acid (P46)
Figure imgf000185_0003
[0482] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with tert-butyl 2-(2-hydroxyethoxy)acetate (CAS: 287174-32-7), payload P46 (3.2 mg, 12% yield) was obtained as a white solid (tBu group was lost during the reaction). ESI m/z: 472.3 (M + H)+. [0483] Payload P48 [0484] 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]ethan-1-ol (P48)
Figure imgf000186_0001
[0485] Following the general procedure VIII starting from benzyl chloride P3-1 reacting with ethylene glycol (CAS: 107-21-1), payload P48 (5 mg, 10% yield, TFA salt) was obtained as an white solid. ESI m/z: 414.3 (M + H)+. EXAMPLE 25 [0486] Payload P9 [0487] 5-{[4-(13-Azido-2,5,8,11-tetraoxatridecan-1-yl)-2-methoxyphenyl]methyl}-N4-pentyl- 5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P9-3) [0488]
Figure imgf000186_0002
[0489] Following the general procedure VIII starting from benzyl bromide P4-1 reacting with 11-azido-3,6,9-trioxaundecanol P9-2 (CAS: 86770-76-4), compound P9-3 (3.0 g, 45% yield, TFA salt) was obtained as a yellow solid. ESI m/z: 571.5 (M + H)+. [0490] 5-{[4-(13-Amino-2,5,8,11-tetraoxatridecan-1-yl)-2-methoxyphenyl]methyl}-N4- pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P9) [0491]
Figure imgf000186_0003
[0492] To a solution of compound P9-3 (1.7 g, 3.0 mmol) in methanol (50 mL) was added palladium on carbon (containing 10wt% palladium, 0.20 g) under nitrogen. The reaction mixture was stirred at room temperature under hydrogen atmosphere for 2 hours, which was monitored by LCMS. The resulting suspension was filtered through Celite and the filtrate was concentrated in vacuo. The residue was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to give P9 (0.40 g, 20% yield, di-TFA salt) as a light yellow solid. ESI m/z: 545.5 (M + H)+, 273.4 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 12.54 (s, 1H), 7.82 (br s, 3H), 7.46 (s, 2H), 7.40 (d, J = 3.2 Hz, 1H), 7.33 (t, J = 5.6 Hz, 1H), 7.01 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.58 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.2 Hz, 1H), 5.54 (s, 2H), 4.46 (s, 2H), 3.83 (s, 3H), 3.70- 3.45 (16H, partially covered by water peak), 3.04-2.83 (m, 2H), 1.51-1.40 (m, 2H), 1.27-1.20 (m, 2H), 1.20-1.04 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.7 ppm. EXAMPLE 26 [0493] Payload P10 and P43 [0494] 1,1-Dimethylethyl 4-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]-1- piperazinecarboxylate P10-2 was synthesized according to Angew. Chem., Int. Ed., 2012, 51(48), 12000-12004. [0495] tert-Butyl 4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}- 3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazine-1-carboxylate (P43)
Figure imgf000187_0001
[0496] Following the general procedure VIII starting from benzyl bromide P4-1 reacting with P10-2, payload P43 was obtained as a yellow solid. ESI m/z: 358.3 (M/2 + H)+. [0497] 5-({2-Methoxy-4-[13-(piperazin-1-yl)-2,5,8,11-tetraoxatridecan-1-yl]phenyl}methyl)- N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P10)
Figure imgf000187_0002
[0498] Following the general procedure IX starting from P43, payload P10 (22 mg, 16% yield, di-TFA salt) was obtained as a yellow solid. ESI m/z: 308.0 (M/2 + H)+. 1H NMR (400 MHz, DMSOd6) δ 12.67 (s, 1H), 9.39 (br s, 2H), 7.50 (br s, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.34 (t, J = 5.4 Hz, 1H), 7.01 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 5.54 (s, 2H), 4.46 (s, 2H), 3.83 (s, 3H), 3.75-3.25 (m, 26H, covered in water peak), 1.50-1.42 (m, 2H), 1.26-1.19 (m, 2H), 1.19-1.01 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -74.1 ppm. EXAMPLE 27 [0499] Payload P17 [0500] (2R)-3-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]propane-1,2-diol (P17)
Figure imgf000188_0001
[0501] Following the general procedures VIII and IX subsequently starting from benzyl bromide P4-1 reacting with S-glycerol acetonide P17-2, payload P17 (9 mg, 7% yield, TFA salt) was obtained as a light yellow solid. ESI m/z: 444.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.36 (br s, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.40 (s, 2H), 7.32 (t, J = 5.6 Hz, 1H), 7.04 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 5.53 (s, 2H), 4.72 (br s, 1H), 4.54 (br s, 1H), 4.46 (s, 2H), 3.83 (s, 3H), 3.71-3.25 (m, 7H, covered in water peak), 1.51-1.42 (m, 2H), 1.35-1.18 (m, 2H), 1.16-1.02 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 28 [0502] Payload P18 [0503] (2S)-3-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]propane-1,2-diol (P18)
Figure imgf000188_0002
[0504] Following the general procedures VIII and IX subsequently starting from benzyl bromide P4-1 reacting with R-glycerol acetonide P18-2, payload P18 (9 mg, 7% yield, TFA salt) was obtained as a light yellow solid. ESI m/z: 444.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.31 (s, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.38 (s, 2H), 7.31 (t, J = 5.6 Hz, 1H), 7.04 (s, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.58 (d, J = 8.0 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.53 (s, 2H), 4.46 (s, 2H), 3.83 (s, 3H), 3.71-3.25 (m, 9H, covered in water peak), 1.51-1.42 (m, 2H), 1.29-1.20 (m, 2H), 1.20-1.03 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 29 [0505] Payload P19 [0506] 2-Amino-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]propan-1-ol (P19)
Figure imgf000189_0001
[0507] Following the general procedures VIII and IX subsequently starting from benzyl bromide P4-1 reacting with N-Boc-serinol P19-2, payload P19 (13 mg, 6% yield, di-TFA salt) was obtained as a white solid. ESI m/z: 443.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.48 (br s, 1H), 7.96 (s, 3H), 7.45 (s, 2H), 7.43-7.35 (m, 2H), 7.05 (s, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 6.20 (d, J = 2.8 Hz, 1H), 5.54 (s, 2H), 5.32 (br s, 1H), 4.50 (s, 2H), 3.84 (s, 3H), 3.62-3.23 (7H, partially covered by water peak), 1.51-1.44 (m, 2H), 1.29-1.20 (m, 2H), 1.20-1.05 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 30 [0508] Payload P41 [0509] tert-butyl N-[2-(2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}-3-methoxyphenyl)methoxy]ethoxy}ethoxy)ethyl]carbamate (P41-3)
Figure imgf000189_0002
[0510] Following the general procedures VIII starting from benzyl bromide P4-1 reacting with N-Boc-PEG3-OH (P41-2, CAS: 139115-92-7), compound P41-3 (51 mg, 71% yield) was obtained as a white solid. Rt in LC: 2.05 min.; ESI m/z: 601.3 (M + H)+. [0511] 5-{[4-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)-2-methoxyphenyl]methyl}-N4- pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P41)
Figure imgf000190_0001
[0512] Following the general procedures IX starting from P41-3, compound P41 (15 mg, 36% yield) was obtained as a white solid. Rt in LC: 1.38 min.; ESI m/z: 501.1 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 12.63 (s, 1H), 9.07 (s, 2H), 7.50 (s, 2H), 7.44 (t, J = 5.5 Hz, 1H), 7.40 (d, J = 3.0 Hz, 1H), 7.26 (d, J = 1.0 Hz, 1H), 6.98 (d, J = 7.8 Hz, 1H), 6.62 (d, J = 7.8 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.57 (s, 2H), 4.64 (s, 1H), 4.14 (s, 2H), 3.85 (s, 3H), 3.67 (t, J = 5.1 Hz, 2H), 3.56 (s, 4H), 3.50-3.41 (m, 8H), 3.06 (br, 2H), 1.53-1.43 (m, 2H), 1.29-1.19 (m, 2H), 1.17-1.08 (m, 2H), 0.83 (t, J = 7.2 Hz, 3H) ppm. EXAMPLE 31 [0513] Payload P42 [0514] tert-butyl N-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]-N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}carbamate ()
Figure imgf000190_0002
[0515] Following the general procedures VIII starting from benzyl bromide P4-1 reacting with N-Boc-PEG3-OH (P41-2), except using 4 equiv. of sodium hydride (60% in mineral) instead of 2.5 equiv., compound P41-3 (80 mg, 36% yield) and compound P42-3 (20 mg, 9% yield) were obtained separately as white solids after purification by prep-HPLC (5-95% acetonitrile in aq. ammonium bicarbonate (10 mM)). [0516] For 41-3: Rt in LC: 2.05 min.; ESI m/z: 601.3 (M + H)+. [0517] For 42-3: Rt in LCMS: 1.84 min., ESI m/z: 601.3 (M + H)+. [0518] 2-[2-(2-{[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]amino}ethoxy)ethoxy]ethan-1-ol (P42)
Figure imgf000191_0001
[0519] Following the general procedures IX starting from P42-3, compound P42 (10 mg, 13% yield) was obtained as a white solid. Rt in LC: 1.28 min.; ESI m/z: 501.1 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 12.63 (s, 1H), 9.07 (s, 2H), 7.50 (s, 2H), 7.44 (t, J = 5.5 Hz, 1H), 7.40 (d, J = 3.0 Hz, 1H), 7.26 (s, 1H), 6.98 (d, J = 7.8 Hz, 1H), 6.62 (d, J = 7.8 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.57 (s, 2H), 4.64 (s, 1H), 4.14 (s, 2H), 3.85 (s, 3H), 3.67 (t, J = 5.1 Hz, 2H), 3.56 (br, 4H), 3.50-3.42 (m, 6H), 3.06 (br, 2H), 1.53-1.42 (m, 2H), 1.33-1.18 (m, 2H), 1.16-1.06 (m, 2H), 0.83 (t, J = 7.2 Hz, 3H) ppm. EXAMPLE 32 [0520] Payload P11 [0521] 2-Amino-N-{[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}- 3-methoxyphenyl)methoxy]methyl}acetamide (P11)
Figure imgf000191_0002
[0522] To a solution of P1 (0.30 g, 0.81 mmol) in anhydrous THF (5 mL) were added potassium tert-butoxide (0.18 g, 1.6 mmol) and compound P11-1 (CAS: 1599440-06-8) (0.45 g, 1.2 mmol) at 0 oC under nitrogen atmosphere, and the reaction mixture was stirred at 0 oC for an hour, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% methanol in aq. TFA (0.01%)) to give compound P11-2 (0.15 g, ESI m/z: 678.5 (M + H)+) as a yellow solid. [0523] To a solution of compound P11-2 (20 mg, 30 µmol) in DMF (1 mL) was added piperidine (13 mg, 0.15 mmol), and the reaction mixture was stirred at room temperature for 2 hours until Fmoc was totally removed according to LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (5 mg, 8% yield from P1, TFA salt) as a yellow solid. ESI m/z: 456.1 (M + H)+.1H NMR (400 MHz, MeODd4) δ 7.50 (t, J = 5.4 Hz, 0.5H) 7.48 (d, J = 3.0 Hz, 1H), 7.37 (t, J = 5.4 Hz, 1H), 7.03 (s, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 6.37 (d, J = 3.0 Hz, 1H), 5.37 (s, 2H), 5.12 (s, 2H), 4.60 (s, 2H), 3.82 (s, 3H), 3.70 (s, 2H), 3.58-3.51 (m, 2H), 1.73-1.63 (m, 2H), 1.31-1.23 (m, 2H), 1.17-1.07 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, MeODd4) δ -73.6 ppm. EXAMPLE 33 [0524] Payload P12 [0525] 5-[(4-{[(4-Aminophenyl)methoxy]methyl}-2-methoxyphenyl)methyl]-N4-pentyl-5H- pyrrolo[3,2-d]pyrimidine-2,4-diamine (P12)
Figure imgf000192_0001
[0526] To a solution of P1 (20 mg, 54 µmol) in DMF (3 mL) were added N-Boc-4- (bromomethyl)aniline P12-1 (19 mg, 65 µmol), cesium carbonate (35 mg, 0.11 mmol and potassium iodide (3 mg, 18 µmol), and the reaction mixture was stirred at 50 oC for 18 hours, which was monitored by LCMS. The mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound P12-2 (5 mg, ESI m/z: 575.5 (M + H)+) as a yellow solid, which was dissolved in DCM (2.5 mL). To the solution was added TFA (0.5 mL), and the reaction mixture was stirred at room temperature for 3 hours until Boc was totally removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (5-95% acetonitrile in aq. TFA (0.01%)) to give P12 (2 mg, 6% yield from P1, TFA salt) as a white solid. ESI m/z: 475.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.81 (s, 2H), 7.42 (t, J = 5.4 Hz, 1H), 7.40 (d, J = 3.0 Hz, 1H), 7.03 (d, J = 7.2 Hz, 2H), 7.02 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.73-6.61 (m, 3H), 6.44 (d, J = 3.0 Hz, 1H), 5.52 (s, 2H), 5.18 (s, 2H), 4.47 (s, 2H), 3.81 (s, 3H), 3.48-3,45 (m, 2H, partially covered in water peak), 1.53-1.45 (m, 2H), 1.27-1.21 (m, 2H), 1.17-1.10 (m, 2H), 0.82 (t, J = 7.3 Hz, 3H) ppm. (The proton of TFA and 2 protons of aniline were not revealed.) 19F NMR (376 MHz, DMSOd6) δ -73.9 ppm. EXAMPLE 34 [0527] Payload P7 [0528] Methyl 4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3-methoxybenzoate (P7-2)
Figure imgf000193_0001
[0529] To a solution of compound P7-1 (CAS: 79236-96-7) (0.10 g, 0.51 mmol) in pyridine (5 mL) were added tert-butylchlorodiphenylsilane (TBDPS-Cl) (0.14 g, 0.51 mmol) and DMAP (4 mg, 33 µmol), and the reaction mixture was stirred at room temperature for 16 hours, which was monitored by LCMS. The resulting mixture was extracted with ethyl acetate twice and the combined organic solution was washed with brine , dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-35% ethyl acetate in petroleum ether) to give compound P7-2 (0.18 g, 81% yield) as a white solid. ESI m/z: 457.3 (M + Na)+. [0530] (4-{[(tert-Butyldiphenylsilyl)oxy]methyl}-3-methoxyphenyl)methanol (P7-3)
Figure imgf000193_0002
[0531] To a stirred suspension of lithium aluminum tetrahydride (LAH, 8.7 mg, 0.23 mmol) in anhydrous THF (4 mL) was added dropwise a solution of ester P7-2 (0.10 g, 0.23 mmol) in anhydrous THF (1 mL) for 5 minutes at 0 oC under nitrogen protection. The reaction mixture was stirred at 0 oC for 15 minutes and was then stirred at room temperature for 20 minutes until the ester was totally reduced according to LCMS. The resulting mixture was cooled to 0 oC and carefully quenched with two drops of sat. aq. sodium bicarbonate and two drops of water. The mixture was filtered and the filtrate was extracted with ethyl acetate (x 2). The combined organic solution was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel flash chromatography (0- 35% ethyl acetate in petroleum ether) to give P7-3 (60 mg, 64% yield) as colorless oil. ESI m/z: 429.3 (M + Na)+. [0532] {[4-(Bromomethyl)-2-methoxyphenyl]methoxy}(tert-butyl)diphenylsilane (P7-4)
Figure imgf000193_0003
[0533] To a cooled solution of compound P7-3 (2.0 g, 4.9 mmol) in diethyl ether (30 mL) was added phosphorus tribromide (0.67 g, 2.5 mmol) under argon protection, and the reaction mixture was stirred at 0 oC for 3 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0- 100% acetonitrile in water) to give P7-4 (1.0 g, 43% yield) as a solid. ESI m/z: 491.1 (M + Na)+. [0534] tert-Butyl N-(2-{2-[(4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3- methoxyphenyl)methoxy]ethoxy}ethyl)carbamate (P7-6)
Figure imgf000194_0001
[0535] To a cooled (0 oC) solution of 2-(2-Boc-aminoethoxy)ethanol P7-5 (CAS: 139115-91- 6) (34 mg, 0.16 mmol) in anhydrous THF (4 mL) was added sodium hydride (60% in mineral, 9 mg, 0.22 mmol) in two portions under argon atmosphere. The resulting suspension was stirred at 0 oC under argon for 40 minutes. To the mixture were then added TBAI (4.0 mg, 11 µmol) and a solution of P7-4 (50 mg, 0.11 mmol) in THF (1 mL), and the reaction mixture was stirred at 0 oC for half an hour and was then stirred at room temperature for 6 hours, which was monitored by LCMS. The resulting mixture was carefully quenched with water and the volatile were removed in vacuo. The residue was purified by reversed phase flash chromatography (0-100% acetonitrile in water) to give P7-6 (19 mg, 30% yield) as colorless oil. ESI m/z: 616.3 (M + H)+. [0536] tert-Butyl N-[2-(2-{[4-(hydroxymethyl)-3- methoxyphenyl]methoxy}ethoxy)ethyl]carbamate (P7-7)
Figure imgf000194_0002
[0537] To a solution of P7-6 (0.20 g, 0.34 mmol) in anhydrous THF (5 mL) was added a solution of TBAF (1 M in THF, 0.68 mL, 0.68 mmol), and the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.1%)) to give P7-7 (80 mg, 66% yield) as colorless oil. ESI m/z: 378.3 (M + Na)+. [0538] tert-Butyl N-[2-(2-{[4-(bromomethyl)-3- methoxyphenyl]methoxy}ethoxy)ethyl]carbamate (P7-8)
Figure imgf000195_0001
[0539] To a cooled (0 oC) solution of P7-7 (80 mg, 0.23 mmol) in diethyl ether (8 mL) was added phosphorus tribromide (62 mg, 0.23 mmol) under argon, and the reaction mixture was stirred at 0 oC for an hour. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in water) to give P7-8 (50 mg, 52% yield) as oil. ESI m/z: 440.1 (M + Na)+. [0540] tert-Butyl N-[2-(2-{[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxyphenyl]methoxy}ethoxy)ethyl]carbamate (P7-9)
Figure imgf000195_0003
[0541] To a mixture of P7-8 (50 mg, 0.12 mmol) in DMF (4 mL) were added intermediate 6a (20 mg, 0.12 mmol), potassium carbonate (30 mg, 0.24 mmol) and potassium iodide (5 mg, 30 µmol), and the suspension was stirred at room temperature for 18 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.1%)) to give P7-9 (30 mg, 49% yield) as a white solid. ESI m/z: 506.2 (M + H)+. [0542] tert-Butyl N-(2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}-3-methoxyphenyl)methoxy]ethoxy}ethyl)carbamate (P7-10)
Figure imgf000195_0002
[0543] To a microwave tube was charged with P7-9 (60 mg, 0.12 mmol), 1-pentanamine 8a (31 mg, 0.36 mmol), DIPEA (93 mg, 0.72 mmol) and DMSO (4 mL), and the tube was sealed. The reaction mixture was stirred at 120 oC for 8 hours, which was monitored by LCMS. After cooled, the mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.1%)) to give P7-10 (30 mg, 45% yield) as a white solid. ESI m/z: 557.4 (M + H)+. [0544] 5-[(4-{[2-(2-Aminoethoxy)ethoxy]methyl}-2-methoxyphenyl)methyl]-N4-pentyl-5H- pyrrolo[3,2-d]pyrimidine-2,4-diamine (P7)
Figure imgf000196_0001
[0545] To a solution of P7-10 (20 mg, 36 µmol) in DCM (6 mL) was added TFA (1 mL), and the reaction mixture was stirred at room temperature for 4 hours until Boc was totally removed in vacuo according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give P7 (10 mg, 49% yield, di-TFA salt) as a white solid. ESI m/z: 457.4 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 12.36 (br s, 1H), 7.79 (br s, 3H), 7.41 (s, 2H), 7.40 (d, J = 3.0 Hz, 1H), 7.35 (t, J = 5.6 Hz, 1H), 7.01 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 7.6 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.54 (s, 2H), 4.47 (s, 2H), 3.83 (s, 3H), 3.64-3.56 (m, 6H), 3.48-3.44 (m, 2H), 3.04-2.93 (m, 2H), 1.51-1.40 (m, 2H), 1.29-1.17 (m, 2H), 1.14-1.05 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. EXAMPLE 35 [0546] Payload P14 [0547] Ethyl 2-[(4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3-methoxyphenyl)methoxy]acetate (P14-6)
Figure imgf000196_0002
[0548] To a cooled (0 oC) solution of compound P7-3 (0.10 g, 0.23 mmol) in anhydrous DMF (4 mL) was added sodium hydride (60% in mineral, 26 mg, 0.39 mmol) under argon protection, and the suspension was stirred at 0 oC for 30 minutes before the addition of a solution of ethyl bromoacetate (0.15 g, 0.39 mmol) in anhydrous DMF (1 mL). The reaction mixture was stirred at 0 oC for 25 minutes and then stirred at room temperature for 16 hours, which was monitored by LCMS. The resulting mixture was quenched carefully with water at 0 oC and extracted with ethyl acetate twice. The combined organic solution was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel flash chromatography (0-25% ethyl acetate in petroleum ether) to give P14-6 (40 mg, 35% yield) as colorless oil. ESI m/z: 515.2 (M + Na)+. [0549] Ethyl 2-{[4-(hydroxymethyl)-3-methoxyphenyl]methoxy}acetate (P14-7)
Figure imgf000197_0001
[0550] Following the similar procedure as P7-7 except substituting P14-6 (0.20 g, 0.40 mmol) for P7-6, compound P14-7 (60 mg, 59% yield) was obtained as colorless oil. ESI m/z: 277.3 (M + Na)+.1H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 7.5 Hz, 1H), 6.96 (s, 1H), 6.91 (d, J = 7.5 Hz, 1H), 4.68 (s, 2H), 4.63 (s, 2H), 4.24 (q, J = 7.1 Hz, 2H), 4.09 (s, 2H), 3.89 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H) ppm. [0551] Ethyl 2-{[4-(bromomethyl)-3-methoxyphenyl]methoxy}acetate (P14-8)
Figure imgf000197_0002
[0552] Following the similar procedure as P7-8 except substituting P14-7 (30 mg, 0.12 mmol) for P7-7, compound P14-8 (20 mg, 53% yield) was obtained as a white solid. ESI m/z: 340.3 (M + Na)+. [0553] Ethyl 2-{[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxyphenyl]methoxy}acetate (P14-9)
Figure imgf000197_0003
[0554] Following the similar procedure as P7-9 except substituting P14-8 (50 mg, 0.16 mmol) for P7-8, compound P14-9 (30 mg, 47% yield) was obtained as a white solid. ESI m/z: 405.2 (M + H)+. [0555] Ethyl 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]acetate (P14)
Figure imgf000198_0001
[0556] Following the similar procedure as P7-10 except substituting P14-9 (0.10 g, 0.25 mmol) for P7-9, payload P14 (60 mg, 42% yield, TFA salt) was obtained as a white solid. ESI m/z: 456.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.38 (s, 1H), 7.42 (d, J = 3.0 Hz, 3H), 7.33 (t, J = 5.6 Hz, 1H), 7.05 (s, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.57 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.54 (s, 2H), 4.52 (s, 2H), 4.20-4.05 (m, 4H), 3.84 (s, 3H), 3.48-3.41 (m, 2H), 1.50-1.40 (m, 2H), 1.25-1.17 (m, 5H), 1.14-1.04 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 36 [0557] Payload P13 [0558] 2-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]acetic acid (P13)
Figure imgf000198_0002
[0559] To a solution of P14 (20 mg, 44 µmol) in isopropanol (3 mL) was added aq. sodium hydroxide (1N, 60 µL). The reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was neutralized by TFA until pH < 7. The mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.1%)) to give P13 (10 mg, 42% yield, TFA salt) as a white solid. ESI m/z: 428.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.20 (br s, 1H), 7.41 (d, J = 2.8 Hz, 1H), 7.23 (s, 3H), 7.05 (s, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.55 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 2.8 Hz, 1H), 5.53 (s, 2H), 4.51 (s, 2H), 4.04 (s, 2H), 3.84 (s, 3H), 3.46-3.40 (m, 2H), 1.50-1.38 (m, 2H), 1.24- 1.18 (m, 2H), 1.13-1.04 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. EXAMPLE 37 [0560] Payload P15 [0561] 2-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]-N,N-dipropylacetamide (P15)
Figure imgf000199_0001
[0562] To a solution of P13 (3.0 mg, 5.5 µmol) in DMF (2.0 mL) were added HATU (4.0 mg, 10 µmol), dipropylamine (1.0 mg, 10 µmol) and DIPEA (2 mg, 16 µmol), and the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give P15 (2 mg, 58% yield, TFA salt) as a white solid. ESI m/z: 511.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.21 (d, J = 3.0 Hz, 1H), 7.03 (s, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 5.96 (d, J = 2.9 Hz, 1H), 5.64 (t, J = 5.3 Hz, 1H), 5.39 (s, 2H), 5.29 (s, 2H), 4.47 (s, 2H), 4.14 (s, 2H), 3.86 (s, 3H), 3.29-3.25 (m, 2H), 3.20-3.10 (m, 4H), 1.53-1.32 (m, 6H), 1.25-1.19 (m, 2H), 1.13-1.00 (m, 2H), 0.85-0.74 (m, 9H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.6 ppm. EXAMPLE 38 [0563] Payload P16 [0564] 2-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]-1-(piperazin-1-yl)ethan-1-one (P16)
Figure imgf000199_0002
[0565] Following the similar procedure as P15 except substituting N-Boc-piperazine for dipropylamine reaction with P13 (30 mg, 35 µmol), compound P16-1 (20 mg, 48% yield, ESI m/z: 596.2 (M + H)+) was obtained as a white solid, which was dissolved in DCM (6 mL). To the solution was added TFA (1 mL), and the reaction mixture was stirred at room temperature for 4 hours until Boc was totally removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.01%)) to give P16 (10 mg, 22% yield from P13, di-TFA salt) as a white solid. ESI m/z: 496.4 (M + H)+. 1H NMR (400 MHz, DMSO d6) δ 12.70 (br s, 1H), 9.10 (br s, 2H), 7.51 (s, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.36 (t, J = 5.6 Hz, 1H), 7.05 (s, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.58 (d, J = 7.7 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 5.55 (s, 2H), 4.49 (s, 2H), 4.25 (s, 2H), 3.84 (s, 3H), 3.63 (br s, 4H), 3.48-3.44 (m, 2H), 3.11 (br s, 4H), 1.53-1.42 (m, 2H), 1.27-1.17 (m, 2H), 1.15-1.05 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.7 ppm. EXAMPLE 39 [0566] Linker-payload LP1 [0567] 1-(2-{4-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]piperazin-1-yl}ethyl)-2,5-dihydro-1H-pyrrole-2,5-dione (LP1)
Figure imgf000200_0001
[0568] To a stirred solution of payload P3 (0.10 g, 0.23 mmol) in methanol (5 mL) were added 2-maleimidoacetaldehyde (S1a, CAS: 188985-04-8) (0.13 g, 0.92 mmol) and sodium cyanoborohydride (0.19 g, 2.99 mmol) at room temperature, and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting solution was directly purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to give LP1 (10 mg, 6% yield, TFA salt) as a white solid. ESI m/z: 561.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.63 (s, 1H), 7.50 (s, 2H), 7.42 (d, J = 3.0 Hz, 1H), 7.38 (t, J = 5.5 Hz, 1H), 7.17 (s, 1H), 7.04 (s, 2H), 6.92 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 7.7 Hz, 1H), 6.22 (d, J = 3.0 Hz, 1H), 5.56 (s, 2H), 4.06 (s, 2H), 3.85 (s, 3H), 3.77-3.17 (6H, covered by water peak), 3.13-2.66 (m, 8H), 1.50-1.38 (m, 2H), 1.28-1.15 (m, 2H), 1.13-1.04 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. EXAMPLE 40 [0569] Linker-payload LP2 [0570] 1-(3-{4-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]piperazin-1-yl}-3-oxopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione (LP2)
Figure imgf000200_0002
[0571] To a solution of payload P3 (0.10 g, 0.23 mmol) in DMF (5 mL) were added N- Succinimidyl 3-maleimidopropionate (BMPS, CAS: 55750-62-4) (0.12 g, 0.46 mmol) and DIPEA (0.15 g, 1.2 mmol), and the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting solution was directly purified by prep- HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to give LP2 (20 mg, 15% yield, TFA salt) ESI m/z: 589.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.54 (s, 1H), 7.48 (s, 2H), 7.43 (d, J = 3.0 Hz, 1H), 7.41-7.37 (t, J = 5.7 Hz, 1H), 7.20 (s, 1H), 7.02 (s, 2H), 6.94 (d, J = 7.5 Hz, 1H), 6.58 (d, J = 7.7 Hz, 1H), 6.23 (d, J = 3.0 Hz, 1H), 5.58 (s, 2H), 4.19 (br s, 2H), 3.86 (s, 3H), 3.61 (t, J = 7.5 Hz, 2H), 3.55-3.25 (4H, covered by water peak), 3.17-2.85 (br s, 6H), 2.64 (t, J = 7.5 Hz, 2H), 1.50-1.42 (m, 2H), 1.25-1.18 (m, 2H), 1.13-1.06 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.7 ppm. EXAMPLE 41 [0572] Linker-payload LP3 [0573] (2-{4-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]piperazin-1-yl}ethyl)aminyl (LP3-2)
Figure imgf000201_0001
[0574] To a stirred solution of payload P3 (43 mg, 98 µmol) in methanol (5 mL) were added N-Boc-2-aminoacetaldehyde (LP3-1, CAS: 89711-08-0) (48 mg, 0.30 mmol) and sodium cyanoborohydride (63 mg, 1.0 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.05%)) to give Boc-LP3- 2 (45 mg, ESI m/z: 581.5 (M + H)+) as a light yellow solid, which was dissolved in DCM (5 mL). To the solution was added hydrochloride in dioxane (4 N, 5 mL) and the solution was stirred at room temperature for an hour until Boc was totally removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP3-2 (35 mg, 57% yield, 4HCl salt (estimated)) as a yellow solid. ESI m/z: 481.4 (M + H)+. [0575] N-(2-{4-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]piperazin-1-yl}ethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (LP3)
Figure imgf000201_0002
[0576] To a solution of LP3-2 (35 mg, HCl salt, 56 µmol (calc.)) in DMF (1 mL) were added cyclooctyne-O-NHS ester (S1d, CAS: 1425803-45-7) (27 mg, 96 µmol) and DIPEA (70 mg, 0.56 mmol). The reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP3 (25 mg, 34% yield from P3, TFA salt) as a white solid. ESI m/z: 645.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.59 (s, 1H), 7.88 (t, J = 5.3 Hz, 1H), 7.49 (s, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.39 (t, J = 5.6 Hz, 1H), 7.11 (s, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.56 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.55 (s, 2H), 4.29-4.28 (m, 1H), 3.91-3.35 (m, 13H, covered or partially covered by water peak), 3.02-2.97 (br, 8H), 2.28-1.37 (m, 12H), 1.27-1.16 (m, 2H), 1.14-1.03 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -74.06 ppm. EXAMPLE 42 [0577] Linker-payload LP4 [0578] (2R)-2-Amino-3-{[1-(2-{4-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin- 5-yl]methyl}-3-methoxyphenyl)methyl]piperazin-1-yl}ethyl)-2,5-dioxopyrrolidin-3- yl]sulfanyl}propanoic acid (LP4-1)
Figure imgf000202_0001
[0579] To a solution of cysteine (41 mg, 0.34 mmol) in aq. hydrochloride (2 N, 0.50 mL) was added sat. aq. sodium bicarbonate until pH = 7.0. To the aqueous solution was added a solution of LP1 (20 mg, 36 µmol) in acetonitrile (3 mL), the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP4-1 (15 mg, 61% yield, TFA salt) as a white solid. ESI m/z: 682.4 (M + H)+. 1H NMR (400 MHz, MeODd4) δ 7.38 (d, J = 3.0 Hz, 1H), 7.13 (d, J = 0.9 Hz, 1H), 6.93 (d, J = 7.7 Hz, 1H), 6.74 (d, J = 7.7 Hz, 1H), 6.25 (d, J = 3.0 Hz, 1H), 5.53 (s, 2H), 4.18-4.10 (m, 1H), 3.94 (s, 3H), 3.92-3.87 (m, 1H), 3.66 (s, 2H), 3.54 (t, J = 7.1 Hz, 2H), 3.50-3.44 (m, 1H), 3.42- 3.36 (m, 1H), 3.22-3.14 (m, 1H), 3.04-2.95 (m, 1H), 2.93-2.51 (m, 12H), 1.56-1.47 (m, 2H), 1.36-1.28 (m, 2H), 1.23-1.14 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H) ppm. 19F NMR (376 MHz, MeODd4) δ -76.9 ppm. [0580] 3-{[(2R)-2-Amino-2-carboxyethyl]sulfanyl}-3-[(2-{4-[(4-{[2-amino-4-(pentylamino)- 5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methyl]piperazin-1- yl}ethyl)carbamoyl]propanoic acid (LP4-2)
Figure imgf000203_0001
[0581] A solution of LP4-1 (15 mg, 19 µmol) in acetonitrile (1.0 mL) was diluted with PBS buffer (pH 7.4, 2.0 mL), and to the solution was added aq. sodium hydroxide (1 M, 0.5 mL). The reaction mixture was then stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. ammonium bicarbonate (10 mM)) to give LP4-2 (5 mg, 38% yield) as a white solid. ESI m/z: 700.2 (M + H)+. [0582] 3-[(2-{4-[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methyl]piperazin-1-yl}ethyl)carbamoyl]-3-{[(2R)-2-carboxy-2-[2-(cyclooct-2- yn-1-yloxy)acetamido]ethyl]sulfanyl}propanoic acid (LP4)
Figure imgf000203_0002
[0583] To a solution of LP4-2 (5 mg, 7.2 µmol) in DMF (1.0 mL) were added cyclooctyne- O-NHS ester (S1d, CAS: 1425803-45-7) (2.5 mg, 8.6 µmol) and DIPEA (1.5 mg, 12 µmol), and the mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (5-95% acetonitrile in aq. ammonium bicarbonate (10 mM)) to give LP4 (5 mg, 80% yield) as a white solid. ESI m/z: 864.2 (M + H)+. EXAMPLE 43 [0584] Linker-payload LP5 [0585] 2-Amino-N-{[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}- 3-methoxyphenyl)methoxy]methyl}acetamide (LP5-2)
Figure imgf000204_0001
[0586] To a solution of compound P1 (0.30 g, 0.81 mmol) in anhydrous THF (5 mL) were added compound LP5-1 (CAS: 1599440-06-8) (0.45 g, 1.2 mmol) and potassium tert-butoxide (0.18 g, 1.6 mmol) at 0 oC under nitrogen protection, and the reaction mixture was stirred at 0 oC for an hour, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% methanol in aq. formic acid (0.01%)) to give Fmoc-LP5-2 (0.15 g, ESI m/z: 678.4 (M + H)+) as a yellow solid, which was dissolved in DMF. To the solution was added piperidine (94 mg, 1.1 mmol), and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP-2 (80 mg, 17% yield from P1, TFA salt) as a yellow solid. ESI m/z: 456.4 (M + H)+. [0587] 2-[2-(2-{1-[2-(Cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15- amido}acetamido)acetamido]acetic acid (LP5-4)
Figure imgf000204_0002
[0588] To a solution of H-Gly-Gly-Gly-OH (CAS: 556-33-2) (0.17 g, 0.91 mmol) and sodium bicarbonate (0.12 g, 1.4 mmol) in water (2 mL) was added a solution of LP5-3 (CAS: 2101206- 50-0, synthesized as described in WO2020146541) (0.30 g, 0.70 mmol) in THF (2 mL). The reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound LP5-4 (0.25 g, 59% yield) as a yellow solid. ESI m/z: 601.4 (M + H)+. [0589] N-[({[({[({[(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)methoxy]methyl}carbamoyl)methyl]carbamoyl}methyl)carbamoyl]methyl}carb amoyl)methyl]-1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15- amide (LP5)
Figure imgf000205_0001
[0590] To a solution of LP5-4 (13 mg, 0.10 mmol) in anhydrous DMF (2 mL) was added HATU (57 mg, 0.15 mmol), and the reaction mixture was stirred at room temperature for 15 minutes. To the mixture were then added LP5-2 (45 mg, 0.10 mmol) and DIPEA (39 mg, 0.30 mmol), and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (0-100% acetonitrile in aq. ammonium bicarbonate (10 mM)) to give LP5 (10 mg, 18% yield) as a white solid. ESI m/z: 519.9 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 8.21-8.08 (m, 4H), 8.05 (t, J = 5.9 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.24 (d, J = 3.0 Hz, 1H), 7.01 (s, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.47 (d, J = 7.7 Hz, 1H), 6.08 (d, J = 6.6 Hz, 1H), 6.06 (d, J = 3.0 Hz, 1H), 5.78 (t, J = 5.3 Hz, 1H), 5.39 (s, 2H), 5.19 (t, J = 5.6 Hz, 1H), 4.64 (t, J = 5.8 Hz, 2H), 4.45 (d, J = 5.4 Hz, 2H), 4.31-4.21 (m, 1H), 3.85 (s, 3H), 3.79-3.70 (m, 7H), 3.67 (d, J = 5.7 Hz, 1H), 3.60 (t, J = 6.5 Hz, 2H), 3.53-3.45 (m, 12H), 3.42-3.21 (8H, covered or partially covered by water peak), 2.39 (t, J = 6.5 Hz, 2H), 2.27-2.14 (m, 2H), 2.11-2.03 (m, 1H), 1.96-1.89 (m, 1H), 1.88- 1.81 (m, 1H), 1.80-1.69 (m, 2H), 1.65-1.52 (m, 2H), 1.46-1.34 (m, 3H), 1.20 (d, J = 7.2 Hz, 2H), 1.15-1.05 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm. EXAMPLE 44 [0591] General procedure X to synthesize amides from NHS linkers reacting with amines
Figure imgf000205_0002
[0592] To a solution of amine (1.0 equiv.) in DMF (10 mg/mL) were added Y-OSu (1.0 equiv.) and DIPEA (3.0 equiv.), and the mixture was stirred at room temperature for 1-2 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified directly by reserved phase flash chromatography (0-100% acetonitrile in ammonium bicarbonate (10 mM)) to give corresponding linker-payloads as a white solid. EXAMPLE 45 [0593] Linker-payload LP6A [0594] N-[1-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]-2-(piperazin-1-yl)acetamide (LP6-2)
Figure imgf000206_0001
[0595] To a solution of 1-Boc-4-carboxymethyl piperazine (LP6-1, CAS 156478-71-6) (16 mg, 66 µmol) in DMF (2 mL) were added P9 (30 mg, 55 µmol), DIPEA (14 µL, 83 µmol) and HATU (25 mg, 66 µmol), and the reaction mixtue was stirred at room temperature for 20 hours, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to provide crude Boc- LP6-2 (90 mg, ESI m/z: 386.4 (M/2 + H)+ as yellow oil, which was dissolved in DCM (3 mL). To the solution was added TFA (1 mL), and the reaction mixture was stirred at room temperature for half an hour, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)) to give LP6-2 (45 mg, 67% yield, TFA salt) as yellow oil. ESI m/z: 336.4 (M/2 + H)+. [0596] N-[1-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]-2-{4-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethyl]piperazin-1-yl}acetamide (LP6A)
Figure imgf000206_0002
[0597] To a stirred solution of payload LP6-2 (45 mg, 44 µmol, TFA salt) in methanol (10 mL) were added S1a (40 mg, 0.20 mmol) and sodium cyanoborohydride (42 mg, 0.67 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting solution was purified twice by prep-HPLC (5- 95% acetonitrile in aq. TFA (0.05%)) to give LP6A (11 mg, 27% yield, TFA salt) as a light yellow solid. ESI m/z: 397.8 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 12.42 (s, 1H), 8.29 (br s, 1H), 7.43 (s, 2H), 7.40 (d, J = 3.0 Hz, 1H), 7.33 (t, J =5.6 Hz, 1H), 7.06 (s, 2H), 7.01 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.2 Hz, 1H), 5.53 (s, 2H), 4.46 (s, 2H), 3.83 (s, 3H), 3.76-2.69 (32H, covered or partially covered by water peak), 1.49-1.41 (m, 2H), 1.27-1.17 (m, 2H), 1.12-1.04 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.9 ppm. EXAMPLE 46 [0598] Linker-payload LP6B [0599] N-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]-2-[4-(4-{2-azatricyclo[10.4.0.0⁴,⁹]hexadeca- 1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanoyl)piperazin-1-yl]acetamide (LP6B)
Figure imgf000207_0001
[0600] Following the general procedure X starting from LP6-2 (40 mg, 60 µmol) reacting with S1c (CAS: 1353016-71-3, 24 mg, 60 µmol), linker-payload LP6B (21 mg, 37% yield) was obtained as a white solid. ESI-MS (M + H)+: 958.5.1H NMR (400 MHz, DMSOd6) δ 7.78-7.75 (m, 2H), 7.61 (d, J = 6.4 Hz, 1H), 7.49-7.44 (m, 3H), 7.37-7.30 (m, 3H), 7.22 (d, J = 2.8 Hz, 1H), 7.00 (s, 1H), 6.77 (d, J = 7.6 Hz, 1H), 6.42 (d, J = 7.6 Hz, 1H), 5.97 (d, J = 2.8 Hz, 1H), 5.74-5.73 (m, 1H), 5.43-5.39 (m, 3H), 5.04-5.00 (m, 1H), 4.43 (s, 2H), 3.85 (s, 3H), 3.63-3.48 (m, 15H), 3.29-3.22 (m, 8H), 2.88 (s, 2H), 2.67-2.60 (m, 1H), 2.32-2.19 (m, 6H), 2.01-1.99 (m, 1H), 1.77-1.72 (m, 1H), 1.40-1.33 (m, 2H), 1.20-1.15 (m, 2H), 1.09-1.02 (m, 2H), 0.81-0.75 (m, 3H) ppm. EXAMPLE 47 [0601] Linker-payload LP7A [0602] {4-[(2S)-2-[(2S)-2-Amino-3-methylbutanamido]-5- (carbamoylamino)pentanamido]phenyl}methyl N-[1-(4-{[2-amino-4-(pentylamino)-5H- pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]carbamate (LP7-1)
Figure imgf000207_0002
[0603] To a mixture of compound P9 (30 mg, 55 µmol) and Fmoc-vcPAB-PNP (CAS: 863971-53-3) (43 mg, 55 µmol) in DMF (2 mL) was added DIPEA (11 mg, 83 µmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. To the solution was then added piperidine (0.2 mL), and the reaction mixture was stirred at room temperature for an hour until Fmoc was totally removed according to LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP7-1 (30 mg, 51% yield, TFA salt) as a white solid. ESI m/z: 475.9 (M/2 + H)+. [0604] {4-[(2S)-5-(Carbamoylamino)-2-[(2S)-2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)acetamido]-3-methylbutanamido]pentanamido]phenyl}methyl N-[1-(4-{[2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11- tetraoxatridecan-13-yl]carbamate (LP7A)
Figure imgf000208_0001
[0605] Following the general procedure X starting from LP7-1 (30 mg, 28 µmol, TFA salt) reacting with N-succinimidyl maleimidoacetate (S1b) (8.0 mg, 31 µmol), linker-payload LP7A (10 mg, 30% yield, TFA salt) was obtained as a white solid. ESI m/z: 1087.6 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.18 (s, 1H), 10.01 (s, 1H), 8.26 (d, J = 8.9 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.36 (s, 2H), 7.32 (d, J = 5.8 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H), 7.24-7.18 (m, 1H), 7.09 (s, 2H), 7.02 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 5.99 (br s, 1H), 5.53 (s, 2H), 5.40 (br s, 1H), 4.93 (s, 2H), 4.45 (s, 2H), 4.40-4.34 (m, 1H), 4.27-4.23 (m, 1H), 4.13 (s, 2H), 3.83 (s, 3H), 3.59-3.30 (17H, partially covered by water peak), 3.17-3.08 (m, 2H), 3.06-2.88 (m, 2H), 2.03-1.91 (m, 1H), 1.74-1.51 (m, 2H), 1.51-1.40 (m, 4H), 1.27-1.15 (m, 2H), 1.14-1.02 (m, 2H), 0.90-0.76 (m, 9H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.9 ppm. [0606] (2R)-2-amino-3-{[1-({[(1S)-1-{[(1S)-1-({4-[({[1-(4-{[2-amino-4-(pentylamino)-5H- pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]carbamoyl}oxy)methyl]phenyl}carbamoyl)-4-(carbamoylamino)butyl]carbamoyl}-2- methylpropyl]carbamoyl}methyl)-2,5-dioxopyrrolidin-3-yl]sulfanyl}propanoic acid (Qc-LP7A)
Figure imgf000209_0001
[0607] Following the similar procedure as LP4-1 except starting from LP7A, Qc-LP7A (10 mg, 23% yield, TFA salt) was obtained as a white solid. ESI m/z: 1209.3 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 10.05-10.03 (m, 1H), 8.30-8.25 (m, 3H), 7.58 (d, J = 8.0 Hz, 2H), 7.41- 7.40 (m, 3H), 7.34-7.21 (m, 4H), 7.02 (s, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 8.4 Hz, 1H), 6.20 (d, J = 2.8 Hz, 1H), 6.03-6.01 (m, 1H), 5.53 (s, 2H), 5.46-5.44 (m, 1H), 4.93 (br, 2H), 4.45 (br, 2H), 4.39-4.37 (m, 1H), 4.28-4.17 (m, 3H), 4.12 (br, 2H), 3.83 (s, 3H), 3.53-3.38 (m, 17H), 3.28-3.21 (m, 2H), 3.14-3.09 (m, 2H), 3.05-2.94 (m, 3H), 2.64-2.51 (m, 2H), 2.01-1.97 (m, 1H), 1.67-1.58 (m, 2H), 1.45-1.38 (m, 4H), 1.21-1.17 (m, 2H), 1.13-1.11 (m, 2H), 0.86- 0.79 (m, 9H) ppm.
Figure imgf000209_0002
[0608] 3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-3-[({[(1S)-1-{[(1S)-1-({4-[({[1-(4-{[2- amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11- tetraoxatridecan-13-yl]carbamoyl}oxy)methyl]phenyl}carbamoyl)-4- (carbamoylamino)butyl]carbamoyl}-2-methylpropyl]carbamoyl}methyl)carbamoyl]propanoic acid (Qo-LP7A) [0609] Following the similar procedure as LP4-2 except starting from Qc-LP7A, Qo-LP7A (9.8 mg, 39% yield) was obtained as a white solid. ESI m/z: 1227.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 10.00-9.82 (m, 1H), 9.03-9.01 (m, 1H), 8.54-8.52 (m, 1H), 8.41-8.39 (m, 1H), 8.20-8.10 (m, 1H), 7.67-7.62 (m, 2H), 7.31-7.23 (m, 4H), 7.01 (s, 1H), 6.80-6.79 (m, 1H), 6.48- 6.22 (m, 4H), 6.03-6.01 (m, 1H), 5.60-5.44 (m, 4H), 4.91 (br, 2H), 4.44 (br, 2H), 4.26-4.22 (m, 1H), 4.07-4.03 (m, 1H), 3.84 (s, 3H), 3.71-3.66 (m, 2H), 3.53-3.37 (m, 19H), 3.11-3.04 (m, 4H), 2.99-2.94 (m, 2H), 2.67-2.57 (m, 2H), 2.41-2.37 (m, 1H), 2.07-1.96 (m, 1H), 1.75-1.67 (m, 2H), 1.46-1.35 (m, 4H), 1.23-1.16 (m, 2H), 1.09-1.05 (m, 2H), 0.86-0.78 (m, 9H) ppm. EXAMPLE 48 [0610] Linker-payload LP7B [0611] {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0⁴,⁹]hexadeca-1(12),4(9),5,7,13,15- hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-3- methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-[1-(4-{[2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11- tetraoxatridecan-13-yl]carbamate (LP7B)
Figure imgf000210_0001
[0612] To a solution of payload P9 (40 mg, 74 µmol) in DMF (2 mL) were added DIBAC- PEG4-vcPAB-PNP (S1h, CAS: 2226472-28-0) (80 mg, 74 µmol), HOBt (16 mg, 0.12 mmol) and DIPEA (21 mg, 0.16 mmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (5-95% acetonitrile in aq. ammonium bicarbonate (10 mM)) to give linker-payload LP7B (40 mg, 36% yield) as a white solid. ESI m/z: 743.9 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 10.01 (s, 1H), 8.16 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.79 (t, J = 5.6 Hz, 1H), 7.69-7.67 (m, 1H), 7.63-7.58 (m, 3H), 7.52-7.45 (m, 3H), 7.40-7.23 (m, 7H), 7.00 (s, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.43 (d, J = 7.6 Hz, 1H), 5.99 (d, J = 2.8 Hz, 2H), 5.43-5.40 (m, 2H), 5.04-5.01 (m, 1H), 4.93 (br s, 2H), 4.44 (br s, 2H), 4.38-4.37 (m, 1H), 4.25-4.21 (m, 1H), 3.85 (s, 3H), 3.63-3.55 (m, 3H), 3.52-3.44 (m, 26H), 3.31-3.22 (m, 6H), 3.14-2.92 (m, 7H), 2.68-2.67 (m, 2H), 2.40-2.20 (m, 5H), 2.03-1.93 (m, 2H), 1.79-1.57 (m, 4H), 1.41-1.34 (m, 2H), 1.23-1.15 (m, 2H), 1.09-1.03 (m, 2H), 0.86-0.78 (m, 9H) ppm. EXAMPLE 49 [0613] Linker-payload LP7C [0614] {4-[(2S)-2-[(2S)-2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-3- methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-[1-(4-{[2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11- tetraoxatridecan-13-yl]carbamate (LP7C)
Figure imgf000211_0001
[0615] Following the general procedure X starting from LP7-1 (30 mg, 28 µmol, TFA salt) reacting with N-Fmoc-PEG4-OSu (S1f) (18 mg, 31 µmol), the reaction solution of Fmoc-LP7C (ESI m/z: 1419.7 (M + H)+) in DMF (2 mL) was obtained, to which was added piperidine (0.2 mL, excess). The reaction mixture was stirred at room temperature for an hour until Fmoc was totally removed, which was monitored by LCMS. The resulting mixture was directly separated by prep-HPLC (0-100% acetonitrile in aq. TFA (0.01%)) to give linker-payload LP7C (20 mg, 60% yield) as a white solid. ESI m/z: 599.6 (M/2 + H)+.1H NMR (500 MHz, DMSOd6) δ 12.33 (s, 1H), 10.01 (s, 1H), 8.15 (d, J = 7.1 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.77 (s, 3H), 7.59 (d, J = 8.2 Hz, 2H), 7.41 (s, 2H), 7.31 (d, J = 15.1 Hz, 1H), 7.28-7.22 (m, 3H), 7.02 (s, 1H), 6.82 (d, J = 7.5 Hz, 1H), 6.55 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 2.6 Hz, 1H), 6.04-6.00 (m, 1H), 5.53 (s, 2H), 5.45 (s, 1H), 4.93 (s, 2H), 4.45 (s, 2H), 4.38 (d, J = 4.7 Hz, 1H), 4.29-4.14 (m, 1H), 3.83 (s, 3H), 3.65-3.50 (m, 28H), 3.41-3.30 (m, 4H), 3.17-3.07 (m, 2H), 3.01-2.94 (m, 4H), 2.44-2.26 (m, 3H), 1.95-1.90 (m, 1H), 1.79-1.49 (m, 2H), 1.54-1.33 (m, 3H), 1.24-1.18 (m, 2H), 1.09-1.06 (m, 2H), 0.92-0.70 (m, 9H) ppm. EXAMPLE 50 [0616] Linker-payload LP7D [0617] 2,5-dioxopyrrolidin-1-yl 1-{2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetamido}- 3,6,9,12-tetraoxapentadecan-15-oate (S1g)
Figure imgf000211_0002
[0618] To a mixture of compound S1e (CAS: 1644644-96-1, 0.46 g, 1.4 mmol) and amino- PEG4-acid (CAS: 663921-15-1, 0.34 g, 1.3 mmol) in DMF (5 mL) was added DIPEA (0.45 g, 3.5 mmol), and the reaction mixture was stirred at 15 oC for 2 hours, which was monitored by LCMS. The resulting mixture was quenched with water (20 mL) and extracted with DCM (3 x 20 mL). The combined organic solution was washed with brine (20 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by prep-HPLC (5-95% acetonitrile in aq. formic acid (0.1%)) twice to give the acid as a red solid (107 mg, ESI m/z: 500.2 (M + Na)+, 478.3 (M + H)+), which was dissolved in DCM (4 mL). To the solution were added HOSu (48 mg, 0.42 mmol) and EDCI (80 mg, 0.42 mmol), and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (30-50% acetonitrile in aq. TFA (0.01%)) to give compound S1g (76 mg, 10% yield) as a red solid. ESI m/z: 597.2 (M + Na)+. [0619] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-3-methyl-2-(1-{2-[4-(6-methyl-1,2,4,5-tetrazin- 3-yl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)butanamido]pentan- amido]phenyl}methyl N-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]carbamate (LP7D)
Figure imgf000212_0001
[0620] Following the general procedure X starting from LP7-1 (40 mg, 38 µmol, TFA salt) reacting with compound S1g (24 mg, 42 µmol), linker-payload LP7D (12 mg, 23% yield) was obtained as a red solid. ESI m/z: 705.4 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 12.11 (s, 1H), 9.99 (s, 1H), 8.40 (d, J = 8.3 Hz, 2H), 8.27 (t, J = 5.5 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.36-7.30 (m, 3H), 7.27 (d, J = 8.5 Hz, 2H), 7.22 (t, J = 7.7 Hz, 1H), 7.01 (s, 1H), 6.82 (d, J = 7.7 Hz, 1H), 6.56 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 6.00 (t, J = 8.5 Hz, 1H), 5.53 (s, 2H), 5.43 (s, 2H), 4.93 (s, 2H), 4.45 (s, 2H), 4.40-4.37 (m, 1H), 4.24-4.21 (m, 1H), 3.83 (s, 3H), 3.57-3.56 (m, 6H), 3.52-3.48 (m, 24H), 3.33 (s, 2H), 3.23 (d, J = 5.7 Hz, 2H), 3.14-3.10 (m, 2H), 2.99 (s, 3H), 2.95-2.91 (m, 1H), 2.40-2.37 (m, 1H), 2.02-1.92 (m, 3H), 1.70-1.67 (m, 1H), 1.61-1.58 (m, 1H), 1.48-1.40 (m, 4H), 1.24-1.17 (m, 4H), 1.13-1.02 (m, 4H), 0.86-0.78 (m, 9H) ppm. EXAMPLE 51 [0621] Linker-payload LP10A [0622] N-[1-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)acetamide (LP10A)
Figure imgf000213_0001
[0623] Following the general procedure X starting from P9 (20 mg, 36 µmol) reacting with S1b (10 mg, 40 µmol), linker-payload LP10A (16 mg, 56% yield, TFA salt) was obtained as a white solid. ESI m/z: 682.5 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.19 (s, 1H), 8.23 (t, J = 5.1 Hz, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.37 (s, 2H), 7.34 (m, 1H), 7.09 (s, 2H), 7.02 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.53 (s, 2H), 4.46 (s, 2H), 4.01 (s, 2H), 3.83 (s, 3H), 3.59-3.33 (16H, partially covered by water peak), 3.23-3.15 (m, 2H), 1.50-1.38 (m, 2H), 1.26–1.16 (m, 2H), 1.12-1.01 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm. 19F NMR (376 MHz, DMSOd6) δ -73.8 ppm. EXAMPLE 52 [0624] Linker-payload LP10B [0625] N-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]-4-{2-azatricyclo[10.4.0.0⁴,⁹]hexadeca- 1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanamide (LP10B)
Figure imgf000213_0002
[0626] Following the general procedure X starting from P9 (40 mg, 74 µmol) reacting with S1c (30 mg, 75 µmol), linker-payload LP10B (23 mg, 37% yield) was obtained as a white solid. ESI m/z: 832.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.79 (t, J = 5.6 Hz, 1H), 7.69- 7.67 (m, 1H), 7.63-7.61 (m, 1H), 7.51-7.45 (m, 3H), 7.39-7.28 (m, 3H), 7.22 (d, J = 2.8 Hz, 1H), 7.00 (s, 1H), 6.77 (d, J = 7.6 Hz, 1H), 6.41 (d, J = 7.6 Hz, 1H), 5.97 (d, J = 2.4 Hz, 1H), 5.71 (t, J = 5.2 Hz, 1H), 5.39 (s, 3H), 5.04-5.01 (m, 1H), 4.43 (s, 2H), 3.85 (s, 3H), 3.62-3.43 (m, 13H), 3.28-3.27 (m, 5H), 3.10-3.04 (m, 2H), 2.60-2.54 (m, 1H), 2.27-2.20 (m, 1H), 2.03- 1.97 (m, 1H), 1.78-1.73 (m, 1H), 1.40-1.33 (m, 2H), 1.23-1.16 (m, 2H), 1.09-1.03 (m, 2H), 0.79 (t, J =7.2 Hz, 3H) ppm. EXAMPLE 53 [0627] Linker-payload LP11A [0628] 1-(2-{4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazin-1-yl}ethyl)-2,5-dihydro-1H-pyrrole- 2,5-dione (LP11A)
Figure imgf000214_0001
[0629] Following the similar procedure as LP6A except using P10 (0.13 g, 0.22 mmol) instead of P9, linker-payload LP11A (42 mg, 26% yield) was obtained as a light-yellow solid. ESI m/z: 369.4 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.53 (s, 1H), 7.46 (br s, 2H), 7.40 (d, J = 6.8 Hz, 1H), 7.34 (t, J = 5.6 Hz, 1H), 7.03 (s, 2H), 7.01 (s, 1H), 6.82 (d, J = 7.2 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 2.4 Hz, 1H), 5.53 (s, 2H), 4.45 (s, 2H), 3.83 (s, 3H), 3.70 (t, J = 5.2 Hz, 2H), 3.74-3.39 (m, 24H), 3.25-3.18 (m, 2H), 2.68-2.58 (m, 2H), 1.49-1.40 (m, 2H), 1.26-1.17 (m, 2H), 1.12-1.04 (m, 2H), 0.80 (t, J = 7.2Hz, 3H) ppm. [0630] Linker-payload LP11A alternative synthesis [0631] tert-butyl 4-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)piperazine-1-carboxylate (11A-2)
Figure imgf000214_0002
[0632] To a solution of N-Boc-piperazine (2.2 g, 12 mmol) in acetonitrile (90 mL) was added K2CO3 (6.0 g, 43 mmol), and the mixture was stirred at 80 oC for 10 minutes. To the mixture was added a solution of compound 11A-1 (3.0 g, 12 mmol) in acetonitrile (10 mL) in 10 minutes at 80 oC, and the mixture was then stirred at this temperature for 12 hours, which was monitored by LCMS. After cooled to room temperature, the resulting suspension was filtered and the filtrate was concentrated. The residue was purified by silica gel flash chromatography (DCM/MeOH, v/v = 10) to give 11A-2 (2.8 g, 66% yield) as yellow oil. ESI m/z: 363.1 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 5.76 (s, 1H), 3.51-3.47 (m, 12H), 3.42-3.40 (m, 2H), 3.28-3.27 (m, 4H), 2.48-2.45 (m, 2H), 2.36-2.34 (m, 4H), 1.39 (s, 9H) ppm. [0633] 4-(13-{4-[(tert-butoxy)carbonyl]piperazin-1-yl}-2,5,8,11-tetraoxatridecan-1-yl)-2- methoxybenzoic acid (11A-4)
Figure imgf000215_0001
[0634] To a solution of compound 11A-2 (3.2 g, 8.8 mmol) in dry THF (100 mL) was added sodium hydride (60% in mineral oil, 0.71 g, 18 mmol) in one portion at 0 oC under nitrogen, and the reaction suspension was stirred at 0 oC under nitrogen for 10 minutes. To the mixture were then added TBAI (0.23 g, 0.88 mmol) and compound 11A-3 (2.3 g, 8.8 mmol), and the reaction mixture was stirred at 0 oC for 3 hours, which was monitored by LCMS. The resulting mixture was quenched with water (5 mL) at 0 oC. The volatiles were removed in vacuo, and the mixture was dissolved in methanol (10 mL). To the solution was added sodium hydroxide (1.1 g, 27 mmol), and the mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residual aqueous solution was acidified with diluted HCl (1 M) to pH7. The solution was directly separated by reserved phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give compound 11A- 4 (2.9 g, 64% yield) as yellow oil. ESI m/z: 527.3 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 7.27 (d, J = 7.6 Hz, 1H), 6.86 (s, 1H), 6.78 (d, J = 7.2 Hz, 1H), 4.45 (s, 2H), 3.71 (s, 3H), 3.58- 3.48 (m, 14H), 3.27-3.26 (m, 4H), 2.47-2.44 (m, 2H), 2.35-2.33 (m, 4H), 1.38 (s, 9H) ppm. [0635] tert-butyl 4-{1-[4-(hydroxymethyl)-3-methoxyphenyl]-2,5,8,11-tetraoxatridecan-13- yl}piperazine-1-carboxylate (11A-5)
Figure imgf000215_0002
[0636] To a solution of compound 11A-4 (0.10 g, 0.19 mmol) in dry THF (10 mL) were added 4-methylmorpholine (58 mg, 0.57 mmol) and Isobutyl chloroformate (39 mg, 0.29 mmol) at 0 oC under nitrogen, and the mixture was stirred at 0 oC under nitrogen for an hour. To the reaction mixture were then added sodium borohydride (22 mg, 0.57 mmol) and THF (2 mL) at 0 oC, and the resulting mixture was allowed to warm to room temperature and stirred at room temperature under nitrogen for an hour, which was monitored by LCMS. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic solution was washed with brine (150 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by reserved phase flash chromatography (5- 95% acetonitrile in aq. TFA (0.05%)) to give compound 11A-5 (61 mg, 63% yield) as yellow oil. ESI m/z: 513.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.32 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 6.0 Hz, 1H), 4.98-4.95 (m, 1H), 4.47 (br, 4H), 3.77 (s, 3H), 3.58-3.46 (m, 14H), 3.28-3.26 (m, 4H), 2.47-2.44 (m, 2H), 2.35-2.32 (m, 4H), 1.38 (s, 9H) ppm. [0637] tert-butyl 4-{1-[4-(chloromethyl)-3-methoxyphenyl]-2,5,8,11-tetraoxatridecan-13- yl}piperazine-1-carboxylate (11A-6)
Figure imgf000216_0001
[0638] To a solution of compound 11A-5 (99 mg, 0.19 mmol) in dry DCM (10 mL) were added mesyl chloride (43 mg, 0.38 mmol) and DIPEA (74 mg, 0.57 mmol) at 0 oC, and the mixture was stirred at room temperature under nitrogen for 12 hours, which was monitored by LCMS. The resulting mixture was quenched with water (20 mL) at 0 oC, and was extracted with DCM (50 mL x 2). The combined organic solution was washed with brine (150 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound 11A-6 (67 mg, 65% yield) as yellow oil. ESI m/z: 531.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 7.35 (d, J = 7.6 Hz, 1H), 6.99 (s, 1H), 6.90 (d, J = 8.0 Hz, 1H), 4.69 (br, 2H), 4.50 (br, 2H), 3.84 (s, 3H), 3.59- 3.55 (m, 4H), 3.52-3.46 (m, 10H), 3.28-3.27 (m, 4H), 2.47-2.44 (m, 2H), 2.34-2.33 (m, 4H), 1.38 (s, 9H) ppm. [0639] tert-butyl 4-{1-[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3- methoxyphenyl]-2,5,8,11-tetraoxatridecan-13-yl}piperazine-1-carboxylate (11A-8)
Figure imgf000216_0002
[0640] To a solution of compound 11A-6 (20 mg, 38 µmol) in DMF (3 mL) were successively added compound 11A-7 (6.4 mg, 38 µmol), potassium iodide (6.3 mg, 38 µmol) and potassium carbonate (16 mg, 0.12 mmol), and the reaction mixture was stirred at 80 oC under nitrogen for 3 hours, which was monitored by LCMS. After cooled to room temperature, the reaction mixture was diluted with ethyl acetate (50 mL) and water (20 mL). The aqueous layer was extracted with ethyl acetate (50 mL). The combined organic solution was washed with brine (150 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound 11A- 8 (14 mg, 56% yield) as yellow oil. ESI m/z: 663.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 8.36 (s, 1H), 7.29 (d, J = 3.2 Hz, 1H),7.00 (s, 1H), 6.79 (d, J = 7.6 Hz, 1H), 6.69 (br, 1H), 6.46 (d, J = 8.0 Hz, 1H), 6.39-6.37 (m, 1H), 6.03 (d, J = 2.8 Hz, 1H), 5.45 (s, 2H), 4.44 (br, 2H), 3.84 (s, 3H), 3.55-3.45 (m, 16H), 3.26-3.25 (m, 4H), 2.45-2.42 (m, 2H), 2.34-2.31 (m, 4H), 1.42-1.40 (m, 2H), 1.37 (s, 9H), 1.23-1.17 (m, 2H), 1.10-1.04 (m, 2H), 0.80 (t, J = 7.2 Hz, 3H) ppm. [0641] 1-(2-{4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazin-1-yl}ethyl)-2,5-dihydro-1H-pyrrole- 2,5-dione (LP11A)
Figure imgf000217_0001
[0642] Following the same procedures from P43 to P10 as described in scheme 6 and from P10 to LP11A as described in scheme 14A, linker-payload LP11A (0.26 g, 42% yield) was obtained as a white solid. ESI m/z: 369.3 (M/2 + H)+. [0643] (2R)-2-amino-3-{[1-(2-{4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2- d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazin-1- yl}ethyl)-2,5-dioxopyrrolidin-3-yl]sulfanyl}propanoic acid (Qc-LP11A)
Figure imgf000217_0002
[0644] Following the similar procedure as LP4-1 except starting from LP11A, Qc-LP11A (8.4 mg, 34% yield, formic acid salt) was obtained as a white solid. ESI m/z: 858.3 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 8.28 (s, 2H), 7.32-7.30 (m, 1H), 7.01 (s, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.47 (d, J = 7.6 Hz, 1H), 6.05 (s, 1H), 5.47 (s, 2H), 4.45 (br, 2H), 3.84 (s, 3H), 3.53- 3.36 (m, 27H), 2.37-2.33 (m, 12H), 1.43-1.39 (m, 2H), 1.23-1.18 (m, 2H), 1.08-1.05 (m, 2H), 0.80 (t, J = 7.6 Hz, 3H) ppm. [0645] 3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-3-[(2-{4-[1-(4-{[2-amino-4-(pentylamino)- 5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]piperazin-1-yl}ethyl)carbamoyl]propanoic acid (Qo-LP11A)
Figure imgf000218_0001
[0646] Following the similar procedure as LP4-2 except starting from Qc-LP7A, Qo-LP11A (83 mg, 62% yield) was obtained as a white solid. ESI m/z: 438.7 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 7.79-7.77 (m, 1H), 7.34-7.32 (m, 1H), 7.01 (s, 1H), 6.83(d, J = 7.2 Hz, 1H), 6.52-6.49 (m, 1H), 6.11-6.07 (m, 1H), 5.50 (d, J = 4.8 Hz, 2H), 4.47 (br, 2H), 3.84 (s, 3H), 3.54-3.39 (m, 22H), 3.25-3.02 (m, 5H), 2.67-2.66 (m, 1H), 2.41-2.22 (m, 13H), 1.45-1.41 (m, 2H), 1.24-1.17 (m, 2H), 1.10-1.07 (m, 2H), 0.81 (t, J = 7.6 Hz, 3H) ppm. EXAMPLE 54 [0647] Linker-payload LP11B [0648] 1-{4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazin-1-yl}-4-{2- azatricyclo[10.4.0.0⁴,⁹]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}butane-1,4-dione (LP11B)
Figure imgf000218_0002
[0649] Following the general procedure X starting from P10 (15 mg, 24 µmol) reacting with S1c, linker-payload LP11B (7.8 mg, 35% yield) was obtained as a white solid. ESI m/z: 451.2 (M/2 + H)+. EXAMPLE 55 [0650] Linker-payload LP11C [0651] 1-{4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazin-1-yl}-2-(cyclooct-2-yn-1- yloxy)ethan-1-one (LP11C)
Figure imgf000219_0001
[0652] Following the general procedure X starting from P10 (15 mg, 24 µmol) reacting with S1d, linker-payload LP11C (13 mg, 69% yield) was obtained as a white solid. ESI m/z: 389.7 (M/2 + H)+. EXAMPLE 56 [0653] Linker-payload LP11D [0654] 1-{4-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]piperazin-1-yl}-2-[4-(6-methyl-1,2,4,5- tetrazin-3-yl)phenyl]ethan-1-one (LP11D)
Figure imgf000219_0002
[0655] Following the general procedure X starting from P10 (15 mg, 24 µmol) reacting with S1e, linker-payload LP11D (10 mg, 50% yield) was obtained as a red solid. ESI m/z: 413.7 (M/2 + H)+. EXAMPLE 57 [0656] Linker-payload LP7E [0657] (4S)-4-amino-4-{[(1S)-1-{[(1S)-1-({4-[({[1-(4-{[2-amino-4-(pentylamino)-5H- pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]carbamoyl}oxy)methyl]phenyl}carbamoyl)-4-(carbamoylamino)butyl]carbamoyl}-2- methylpropyl]carbamoyl}butanoic acid (LP12-1)
Figure imgf000220_0001
[0658] Following the similar procedure as LP7-1 except using N-Fmoc-EvcPAB-PNP (CAS: 2395887-68-8) instead of N-Fmoc-vcPAB-PNP, linker-payload LP12-1 (43 mg, 40% yield) was obtained as a yellow solid. ESI m/z: 1079.6 (M + H)+.1H NMR (400 MHz, DMSOd6) for Fmoc-LP12-1 δ 12.18 (s, 1H), 11.07 (s, 1H), 10.05 (s, 1H), 8.23-8.21 (m, 1H), 8.14-8.10 (m, 1H), 7.90-7.88 (m, 2H), 7.75-7.70 (m, 3H), 7.63-7.55 (m, 3H), 7.43-7.39 (m, 3H), 7.34-7.31 (m, 3H), 7.27-7.21 (m, 2H), 7.01 (s, 1H), 6.95-6.93 (m, 2H), 6.83-6.81 (m, 1H), 6.56-6.54 (m, 1H), 6.21 (d, J = 2.8 Hz, 1H), 5.99-5.98 (m, 1H), 5.53 (s, 2H), 4.93 (s, 1H), 4.45 (s, 2H), 4.32- 4.20 (m, 4H), 4.11-4.08 (m, 1H), 3.83 (s, 3H), 3.54-3.48 (m, 17H), 3.14-2.91 (m, 4H), 2.68- 2.67 (m, 1H), 2.33-2.32 (m, 1H), 2.28-2.25 (m, 2H), 2.00-1.88 (m, 2H), 1.77-1.56 (m, 4H), 1.48-1.36 (m, 4H), 1.26-1.17 (m, 2H), 1.12-1.06 (m, 2H), 0.86-0.78 (m, 9H) ppm. [0659] (4S)-4-{[(1S)-1-{[(1S)-1-({4-[({[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2- d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]carbamoyl}oxy)methyl]phenyl}carbamoyl)-4-(carbamoylamino)butyl]carbamoyl}-2- methylpropyl]carbamoyl}-4-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido]butanoic acid (LP7E)
Figure imgf000221_0001
[0660] Following the general procedure X starting from LP12-1 (38 mg, 35 µmol) reacting with S1b, linker-payload LP7E (12 mg, 25% yield) was obtained as a white solid. ESI m/z: 1216.6 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.18 (s, 1H), 10.04 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 6.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.42-7.41 (m, 1H), 7.36-7.32 (m, 3H), 7.28-7.25 (m, 2H), 7.23-7.20 (m, 1H), 7.09 (s, 2H), 7.02 (s, 1H), 6.83-6.81 (m, 1H), 6.56 (d, J = 8.0 HZ, 1H), 6.21-6.20 (m, 1H), 6.00-5.90 (m, 1H), 5.53 (s, 2H), 5.44-5.43 (m, 1H), 4.93 (s, 2H), 4.45 (s, 2H), 4.40-4.34 (m, 2H), 4.19-4.15 (m, 1H), 4.08 (d, J = 2.8 Hz, 2H), 3.83 (s, 3H), 3.53-3.44 (m, 14H), 3.14-3.10 (m, 2H), 3.04-3.01 (m, 1H), 2.96- 2.93 (m, 1H), 2.67-2.66 (m, 1H), 2.33-2.32 (m, 1H), 2.25-2.20 (m, 2H), 1.99-1.85 (m, 3H), 1.74-1.58 (m, 4H), 1.48-1.41 (m, 3H), 1.24-1.19 (m, 2H), 1.12-1.06 (m, 2H), 0.86-0.79 (m, 9H) ppm. EXAMPLE 58 [0661] Linker-payload LP14 [0662] (2R)-2-amino-2-{[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]carbamoyl}ethane-1-sulfonic acid (LP14-1)
Figure imgf000221_0002
[0663] To a solution of payload P9 (95 mg, 0.17 mmol) in DMF (5 mL) were added N-Fmoc- cysteine (66 mg, 0.17 mmol), HATU (97 mg, 0.26 mmol) and DIPEA (66 mg, 0.51 mmol), and the reaction mixture was stirred at room temperature for 3 hours, which was monitored by LCMS. The resulting mixture was separated by reversed phase flash chromatography (5-95% acetonitrile in aq. TFA (0.05%)) to give Fmoc-LP14-1 (97 mg, ESI m/z: 918.4 (M + H)+) as a yellow solid, which was dissolved in DMF (2 mL). To the solution was added diethylamine (0.2 mL), and the reaction mixture was stirred at room temperature for an hour until Fmoc was totally removed according to LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give LP14-1 (44 mg, 32% yield, TFA salt) as a yellow solid. ESI m/z: 696.3 (M + H)+. [0664] (2R)-2-{[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]carbamoyl}-2-[2-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)acetamido]ethane-1-sulfonic acid (LP14)
Figure imgf000222_0001
[0665] Following the general procedure X starting from LP14-1 (44 mg, 54 µmol, TFA salt) reacting with S1b, linker-payload LP14 (6 mg, 12% yield, TFA salt) was obtained as a white solid. ESI m/z: 833.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 12.12 (s, 1H), 8.34 (d, J = 6.4 Hz, 1H), 7.85-7.83 (m, 1H), 7.42 (d, J = 2.8 Hz, 1H), 7.35-7.32 (m, 2H), 7.08 (s, 2H), 7.02 (s, 2H), 6.83 (d, J = 7.2 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.21 (d, J = 3.2 Hz, 1H), 5.53 (s, 2H), 4.46 (s, 2H), 4.36-4.34 (m, 1H), 4.11-3.99 (m, 2H), 3.83 (s, 3H), 3.54-3.45 (m, 17H), 3.17-3.12 (m, 2H), 2.86-2.81 (m, 1H), 2.74-2.69 (m, 1H), 1.48-1.44 (m, 2H), 1.25-1.19 (m, 2H), 1.13- 1.07 (m, 2H), 0.81 (t, J =7.2 Hz, 3H) ppm. EXAMPLE 59 [0666] Linker-payload LP8A [0667] (2S)-2-(2-{2-[2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1- yl)acetamido]acetamido}acetamido)-3-phenylpropanoic acid (LP8-1)
Figure imgf000222_0002
[0668] To a solution of H-Gly-Gly-Phe-OH (40 mg, 0.14 mmol) in DMF (2 mL) were added AMAS (LP7-2) (36 mg, 0.14 mmol) and DIPEA (27 mg, 0.21 mmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP8-1 (35 mg, 60% yield) as a white solid. ESI m/z: 417.2 (M + H)+. [0669] 2,5-Dioxopyrrolidin-1-yl (2S)-2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)acetamido]acetamido}acetamido)-3-phenylpropanoate (LP8-2)
Figure imgf000223_0001
[0670] To a solution of compound LP8-2 (35 mg, 84 µmol) in DCM (2 mL) were added EDCI (CAS: 7084-11-9) (32 mg, 0.168 mmol) and N-hydroxysuccinimide (CAS: 6066-82-6) (19 mg, 0.17 mmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP8-2 (25 mg, 60% yield) as a white solid. ESI m/z: 514.3 (M + H)+. [0671] 2-Amino-N-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}- 3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]acetamide (Gly-P9)
Figure imgf000223_0002
[0672] To a solution of Boc-Glycine (CAS: 4530-20-5) (9.0 mg, 51 µmol) in DMF (2 mL) were added HATU (29 mg, 75 µmol), P9 (27 mg, 50 µmol) and DIPEA (13 mg, 0.10 mmol) successively, and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give Boc-Gly-P9 (20 mg, crude, containing Gly-P9) as semi-solid, which was dissolved in DCM (5 mL). To the solution was added TFA (1 mL), and the reaction mixture was stirred at room temperature for 2 hours until Boc was totally removed according to LCMS. The volatiles were removed in vacuo to give crude Gly-P9 (20 mg, TFA salt) as yellow oil, which was used for the next step without further purification. ESI m/z: 301.8 (M/2 + H)+, 602.4 (M + H)+. [0673] (2S)-N-({[1-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]carbamoyl}methyl)-2-(2-{2-[2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetamido]acetamido}acetamido)-3-phenylpropanamide (LP8A)
Figure imgf000224_0001
[0674] To a solution of crude Gly-P9 (20 mg, TFA salt) in DMF (2 mL) were added LP8-2 (20 mg, 39 µmol) and DIPEA (8.0 mg, 62 µmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (0-100% acetonitrile in aq. TFA (0.01%)) to give linker-payload LP8A (9.2 mg, 17% yield from P9, TFA salt) as a white solid. ESI m/z: 501.0 (M/2 + H)+. 1H NMR (400 MHz, DMSOd6) δ 12.21 (s, 1H), 8.46 (t, J = 5.7 Hz, 1H), 8.27 (t, J = 5.9 Hz, 1H), 8.20- 8.09 (m, 2H), 7.77 (t, J = 5.5 Hz, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.38 (s, 2H), 7.34 (t, J = 5.4 Hz, 1H), 7.29-7.15 (m, 5H), 7.09 (s, 2H), 7.02 (s, 1H), 6.82 (d, J = 7.1 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 3.0 Hz, 1H), 5.53 (s, 2H), 4.53-4.45 (m, 1H), 4.45 (s, 2H), 4.10 (s, 2H), 3.83 (s, 3H), 3.79-3.56 (m, 8H), 3.56-3.35 (14H, partially covered by water peak), 3.24-3.17 (m, 2H), 3.05-2.99 (m, 1H), 2.81-2.72 (m, 1H), 1.52-1.39 (m, 2H), 1.28-1.16 (m, 2H), 1.13- 1.03 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. EXAMPLE 60 [0675] Linker-payload LP8B [0676] tert-butyl 2-[(2S)-2-(2-{2-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12- tetraoxapentadecan-15-amido]acetamido}acetamido)-3-phenylpropanamido]acetate (LP8B- 1)
Figure imgf000224_0002
[0677] To a mixture of N-Fmoc-PEG4-acid (0.53 g, 0.91 mmol) in DMF (10 mL) were added H-Gly-Gly-Phe-Gly-OtBu (SEQ ID NO: 16; CAS: 2413428-34-7, see WO2020050406, 0.30 g, 0.76 mmol), HATU (0.43 g, 1.1 mmol) and DIPEA (0.20 g, 1.6 mmol), and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was quenched with water (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic solution was concentrated in vacuo and the residue was separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound LP8B-1 (0.32 g, 50% yield) as yellow oil. ESI m/z: 862.1 (M + H)+. [0678] 2-[(2S)-2-(2-{2-[1-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3,6,9,12- tetraoxapentadecan-15-amido]acetamido}acetamido)-3-phenylpropanamido]acetic acid (LP8B-2)
Figure imgf000225_0001
[0679] To a solution of compound LP8B-1 (0.32 g, 0.38 mmol) in DCM (5 mL) was added TFA (1 mL), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound LP8B-2 (0.20 g, 65% yield). ESI m/z: 806.3 (M + H)+. [0680] 1-amino-N-{[({[(1S)-1-[({[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin- 5-yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]carbamoyl}methyl)- carbamoyl]-2-phenylethyl]carbamoyl}methyl)carbamoyl]methyl}-3,6,9,12- tetraoxapentadecan-15-amide (LP8B)
Figure imgf000225_0002
[0681] To a solution of compound LP8B-2 (0.81 g, 1.0 mmol) in DCM (10 mL) were added HOSu (0.23 g, 2.0 mmol) and EDCI (0.38 g, 2.0 mmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The mixture was quenched with water (10 mL) and extracted with DCM (3 x 10 mL). The combined organic solution was concentrated in vacuo to give the NHS ester as a white solid (0.80 g, ESI m/z: 903.4 (M + H)+), which was used for the next step without further purification. [0682] To a solution of the NHS ester obtained above (17 mg, 19 µmol) in dry DMF were added P9 (10 mg, 18 µmol) and DIPEA (4.0 mg, 31 µmol), and the reaction mixture was stirred at room temperature for 4 hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give Fmoc-LP8B (13 mg, ESI m/z: 667.1 (M/2 + H)+) as a white solid, which was dissolved in DMF (2 mL). To the DMF solution was added piperidine (0.02 mL), and the mixture was stirred at room temperature for an hour until Fmoc was totally removed according to LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give linker-payload LP8B (7.0 mg, 35% yield from P9) as a white solid. ESI m/z: 556.0 (M/2 + H)+.1H NMR (500 MHz, DMSOd6) δ 12.42 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 8.19 (t, J = 5.6 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.03 (t, J = 5.6 Hz, 1H), 7.84-7.73 (m, 3H), 7.46-7.39 (m, 3H), 7.33 (t, J = 5.6 Hz, 1H), 7.25-7.24 (m, 3H), 7.20- 7.16 (m, 1H), 7.02 (s, 1H), 6.82 (d, J = 7.8 Hz, 1H), 6.56 (d, J = 7.7 Hz, 1H), 6.20 (d, J = 2.9 Hz, 1H), 5.53 (s, 2H), 4.51-4.41 (m, 3H), 3.83 (s, 3H), 3.79-3.64 (m, 6H), 3.64-3.52 (m, 14H), 3.52-3.44 (m, 18H), 3.22-3.20 (m, 2H), 3.08-2.92 (m, 4H), 2.87-2.75 (m, 1H), 2.40 (t, J = 6.4 Hz, 2H), 1.47-1.41 (m, 2H), 1.30-1.17 (m, 2H), 1.12-1.07 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm. EXAMPLE 61 [0683] Linker-payload LP9 and LP12 [0684] 2,5-Dioxopyrrolidin-1-yl 2-(2-{2-[2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)acetamido]acetamido}acetamido)acetate (LP9-1) FmocHN
Figure imgf000226_0001
[0685] To a solution of Fmoc-Gly-Gly-Gly-Gly (SEQ ID NO: 24; CAS: 1001202-16-9) (0.12 g, 0.25 mmol) in DMF (4 mL) were added N-hydroxysuccinimide (58 mg, 0.50 mmol) and EDCI (96 mg, 0.50 mmol), and the reaction mixture was stirred at room temperature for an hour. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. ammonium bicarbonate (10 mM)) to give LP9-1 (70 mg, 50% yield) as a white solid. ESI m/z: 566.2 (M + H)+. [0686] 2-Amino-N-[({[({[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5- yl]methyl}-3-methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)methyl]acetamide (LP12)
Figure imgf000227_0001
[0687] To a solution of P9 (50 mg, 92 µmol) in DMF (2 mL) were added LP9-1 (60 mg, 0.11 mmol) and DIPEA (15 mg, 0.12 mmol), and the resulting mixture turned clear slowly and was then stirred at room temperature for an hour, which was monitored by LCMS. The mixture was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give Fmoc-LP12 (50 mg, ESI m/z: 995.5 (M + H)+) as a white solid, which was dissolved in DMF (2 mL). To the solution was added piperidine (20 µL) and the mixture was stirred at room temperature for an hour until Fmoc was totally removed according to LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give LP12 (30 mg, 37% yield from P9, TFA salt) as a white solid. ESI m/z: 773.5 (M + H)+. [0688] N-[({[({[1-(4-{[2-Amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13- yl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)methyl]-2-[2-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)acetamido]acetamide (LP9)
Figure imgf000227_0002
[0689] To a solution of LP12 (30 mg, 34 µmol) in DMF (2 mL) were added AMAS (LP7-2) (10 mg, 40 µmol) and DIPEA (6.0 mg, 46 µmol), and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.01%)) to give LP9 (5.0 mg, 14% yield, TFA salt) as a white solid. ESI m/z: 455.7 (M/2 + H)+.1HNMR (400 MHz, DMSOd6) δ 12.31 (s, 1H), 8.47 (t, J = 5.7 Hz, 1H), 8.24 (t, J = 5.6 Hz, 1H), 8.18 (t, J = 5.6 Hz, 1H), 8.09 (t, J = 5.7 Hz, 1H), 7.85 (t, J = 5.3 Hz, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.39 (s, 2H), 7.34 (t, J = 5.4 Hz, 1H), 7.09 (s, 2H), 7.02 (s, 1H), 6.83 (d, J = 7.7 Hz, 1H), 6.56 (d, J = 7.7 Hz, 1H), 6.21 (d, J = 2.9 Hz, 1H), 5.54 (s, 2H), 4.46 (s, 2H), 4.11 (s, 2H), 3.83 (s, 3H), 3.80-3.64 (m, 8H), 3.58-3.42 (m, 16H), 3.24-3.16 (m, 2H), 1.53-1.37 (m, 2H), 1.30-1.16 (m, 2H), 1.15–1.02 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSOd6) δ -73.5 ppm. EXAMPLE 62 [0690] Linker-payload LP13 [0691] (9H-fluoren-9-yl)methyl N-[(1S)-1-{[(1S)-3-carbamoyl-1-{[4- (hydroxymethyl)phenyl]carbamoyl}propyl]carbamoyl}-3-methylbutyl]carbamate (LP13-1)
Figure imgf000228_0001
[0692] To a solution of Fmoc-Leu-Gln-OH (0.78 g, 1.6 mmol) in DCM (20 mL) and methanol (2 mL) were added (4-aminophenyl)methanol (0.40 g, 3.2 mmol) and EEDQ (1.2 g, 4.9 mmol), and the reaction mixture was stirred at 40 oC in dark for 18 hours, which was monitored by LCMS. The volatiles were removed in vacuo. The residue was triturated in ether and dried under reduced vacuum to provide LP13-1 (0.47 g, 50% yield) as a white solid. ESI m/z: 587.3 (M + H)+.1H NMR (400 MHz, DMSOd6) δ 9.95 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.72 (t, J = 7.6 Hz, 2H), 7.53 (d, J = 8.4 Hz, 3H), 7.45-7.38 (m, 2H), 7.34-7.28 (m, 3H), 7.23 (d, J = 8.5 Hz, 2H), 6.78 (s, 1H), 5.10 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 5.6 Hz, 2H), 4.39-4.28 (m, 2H), 4.24 (d, J = 8.6 Hz, 2H), 4.13-4.04 (m, 1H), 2.12 (s, 2H), 2.00-1.77 (m, 2H), 1.63 (s, 1H), 1.49-1.41 (m, 2H), 0.92-0.82 (m, 6H) ppm. [0693] {4-[(2S)-4-carbamoyl-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4- methylpentanamido]butanamido]phenyl}methyl 4-nitrophenyl carbonate (LP13-2)
Figure imgf000228_0002
[0694] To a solution of compound LP13-1 (0.12 g, 0.21 mmol) in DMF (8 mL) were added bis(4-nitrophenol)carbonate (0.32 g, 1.1 mmol), DMAP (26 mg, 0.21 mmol) and DIPEA (0.14 g, 1.1 mmol), and the reaction mixture was stirred at room temperature for 2 hour, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (5-95% acetonitrile in aq. TFA (0.01%)) to give LP13-2 (0.11 g, 69% yield) as a white solid. ESI m/z: 752.3 (M + H)+. [0695] {4-[(2S)-2-[(2S)-2-amino-4-methylpentanamido]-4-carbamoylbutanamido]- phenyl}methyl N-[1-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3- methoxyphenyl)-2,5,8,11-tetraoxatridecan-13-yl]carbamate (LP13-3)
Figure imgf000229_0001
[0696] To a solution of compound LP13-2 (72 mg, 90 µmol) in DMF (5 mL) were added P9 (68 mg, 0.12 mmol), HOBt (6 mg, 40 µmol) and DIPEA (35 mg, 0.26 mmol). The reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (70% acetonitrile in aq. TFA (0.01%)) to give Fmoc-LP13-3 (70 mg, ESI m/z: 1157.1 (M + H)+) as a white solid, which was dissolved in DMF (3 mL). To the solution was added diethylamine (0.3 mL), and the reaction mixture was stirred at room temperature for 2 hours until Fmoc was totally removed according to LCMS. The resulting mixture was separated by prep-HPLC (5- 95% acetonitrile in aq. TFA (0.01%)) to give LP13-3 (45 mg, 53% yield) as a white solid. ESI m/z: 468.4 (M/2 + H)+. [0697] {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0⁴,⁹]hexadeca-1(12),4(9),5,7,13,15- hexaen-10-yn-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-4- methylpentanamido]-4-carbamoylbutanamido]phenyl}methyl N-[1-(4-{[2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)-2,5,8,11- tetraoxatridecan-13-yl]carbamate (LP13)
Figure imgf000229_0002
[0698] To a solution of LP13-3 (30 mg, 32 µmol) in DMF (2 mL) were added DIPEA (10 mg, 78 µmol) and compound LP13-4 (27 mg, 42 µmol), and the reaction mixture was stirred at room temperature for 2 hours, which was monitored by LCMS. The resulting mixture was directly separated by prep-HPLC (5-95% acetonitrile in aq. TFA (0.01%)) to give LP13 (22 mg, 40% yield) as a white solid. ESI m/z: 735.3 (M/2 + H)+.1H NMR (400 MHz, DMSOd6) δ 9.93 (s, 1H), 8.15 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.77 (t, J = 5.6 Hz, 1H), 7.70-7.64 (m, 1H), 7.64-7.53 (m, 3H), 7.53-7.42 (m, 3H), 7.41-7.16 (m, 8H), 7.00 (s, 1H), 6.78 (d, J = 8.2 Hz, 2H), 6.43 (d, J = 7.8 Hz, 1H), 5.98 (d, J = 2.9 Hz, 1H), 5.78-5.67 (m, 1H), 5.40-5.35 (m, 3H), 5.03 (d, J = 14.0 Hz, 1H), 4.93 (s, 2H), 4.44 (s, 2H), 4.38-4.24 (m, 2H), 3.85 (s, 3H), 3.63- 3.44 (m, 28H), 3.29-3.28 (m, 2H), 3.15-3.04 (m, 4H), 2.62-2.56 (m, 2H), 2.46-2.31 (m, 3H), 2.29-2.06 (m, 4H), 2.04-1.89 (m, 3H), 1.86-1.71 (m, 2H), 1.47-1.41 (m, 2H), 1.40-1.34 (m, 2H), 1.22-1.15 (m, 2H), 1.09-1.02 (m, 2H), 0.89-0.78 (m, 9H) ppm. EXAMPLE 63 [0699] The antibody drug conjugates (ADCs) were prepared via partial reduction of the antibody with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced cysteine residues with maleimide functionalized linker-payload (FIG.5). Specifically, antibodies were partially reduced via addition of 1.5 - 3.0-fold molar excess of TCEP in PBS pH7.4 and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 h at 37 °C. The reduced antibodies were buffer exchanged into PBS with 1% w/v polysorbate 20. Linker-payloads were added at a linker- payload / antibody molar ratio of 5 - 10 and reacted for an additional 2 h at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO). The reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80 °C. The protein concentration was determined via UV spectrophotometer. ADC monomer purity was >90% by SEC. The ADCs were further characterized via hydrophobic interaction chromatography (HIC), and liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) to calculate drug-antibody ratio (DAR). EXAMPLE 64 [0700] Conjugation of TLR7 Agonist to an Antibody [0701] In a specific example shown in the conjugation scheme of FIG.5, 15 mg/mL anti- Her2 human IgG antibody in PBS was partially reduced via addition of 2.5-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP) and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 h at 37 °C. The reduced antibody was buffer exchanged into PBS with 1% w/v polysorbate 20. Linker-payload was added at a linker-payload / antibody molar ratio of 6 and reacted for an additional 2 h at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO). The reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80°C. The protein concentration was determined via UV spectrophotometer. ADC monomer purity was 99.7% by SEC. The ADC was further characterized via hydrophobic interaction chromatography (HIC) and liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) to calculate the drug-antibody ratio (LCMS DAR=1.9; HIC DAR=2.6). Results are shown in the Table 7 below. EXAMPLE 65 [0702] Purification method for antibody-LP11A conjugate through preparative size- exclusion chromatography (SEC). [0703] Preparative SEC purification was performed on a Superdex 200 PG column (16 x 60 cm) installed on an AKTA pure FPLC system (Cytiva) at a flow rate of 1.5 mL/min using an elution buffer of PBS with 5% glycerol and monitored 280 nm, 250 nm, and 330 nm UV absorbance. The preparative SEC result (FIG.6) demonstrated a clean separation of ADC monomer from aggregates (“HMW”) and unconjugated linker payload (“Free drug”). EXAMPLE 66 [0704] Methods for characterizing antibody-TLR7 conjugates. [0705] Analytical size exclusion chromatography (SEC) was performed to determine ADC monomer purity. Sample was run on an ACQUITY Protein BEH SEC column (200A, 1.7 µm, 4.6 mm x 150 mm) installed on an ACQUITY UPLC instrument (Waters), using 10 mM phosphate, 1.0 M sodium perchlorate, 5% v/v isopropanol as mobile phase, at a flow rate of 0.3 mL/min, and monitored UV-vis absorbance at 280 nm using an eλ PDA detector (Waters). The analytical SEC result (FIG.7) indicated 99.7% monomer purity. [0706] Liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) analysis was performed to determine the drug distribution profile and to calculate the average drug-antibody ratio (DAR). Each sample (20 µL at 0.5 mg/mL) was deglycosylated by PNGase F enzyme, followed by reduction by dithiothreitol (DTT), then loaded onto an ACQUITY UPLC Protein BEH C4 column (10K psi, 300A, 1.7 µm, 75 µm x 100 mm; mass spectrum was acquired on a Waters Synapt G2-Si mass spectrometer. [0707] As shown in FIG.8, the deconvoluted mass spectra exhibited light chain species (LC, LC1) and heavy chain species (HC, HC1, HC2, etc.). The average DAR can be calculated from the LC and HC drug-loading. [0708] Analytical Hydrophobic interaction Chromatography (HIC) was also performed to determine the drug distribution profile and to calculate the average DAR. HIC samples were prepared by diluting ~100 µg antibody or ADC with 1.5 M ammonium sulfate, then loaded on a TSKgel Butyl NPR column (100 mm x 4.6 mm, 2.5 µm, Tosoh Bioscience) installed on an ACQUITY UPLC instrument (Waters) using a binary gradient of buffer A (1.5 M ammonium sulfate, 50 mM potassium phosphate) and buffer B (50 mM potassium phosphate, 5% isopropanol) at a flow rate of 0.2 mL/min, and monitored UV-vis absorbance at 280 nm using an eλ PDA detector (Waters). [0709] The analytical HIC of an antibody-LP11A conjugate (FIG.9) revealed a mixture of three species: DAR2 species (51%), DAR4 species (28%) and unconjugated antibody (21%). The average DAR of this ADC is 2.1. EXAMPLE 67 [0710] Toll-like receptor (TLR)-7 is an endosomal localized pattern recognition receptor that responds to pathogen-associated single stranded ribonucleic acid (ssRNA) and plays a key role in innate immune responses. TLR-7 activation induces proinflammatory cytokine and type I interferon (IFN) expression via the activation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and interferon regulatory factor (IRF) signaling pathways. [0711] To test the activity of TLR7 agonist payloads, HEK-Blue hTLR7 cells (InvivoGen) were utilized. HEK-Blue TLR7 cells are a human embryonic kidney HEK293 cell line expressing human TLR7 and a secreted alkaline phosphatase (SEAP) reporter gene under the control of the interferon-beta (IFN-β) minimal promoter fused to five NF-κB and AP-1 binding sites. In parallel to testing these compounds in the HEK-Blue hTLR7 cell report, a cytotoxicity assay was also performed. [0712] For the HEK-Blue TLR7 assay, initially the test compounds and a reference compound, resiquimod, were aliquoted at 10 concentrations with a serial of 3-fold dilution in 96 well plates. Subsequently, 50,000 cells/well were seeded into 96-well plates containing test compounds and then the plates were incubated at 37 °C, 5% CO2 for 24 hours. After the 24- hour incubation, 20 µL of supernatants from each well were transferred to plates containing 180 µL of Quanti-Blue for SEAP reporter detection, and then incubated at 37 °C, 5% CO2 for 1 hour. The optical density was then measured using Flexstation III (Molecular Devices) at 650 nm (OD650). [0713] Cell viability was assessed using CellTiter-Glo (Promega) according to the manufacturer’s manual. Luminescence raw data (RLU, relative light unit) was measured using Flexstation III for cell viability detection. [0714] The raw data for both TLR7 activation and cell viability were analyzed in GraphPad Prism and nonlinear regression curves were fit to obtain EC50 values for TLR7 activation and CC50 values of the compounds. [0715] The agonistic activities of the payloads provided herein were assessed in HEK BLUE hTLR7 cells. In the agonistic activity test in HEK BLUE hTLR7 cells, several compounds provided herein (P13, P8, P15, P5, P6, P9) demonstrated activity with EC50 values ranging from 0.05 µM to 0.805 µM and S/N values ranging from 20.14 to 10.59. Other compounds provided herein did not show obvious agonist activity at the tested concentrations and the EC50 values were higher than the highest test concentration (EC50 > 100 µM). [0716] For cytotoxicity testing in HEK BLUE hTLR7 cells, several compounds provided herein (P15, P5, P9) demonstrated cytotoxicity with CC50 values ranging from 16.31 µM to 90.71 µM. Other compounds provided herein did not show obvious cytotoxicity at the tested concentrations and the CC50 values were higher than the highest test concentration (CC50 > 100 µM). [0717] Table 4. TLR-dependent reporter activity and cytotoxicity of TLR7 linker payload agonists in HEK-blue human TLR7 assay
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
* S/N, agonistic activity signal to noise ratio of hTLR7 agonistic assay. EXAMPLE 68 [0718] To assess the plasma stability of representative mAb2 TLR7 antibody drug conjugates (ADCs) containing linker payloads provided herein, ncADCs were incubated in vitro with plasma from different species and the DAR was evaluated after incubation at 37 °C for up to 13 days. [0719] Each ncADC sample (anti-HER2 Ab-LP1 ADC (comparator), anti-HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC and anti-HER2 Ab-LP7A ADC) diluted in PBS buffer (Irvine Scientific, Cat#9236) was added to pooled mouse plasma (BioIVT, Cat#MSE01PLK2P2N) and IgG depleted human plasma (BiochemMed), independently, at a final concentration of 50 µg/mL, and subsequently incubated at 37 °C. A 100-µL aliquot was removed at the time 0, 24, 36, 72, 168 and 312 hours and then immediately stored frozen at -80 °C until analysis. [0720] Affinity capture of the ncADCs from the plasma samples was carried out on a KingFisher Apex 96 magnetic particle processor (Thermo Electron). First, biotinylated anti- human Fc antibody (Regeneron generated reagent) was immobilized on Dynabeads M280 streptavidin paramagnetic beads (Invitrogen, Cat#60210). Each plasma sample containing TLR7 ncADCs was mixed with 0.5 mg of the beads (Regeneron generated reagent immobilized bead) at room temperature for 2 hours in a 96 well plate. The beads were then washed three times with 500 µL of HBS-EP (GE healthcare, Cat#BR100188), once with 500 µL of water, and then once with 500 µL of 8% acetonitrile in water (VWR Chemicals, Cat#BDH83640.100E). Following the washes, the ncADCs were eluted by incubating the beads with 70 µL of 1% formic acid in 30% acetonitrile / 70% water for 20 minutes at room temperature. Fifty µL eluted samples were further reduced by adding 50 µL 10 mM TCEP (Sigma, Cat 646547-10X1ML) and incubated at 37 °C for 20 minutes in ThermoMixer C. [0721] The eluted ncADCs samples were injected onto a 1 x 50 mm 1.7 µm BEH300 C4 column (Waters Corporation, Cat# 186005589) coupled to a Synapt G2-Si Mass Spectrometer (Waters). The flow rate was 80 µL/min (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile). HPLC gradient eluted ncADC between 2.0-6.5 minutes corresponding to 25-40 % of mobile phase B. [0722] The acquired spectra were deconvoluted using MaxEnt1 software (Waters Corporation) with the following parameters: Mass range: 20-70 kDa; m/z range: 800-2500 Da; Resolution: 1.0 Da/channel; Width at half height: 0.7 Da; Minimum intensity ratios: 33%; Iteration max: 12. [0723] The resulting drug to antibody ratios in mouse and human plasma were calculated and are shown in Table 5 and FIG.1, FIG.2, FIG.3, and FIG.4. [0724] Hydrolysis of the succinimide ring on the linker-payload was observed in anti-HER2 Ab-LP1 ADC (comparator), anti-HER2 Ab-LP6A ADC and anti-HER2 Ab-LP11A ADC with a mass shift of approximate +18Da in mass spectra. No significant change in DAR was observed for anti-HER2 Ab-LP1 ADC (comparator), anti-HER2 Ab-LP6A ADC and anti-HER2 Ab-LP11A ADC over 13 days (7 days for anti-HER2 Ab-LP11A ADC in human). However, the maleimide ring in anti-HER2 Ab-LP7A ADC was partially hydrolyzed on Day 0 and a slight decrease of DAR was detected on day one, which then became stable up to 13 days. [0725] Table 5: Drug to antibody ratio (DAR) for select anti-HER2 mAb2-TLR7 ncADCs in human and mouse plasma in vitro.
Figure imgf000236_0001
EXAMPLE 69 [0726] As shown in FIG.5, anti-HER2 and isotype control antibody drug conjugates (ADCs) were prepared via partial reduction of the antibody with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced cysteine residues with maleimide functionalized linker- payload. Specifically, antibodies were partially reduced via addition of 1.5 - 3.0-fold molar excess of TCEP in PBS pH 7.4 and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 hours at 37 °C. The reduced antibodies were buffer exchanged into PBS with 1% w/v polysorbate 20. Linker-payloads were added at a linker-payload / antibody molar ratio of 5 - 10 and reacted for an additional 2 hours at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO). The reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80 °C. The protein concentration was determined via UV spectrophotometer. ADC monomer purity was >90% by SEC. The ADCs were further characterized via hydrophobic interaction chromatography (HIC), and liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) to calculate drug-antibody ratio (DAR). Results are shown in Tables 6 and 7 and FIG.6, FIG.7, and FIG.8. [0727] Example (anti-HER2 Ab-LP1 ADC) [0728] In a specific example, 15 mg/mL of anti-PSMA antibody in PBS was partially reduced via addition of 2.5-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP) and 2 mM ethylenediaminetetraacetic acid (EDTA) for 2 hours at 37 °C. The reduced antibody was buffer exchanged into PBS with 1% w/v polysorbate 20. A linker-payload LP1 was added at a linker- payload / antibody molar ratio of 6 and reacted for an additional 2 hours at 25 °C in the presence of 12% v/v of dimethyl sulfoxide (DMSO). The reaction mixtures were purified via size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase), formulated in PBS with 5% Glycerol and stored at -80 °C. The protein concentration was determined via UV spectrophotometer. ADC monomer purity was 99.7% by SEC. The ADC was further characterized via liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS) to calculate drug-antibody ratio (DAR=1.9). [0729] Table 6: TLR7 antibody drug conjugates
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
) %( % % % % % % % % % % % % % % %
Figure imgf000244_0001
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Figure imgf000245_0001
C L P A A L 7 1 A 1 A P P 6 L L P 1 7 L P L P L ne 2 T E N E E L P O T E N E E L P O T E N E E L P O T E N E E L P O gi t R E G GI F Y R G G F Y R G F Y R G F Y R O T T I O T T G I O T T G I T T n H R A T O N R T O N R T O N R T O O N A T N A S I O A C T N A S I O A C T N A S I O A C T N A S I O C se t - o c u N f
Figure imgf000246_0001
S 7 . 0 . 6 . 1 . M 1 2 1 2 C L A P 0 A A A L 1 8 6 7 P L P L P L P L ne T N E L E L E L L g E E P O T N E P O T N E P O T N E E O it G GI F Y R E G F Y R E G F Y R E P F Y R O T T G I O T T G I T T G GI T T n R A T O N R A T O N R A T O O N R T O O N A T N A S I O C T N A S I O C T N A S I O A C T N A S I O C se t - o c - u c f u N f EXAMPLE 70 [0731] To screen the activity of TLR7 agonist payloads, TLR7 agonist linker-payloads (LPs), and anti-HER2 mAb2 TLR7 antibody drug conjugates (ADCs) provided herein, HEK-Blue hTLR7 cells (InvivoGen) were utilized. HEK-Blue TLR7 cells are a human embryonic kidney HEK293 cell line expressing human TLR7 and a secreted alkaline phosphatase (SEAP) reporter gene under the control of the interferon-beta (IFN-β) minimal promoter fused to five NF-κB and AP-1 binding sites. Control cells expressing the SEAP reporter and human TLR8 (HEK-Blue hTLR8, InvivoGen), the SEAP reporter and mouse TLR7 (HEK-Blue mTLR8, InvivoGen), the SEAP reporter and human TLR9 (HEK-Blue hTLR9), the SEAP reporter and human TLR3 (HEK-Blue hTLR9), and the SEAP reporter alone (HEK-Blue Null1-k, InvivoGen) were also utilized. To assess activity of anti-HER2mAb2-TLR7 ADCs, HEK-Blue TLR7 cells were engineered to overexpress full-length human antigen 2 via lentiviral mediated transduction and are herein referred to as HEK-Blue TLR7/h-antigen2 cells. HEK-Blue TLR7/h-antigen2 cells were grown for at least 2 weeks under G418 selective pressure to enrich for antigen2 positive cells and surface expression was validated via flow cytometry with mAb2. [0732] To assess payload and LP-dependent reporter activity, 40,000 cells were seeded into 96 well plates in HEK-Blue Detection media. Three-fold serial dilutions of free payloads or LPs were prepared in 100% DMSO, transferred to fresh assay media, and added to the cells at a final constant DMSO concentration of 0.2%. The last well in the plate served as a blank control containing only the assay media and 0.2% DMSO (untreated well) and was plotted as a continuation of the 3-fold serial dilution. Sixteen to twenty-four hours later, SEAP activity was determined in a colorimetric assay according to manufacture instructions in which the optical density at 650 nm (OD650) was measured on an Envision luminometer (PerkinElmer) and EC50 values were determined using a four-parameter logistic equation over a 10-point dose response curve (GraphPad Prism). The signal to noise (S/N) was determined by taking the ratio of the highest OD650 value on the dose response curve to the OD650 value in the untreated wells. [0733] To test the ncADCs for TLR-dependent reporter activity, HEK-Blue hTLR7 or HEK- Blue hTLR7/h-antigen2 cells were seeded in complete growth medium at 40,000 cells per well of a 96-well plate and grown overnight. Three-fold serial dilutions of ncADCs, unconjugated antibodies, or isotype control ncADCs were prepared in dilution media (Opti-MemTM + 0.1% BSA) and added to cells for a final assay medium of 20% dilution medium + 80% growth medium (concentrations were corrected for the DAR (drug antibody ratio) and dosed based on the effective payload concentration). The last well in the plate served as a blank control containing assay media alone and was plotted as a continuation of the 3-fold serial dilution. Free payloads were prepared and added as described above. Following a 96-hour incubation, SEAP activity was measured by incubating 20 µL of cell supernatant with 180 µL of Quanti- blue solution for 1~2 hours at 37 °C. The OD650, EC50, and S/N values were determined as above. [0734] As shown in Table 8, a 24 h incubation with P9 increased human and mouse TLR7- dependent activity with EC50 values of 48.8 nM and 38.1 nM, respectively, and S/N values of 6.1 and 2.0 respectively. P9 did not increase human TLR8 within the tested dose range. A known TLR7 payload, P3, increased human and mouse TLR7-dependent activity with EC50 values of 6.98 nM and 7.27 nM, respectively, and S/N values of 2.7 and 3.2 respectively. P3 increased human TLR8 activity with an EC50 value of 826 nM and S/N of 4.9. P3 with an attached linker (LP1) increased human and mouse TLR7-dependent activity with EC50 values of 25.7 nM and 107 nM, respectively, and S/N values of 5.8 and 2.5 respectively. LP1 increased human TLR8 activity with an EC50 value of 746 µM and S/N of 2.1. A known TLR8 agonist increased human TLR8 activity with an EC50 value of 4.7 nM and S/N of 5.2, but was weakly agonistic on human TLR7 and mouse TLR7 with EC50 values > 10 uM and S/N less than or equal to 1.3. A known dual TLR7/8 agonist increases human TLR8 and mouse TLR7 activity with EC50 values of 311 nM and 335 nM, respectively, and S/N values of 4.7 and 3.0 respectively. None of the tested payloads increased TLR3 or TLR9 activity within the tested dose ranges and were inactive in HEK-Blue null cells.
n a mu
Figure imgf000249_0001
yti u l r vi t B c - 0 A K ) 0 0 0 r E C 5 M 9 . 7 . 4 . 0 0 . 9 . 7 . . 0 9 . e t H n 6 8 8 1 6 6 7 8 6 r E ( 7 1 > 4 9 o p e Rt 7 d n R o t o m 0 3 5 3 8 1 8 6 6 3 1 5 e L o i i d T t u 0 . 0 . 0 . 0 . 1 . 3 . 0 . 2 . 0 . 2 . 5 2 . 5 . 1 . 1 5 0 1 0 . 2 . 1 . 4 . n h a q i 1 0 0 0 0 0 0 0 0 0 2 0 2 1 0 0 0 s 2 e sll e R pe e u l e r D c - 7 B- RL R T L K T E 0 5 ) 3 M . 6 . 0 . 9 . 2 . 3 . 7 . 8 . 7 . 8 . 0 . 3 0 . 0 4 0 . 1 . 2 . 7 . H C 0 E n ( 9 4 9 1 1 1 3 1 3 4 7 5 2 5 5 6 0 1 9 7 4 2 7 7 1 6 0 5 5 4 0 0 1 1 8 0 6 : 1 8 1 1 8 e s 1 e l u b o e a m l d c i o T d n t r mi 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 ] a A u q P P P P P P P P P 1 P 1 P 1 1 1 1 1 5 , 9 t s i P P P P P 3 R e s 7 e 0 [ L T T R
Figure imgf000250_0001
l B- K 0 E 5 ) M H C E n ( 7 d o R o t L T o i m i 8 0 0 0 7 8 4 2 7 1 3 u 0 1 5 2 . 3 6 . 4 6 6 2 2 0 2 1 4 4 6 2 . 3 . 4 . 4 5 8 9 . 2 6 . 8 h t a q . . . i 0 0 0 4 2 . 5 2 7 . 0 . 1 . 5 . 0 . 4 . 1 5 1 7 4 1 4 . 6 2 8 e R s 1 1 ul e r B- K 0 ) 9 0 . 0 . 0 0 8 0 0 9 0 . 0 . 0 0 0 0 E H C 5 M 4 . 8 . . 7 0 E n ( 1 3 1 4 3 6 0 0 0 9 . 5 . 2 7 0 . . 4 8 T 0 2 0 9 4 0 0 0 6 1 9 4 1 1 4 1 4 1 6 4 0 1 1 4 0 9 0 1 N 1 1 3 7 4 9 8 3 1 6 2 8 3 el ci t r A 7 1 8 P 1 9 P 1 0 P 2 1 P 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 2 0 3 1 3 2 3 3 3 4 3 5 3 t s P P P P P P P P P P P P P P P e T
e s e h t n
Figure imgf000251_0001
H E n ( 0 1 . 4 3 e > n a c e n s o d c 7 o o u a d R t L T o i m i c e e t s t a u qi b e y t m e R s ul e c t r a s r e u h B- c g K 0 c i h E 5 ) 0 M 0 0 5 a e H C E n ( 0 3 1 3 hti h t > w n d a h t 7 d e n R o t o i r m 7 3 2 7 6 4 4 m e r t a L T o i i u 6 h t a q . 8 0 . 3 . . 0 9 3 7 9 . 2 3 e t e r 2 . . e g e R i s 5 7 1 0 0 d s u l e r e b a B t d - e K ) o t 0 0 0 0 n r E 0 5 M . 9 8 . 9 9 8 0 9 5 . 7 0 0 d o l p H C E n ( 3 1 2 4 2 6 2 1 8 4 0 1 5 9 5 1 0 1 u e r > o c s s i 0 e d et e 5 l t s u l C ci t n r A 6 8 s i n 8 / t s i e t a v E, 3 7 3 8 3 9 3 1 4 2 4 3 4 w R n w 7 n t 0 t P P P P P P P o n L o o n R L o o n C 5 s e c s e K T g A K T g A = E n a T T = t s N > n i [0736] P9 was attached to several linkers to generate LP6A, LP11A, LP7A, and LP8A and tested in the TLR reporter assays as described for the payloads except that incubation times were for 16h. As shown in Table 9, P9 increased human and mouse TLR7-dependent activity. LPs generated from P9 (LP6A, LPA7, LP8A and LP11A) increased human TLR7 reporter activity. P9-derived LPs also increased mouse TLR7 reporter activity. P9 and associated LPs did not activate human TLR8 within the tested dose range. LP1 increased hTLR7 and mTLR7 activity. LP1 was weakly agonistic toward human TLR8. None of the test articles were active in HEK-Blue null cells within the tested dose range.
e s
Figure imgf000253_0001
yti E ( 0 . 6 0 v 7 8 9 1 8 1 3 5 0 i H 5 7 8 1 1 9 t c C A E r e t r d o p o t o e 7 o m i 0 0 9 1 6 3 0 2 3 0 5 8 2 4 4 7 3 3 6 2 6 8 5 4 4 9 1 6 7 9 0 R R i t t u q . 1 . 0 . 0 . 0 . 2 . 2 . 5 . 3 . 5 . 5 . 2 . 2 . 6 2 . 2 5 . 3 . . 3 n L T a i 1 0 s 1 e d h R e n e r e u p l e B D - ) - K M n 3 . R E ( 2 0 6 9 1 0 0 0 0 L H 0 T 5 0 9 7 2 0 . 1 9 7 0 8 5 1 1 9 3 4 2 1 1 2 0 4 3 1 5 7 1 9 5 0 8 1 7 2 0 6 5 3 1 2 2 : C 4 2 1 5 9 E e l b a d T s ll t e o l m A A B A A C D 2 ] e c s 7 e c i t i u 1 P 2 P 3 P 5 P 6 P 7 P 7 D 7 A 8 B 8 9 P 0 1 1 1 1 1 1 1 1 P 3 7 T r q i L L L L L P P P P L P P P P L R A s L L L L L L L L L 7 0 L e [ T r
e s e h t n I . d e v N/ 0 r e ll S . 1 s u b n o s e a ul w e
Figure imgf000254_0001
e s u l a B w - ) K M 0 e E n ( 0 t 0 o t H 0 5 0 p C 1 E > my s a r n e oit 2 d p ar 5 o o p 2 7 u t t m 7 n e R o i i u 6 n a c L t T a q . R i 0 s e n s o c m e r u d e a u c e t l e b s B - ) e K M y t c t s E n ( 7 . a r e H 0 h C 5 7 4 u c g c i E a h h e t d i h t n o t o w d a 7 o m i 2 9 3 e h n t i r R i u 2 . 6 . 2 . e L t a q 4 3 0 m r t T h R i s e e a t e r e r e u d g l e s a B- ) b K M t o d et E n ( 0 H 0 8 0 8 . n r 5 6 7 1 4 7 1 2 d o l p C 1 u e r E o c s s i e 0 5 u l C ts e l e c i 3 1 4 a 1 v E, 9 0 5 s e T t r P P P C c A L L E n = a t s > n i [0738] The LPs provided herein were conjugated to mAb2 to generate the following anti- HER2-TLR ncADCs: anti-HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC, anti-HER2 Ab- LP7A ADC, and anti-HER2 Ab-LP8A ADC. As shown in Table 10, the mAb2-TLR7 ncADCs increased human TLR7 reporter activity in HEK-Blue hTLR7/HER2 cells with EC50s ranging from 13.2 nM to 16.2 nM and S/N values from 2.2 to 4.9. These same ncADCs were weakly agonistic in HEK-Blue hTLR7 cells with EC50s > 1.0 uM and S/N values less than or equal to 1.3. A known anti-HER2 ncADC (anti-HER2 Ab-LP1 ADC) increased TLR7 reporter activity in HEK-Blue hTLR7/h-HER2 and HEK-Blue hTLR7 cells with EC50 values of 9.59 nM and 465 nM, respectively, and S/N values of 6.4 and 2.9, respectively. P9, the free payload of anti- HER2 Ab-LP6A ADC, anti-HER2 Ab-LP11A ADC, anti-HER2 Ab-LP7A ADC, and anti-HER2 Ab-LP8A ADC increased TLR7 activity in HEK-Blue hTLR7/h-HER2 and HEK-Blue hTLR7 cells with EC50 values of 234nM and 153 nM, respectively and S/N values of 8.3 and 6.9, respectively. The known payload, P3, was agonistic in HEK-Blue hTLR7/h-HER2 and HEK- Blue hTLR7 cells with EC50 values of 21.8 nM and 8.16 nM, respectively, and S/N values of 9.1 and 7.5, respectively. The known LP, LP1, increased hTLR7 activity in HEK-Blue hTLR7/h-HER2 and HEK-Blue hTLR7 cells with EC50 values of 115 nM and 71.8 nM, respectively, and S/N values of 8.2 and 7.0, respectively. Non-binding isotype controls (mAb1) conjugated to TLR7 LPs were weakly cytotoxic in all tested cells with EC50s > 1.0 µM and S/N less than or equal to 2.0. [0739] Table 10: TLR-Dependent Reporter Activity by anti-HER2-TLR7 ncADCs in HEK- blue human TLR7 and HEK-blue human TLR7/HER2 cells
Figure imgf000255_0001
Figure imgf000256_0001
> = EC50 values could not be determined with accuracy because an upper asymptote was not reached, or no response was observed. In these instances, EC50 is reported as greater than the highest tested concentration. EXAMPLE 71 [0740] Protocol for human PBMC TLR7 IFN-α release assay [0741] Protocol: 1. Prepare fresh hPBMC from TPCS and centrifuge cells at 350 g for 7 minutes. 2. Remove supernatant by aspiration and resuspend the cell pellet to 1.6×106 cells/mL. 3. Add 80 μL of cells to each well (2x105 cells/well _2nd round and 1.25x105 cells/well-1st round). 4. Prepare serial dilutions of 5x final concentration of test compounds. 5. Transfer 20 μL of serially diluted compounds to the corresponding wells. 6. Incubate cells for 24 hrs at 37oC, 5% CO2. 7. Centrifuge cells at 350 g for 5 minutes. 8. Collect the cell supernatant and detect IFN-α release by ELISA. [0742] Protocol for human PBMC TLR7 TNF-α release assay [0743] Protocol: 1. Thaw frozen human PBMC and put all cells into 40 mL assay media (RPMI1640 supplemented with 10% HI-FBS and 1% penicillin-streptomycin) in 50 mL conical tubes. 2. Spin human PBMC at 1200 rpm for 4 minutes. 3. Remove supernatant by aspiration and resuspend pellet in assay media to 1.5625×106 cells/mL. 4. Add 80 μL cells to each well (1.25×105 cells/well). 5. Incubate the plate at 37oC 5% CO2 incubator for 24 hours. 6. On the day of the bioassay, prepare serial dilutions of 5× final concentration of test compounds (final concentration start at 10 μM, 4-fold dilution for a total of 9 doses). 7. Transfer 20 μL of serially diluted compounds to the cell plates. 8. Incubate cells for 24 hrs at 37°C, 5% CO2. 9. Spin cells 1200 rpm for 5 minutes. 10. Remove supernatant and freeze at -80°C until ready to detect TNF-α release by ELISA. Table 11. Cytokine Release Data of Payloads
Figure imgf000257_0001
[0744] Evaluation of the ADCs provided herein using in vitro co-culture assays consisting of Her2Pos tumor cells (NCI-N87) and effector cells (hPBMC) resulted in elevated IFN-α and TNF- α cytokine release relative to isotype antibody control ADCs and to unconjugated antibodies. The immunogenicity and pharmacokinetic (PK) profiles of the ADCs provided herein were evaluated using hIgG tolerized mice, thereby reducing the incidence of mouse anti-human antibody (MAHA) responses. The ADCs provided herein showed a favorable PK profile and low frequency of MAHA responses. EXAMPLE 72 [0745] The ADCs provided herein were examined for their anti-tumor efficacy using a series of in vivo mouse models. [0746] Growth and Implantation of N87 Tumor Cells into NSG™ Mice [0747] The N87 gastric carcinoma tumor cell line was expanded in T225 flasks in RPMI1640 culture media supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection. N87 cells were then washed twice and an aliquot of cells was collected for determining cell viability and counts using ViaStain™, a solution containing acridine orange and propidium iodide (AOPI), in combination with the Nexcelom Cellaca MX cell counter. The remaining cells were resuspended in Matrigel Basement Membrane Matrix (50%) prepared in sterile solution and 4-5x106 N87 cells were implanted subcutaneously into immunodeficient Fox Chase SCID® Beige mice across studies. In this model, a single dose (5 mg/kg) of ADCs provided herein eradicated tumors (FIG.10 and FIG.11). In particular, FIG.10 shows that tumor regression was observed after treatment with 5 mg/kg (gray circle) of anti-HER2 Ab- LP6A ADC, while treatment with 1 mg/kg (gray square) anti-HER2 Ab-LP6A ADC resulted in tumor stasis, when compared to saline treated animals (open circle). Regression of N87 gastric tumors was not observed in the N87 xenograft mice treated with 5 mg/kg of isotype control Ab-LP6A ADC (Table 3) (black circle), 0.5 mg/kg (gray triangle) anti-HER2 Ab-LP6A ADC, or 0.1 mg/kg (gray diamond) anti-HER2 Ab-LP6A ADC when compared to saline treated animals (open circle). Data represent mean tumor volumes (mean+/-SEM) over time (post- dose). [0748] Growth and Implantation of JIMT-1 Tumor Cells into NSG™ Mice [0749] The JIMT-1 epithelial breast tumor cell line was expanded in T225 flasks in DMEM culture media supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection. JIMT-1 cells were then washed twice and an aliquot of cells was collected for determining cell viability and counts using ViaStain™, a solution containing acridine orange and propidium iodide (AOPI), in combination with the Nexcelom Cellaca MX cell counter. The remaining cells were resuspended in Matrigel Basement Membrane Matrix (50%) prepared in sterile solution and 2.5x106 JIMT-1 cells were implanted subcutaneously into immunodeficient Fox Chase SCID® Beige mice across studies. In this model, ADCs provided herein were found to delay growth relative to an isotype control ADC, and when combined with a non-competing anti-HER2 antibody, pertuzumab, resulted in tumor regression, suggesting that FcR clustering can be used to enhance therapeutic efficacy (FIG.12). [0750] Growth and Implantation of MC38 Tumor Cells Engineered to Express CD20 into C57BL/6J Mice [0751] The MC38 melanoma tumor cell line expressing a defined tumor was expanded in T225 flasks in DMEM culture media supplemented with penicillin, streptomycin, L-glutamine, sodium pyruvate, 1% HEPES, 1% non-essential amino acids and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection. Tumor cells were then washed twice and an aliquot of cells was collected for determining cell viability and counts using ViaStain™, a solution containing acridine orange and propidium iodide (AOPI), in combination with the Nexcelom Cellaca MX cell counter. Tumor cells were resuspended in Hanks Balanced Salt Solution and 1e6 tumor cells were subcutaneously implanted into each C57BL/6J mouse. An anti-human CD20 ADC was shown to mediate tumor regression in this model (FIG.13). [0752] Xenograft and syngeneic tumor model experimental procedure. [0753] N87 cells were cultured in RMPI, 10% FBS, P/S/G, before implantation, cells were mixed with an equal volume of Matrigel, and 100ul of the mixture (5e6 cells) were implanted subcutaneously into the right flank of 6-8-week-old female SCID-Beige mice (Charles River). [0754] JIMT-1 cells were cultured in DMEM, 10% FBS, P/S/G. Before implantation, cells were mixed with an equal volume of Matrigel, and 200 uL of the mixture (2.5e6 cells) were implanted subcutaneously into the right flank of 6-8-week-old female SCID-Beige mice (Charles River). [0755] MC38.hTAAPos cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200 uL of 1e6 cells were implanted subcutaneously into the right flank of 6-8-week- old female C57BL/6J mice (The Jackson Laboratory). [0756] MC38 cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200 uL of 5e5 cells were implanted subcutaneously into the left flank of 6-8-week-old female C57BL/6J mice for the rechallenge study (The Jackson Laboratory). [0757] MC38.hTAAPos cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200 uL of 1e6 cells were implanted subcutaneously into the right flank of 6-8-week- old female hIFNAR mice (Velocigene). [0758] MC38h.TAAPos cells were cultured in DMEM, 10% FBS, P/S/G, NaPyr, 1% HEPES, 1% NEAA.200ul of 1e6 cells were implanted subcutaneously into the right flank of 6-8-week- old female mice having been humanized for TAA and hCD3 mice (Velocigene). [0759] Tumor size was recorded twice a week and was estimated using the following formula: (length x width2)/2. Mice were randomized and treatment were initiated once tumors reached 100-150 mm3. The treatment groups and dosing used for each study are noted in the figure legends. [0760] Mice whose tumors exceeded 2000 mm3 or exhibited signs of distress at any time during the study were euthanized humanely as per IACUC-approved animal protocols. [0761] FIG.16 depicts results following parental MC38 tumor cell rechallenge in mice having initially cleared MC38.hTAAPos engrafted tumors (the same as used in Fig. 13). On day 60 after MC38.hTAAPos tumor cell inoculation, tumor free mice (black square) were rechallenged with parental MC38 cells without overexpression of human TAA. Compared to control naïve mice (open circle), mice previously treated with anti-CD20 -LP6A conjugate are protected against tumor rechallenge. Data represent mean tumor volumes (mean+/-SEM) over time (post-rechallenge). [0762] FIG. 17 depicts results following treatment of mice having been inoculated with MC38.hTAAPos tumor cells with 3 doses every seven days of anti-CD20-LP11A conjugate in wild type mice (closed symbols with solid lines) and in humanized IFNAR mice (open symbols with dashed lines) that lack the ability to respond to murine type I IFN. Regression of tumor was observed after treatment with 5 mg/kg of anti-CD20-LP11A conjugate (closed triangle) when compared to saline treated animals (closed circle) and isotype control antibody conjugate (closed square). Regression of MC38.hTAAPos tumors was not observed in the humanized IFNAR mice treated with 5 mg/kg of anti-CD20-LP11A conjugate (open triangle); isotype control antibody conjugate (open square) or saline treated animals (open circle). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0763] FIG. 18 depicts results following treatment of mice having been inoculated with MC38.hTAAPos tumor cells with 3 doses every seven days of anti-CD20-LP6A conjugate with or without 5 doses every four days of anti-CD20 x anti-hCD3 bispecific antibody in mice humanized for TAA and human CD3. Regression of tumor was observed after treatment with 2.5 mg/kg of anti-CD20-LP6A conjugate in combination with 2.5 mg/kg anti-CD20 x anti-hCD3 bispecific antibody (black triangle), while treatment with 2.5 mg/kg of anti-CD20 in combination with 2.5 mg/kg isotype control for the bispecific antibody (open triangle), 2.5 mg/kg of anti- CD20x anti-hCD3 bispecific antibody alone (black circle); 2.5 mg/kg of isotype control antibody-(NC-1) in combination with 2.5 mg/kg anti-CD20 x anti-hCD3 bispecific antibody (black square) resulted in tumor growth delay when compared to mice treated with 2.5 mg/kg isotype control for the bispecific antibody (open circle) or 2.5 mg/kg of isotype control antibody in combination with 2.5 mg/kg isotype control for the bispecific antibody (open square). Data represent mean tumor volumes (mean+/-SEM) over time (post-dose). [0764] FIG.19 depicts the ring opening of the imide bond of the antibody-drug conjugates from the conjugation of the cysteine thiol with the maleimide of the linker-payload. Ring- opening of the imide bond under physiological conditions affords two regio-isomers that one is the thiol attached to the alpha carbon and the other is the thiol attached to the beta carbon to the carboxylic acid group, respectively. EXAMPLE 73 [0765] Hepatitis B virus (HBV) is a partially double stranded DNA virus that infects hepatocytes that results in acute or lifelong chronic disease in humans. Mice lack the receptor for HBV infection in their hepatocytes and thus natural infection in mice is not possible. However, recent technologies have emerged in which Adeno-Associated Virus (AAV) has been modified to encode an HBV genome and is used to deliver the HBV genome into mouse hepatocytes resulting in HBV gene expression that can recapitulate chronic hepatitis B (CHB) disease (e.g., high HBV sAg protein in circulation, detectable HBV DNA in serum). This AAV- HBV mouse model can be used to assess different therapeutic interventions to look at sustained HBV sAg reduction of treatment (functional cure) or complete elimination of HBV infected hepatocytes. [0766] To assess whether an anti-HBV mAb conjugated with a TLR7 agonist as an antibody drug conjugate (ADC) could provide better efficacy than an anti-sAg mAb or TLR7 agonist alone in this CHB mouse model, male C57BL/6 mice were transduced with 1.32E11 viral genomes of AAV8-HBV virus intravenously (via IV). Six weeks later, HBV sAg levels in their serum was measured to determine if they exhibited a CHB phenotype. Mice that had HBV sAg levels ≥1μg/mL in the serum were included for mAb efficacy studies. Mice were then treated with an anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS three times, two weeks apart subcutaneously (SC). MAb doses used are outlined in Table 12 from two independent experiments. Mice were then bled weekly or biweekly to measure sAg levels before, during and after mAb treatments. As shown in FIG. 14 and FIG.15, CHB mice treated with the anti-sAg-TLR7 ADCs displayed rapid and sustained reduction in circulating sAg levels compared to anti-sAg or TLR7 agonist treated mice. Table 13 shows sAg concentrations at study end for each treatment group. Mice treated with anti- sAg-TLR7 ADCs displayed lower sAg levels than anti-mAb or TLR7 agonist treated mice in both experimental studies. Furthermore, the majority of anti-sAg-TLR7 ADC-treated mice at the lower dosed arms had undetectable sAg compared to all other treatment groups even 67 days after the last treatment. Table 12: List of mAbs, TLR7 agonist, or mAb-TRL7 ADCs used in CHB studies
Figure imgf000262_0001
N/A, not applicable. N/I, not included in study Table 13: sAg serum levels at study end from two independent experiments
Figure imgf000262_0002
SD= Standard Deviation. a undetectable sAg for experiment 1= 0.0058μg/mL. b undetectable sAg for experiment 2= 0.039μg/mL. N/I, not included in study [0767] These data suggest that chemically linking a TLR agonist to an anti-HBV sAg mAb as an ADC provides enhanced efficacy over the TLR agonist or anti-sAg mAbs by themselves in a CHB mouse model. Moreover, an anti-HBV sAg mAB-TLR7 ADC seems to rapidly reduce HBV sAg levels as compared a 10-fold higher amount of anti-HBV sAg mAb alone, suggesting that lower doses and/or reduced treatment cycles may be used to obtain a functional cure against CHB. [0768] This disclosure is not to be limited in scope by the embodiments disclosed in the examples which are intended as single illustrations of individual aspects, and any equivalents are within the scope of this disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. [0769] Various references such as patents, patent applications, and publications are cited herein, the disclosures of which are hereby incorporated by reference herein in their entireties, including the disclosure of U.S. Provisional Application No.63/429,096. EXAMPLE 74 [0770] The metabolism of QoLP11A was evaluated in human liver S9 with NADPH and UDPGA as follows: [0771] Assay buffer: 100 mM potassium phosphate buffer (K+/Mg2+ buffer, pH7.4):
Figure imgf000263_0001
[0772] Preparation of cofactor solution in the K+/Mg2+ Buffer:
Figure imgf000263_0002
[0773] Dilute initial stock of liver S9 in K+/Mg2+ buffer, pH 7.4 from 20 mg/ml to 2× Liver S9 (2 mg/mL) with alamethicin (50 µg/mL):
Figure imgf000263_0003
[0774] Then pre-incubate the 2 mg/mL liver S9 solution with 50 µg/mL alamethicin on ice for 15 min. [0775] Assay procedure: Prepare 2× LS9/compound solution: T0: add 199 µL 2 mg/mL liver S9 solution + 100 µL of 8 mM NADPH solution + 100 µL of 20 mM UDPGA solution + 1200 µL of ACN, vortex at 1000 rpm for 5 min, then add 1 µL of 4 mM cpd solution. T240: add 199 µL 2 mg/mL liver S9 solution + 100 µL of 8 mM NADPH solution + 100 µL of 20 mM UDPGA solution, prewarm the T240 sample at 37°C for 5 min and 1 µL of 4 mM cpd solution was added. After 240 min incubation, add 1200 µL of ACN and then vortex at 1000 rpm for 5 min. T240-w/o: add 199 µL 2 mg/mL liver S9 solution + 200 µL of buffer, prewarm the T240-w/o sample at 37°C for 5 min and 1 µL of 4 mM cpd solution was added. After 240 min incubation, add 1200 µL of ACN and then vortex at 1000 rpm for 5 min. Protein precipitation: centrifuge quenched samples at 14000 rpm for 5 min. Sample preparation: evaporate an aliquot of 1200 µL of the supernatant under N2 stream until dry. Reconstitute dried extracts with 200 µL of 25% aqueous ACN, vortex for 2 minutes, centrifuge at 14000 rpm for 5 min, and then inject 3 µL of reconstituted supernatant for LC- UV-MS analysis. [0776] Results [0777] Qo-LP11A was transformed into 11 metabolites in the current study, which were named as ME.M. based on the exact mass of metabolites and the eluting time under the current HPLC conditions. [0778] 83% of Qo-LP11A remained after 4 hours incubation. [0779] No glucuronide conjugates were observed. [0780] Verapamil was included in this study as positive control. The glucuronide conjugates of Verapamil were formed and detected in this study and the data was shown below. [0781] A summary, including observed m/z value, retention time and MS peak area of Qo- LP11A and its metabolites in human liver S9 with NADPH and UDPGA are presented in the following Table 14. Table 14
Figure imgf000265_0001
[0782] While not wishing to be bound by any specific theory, it is believed that the metabolic pathways for Qo-LP11A in human liver S9 with NADPH and UDPGA are as shown in FIGS. 20 and 21. [0783] Metabolic studies of a drug candidate shed light on the pharmacological and pharmacokinetic pathways that the compounds described herein follow. Such studies may also provide essential information on drug safety and its potential toxicity. The metabolites identified in FIGS. 20 and 21 offer a starting point to better understand the desired pharmacological therapeutic effects as well as the potential side effects that may come with the administration of the drug molecule. [0784] Under physiological conditions, the imide ring of the linker connecting an antibody and the payload exists as an equilibrium between an opened ring and a closed five-membered imide ring as shown in FIG.19. Those ADC species with an opened ring and a closed five- membered imide ring show the same or comparable activities in the research described herein. EXAMPLE 75 [0785] To assess whether an anti-HBV antibody conjugated with a TLR7 agonist as an antibody drug conjugate (ADC) could break B cell tolerance and elicit an antibody response to the HBV sAg protein in an AAV-HBV mouse model, male C57BL/6 mice were transduced with 1E11 viral genomes of AAV8-HBV virus intravenously (I.V.) and six weeks later measured HBV sAg levels in their serum to determine if they exhibited a CHB (chronic hepatitis B) phenotype. Mice that had HBV sAg levels ≥1μg/mL in the serum were included in the study. Mice were then treated with an anti-HBV sAg mAb (mAb3) conjugated with an TLR7 agonist (LP6A) at either 44μg or 0.44μg per injection or given PBS five times, one week apart subcutaneously (SC). The sequence of mAb3 is shown in Table 15 below. As shown in FIG. 22, CHB mice treated with anti-HBV sAg-TLR7 ADC at both doses display rapid and sustained reduction in circulating HBV sAg levels until end of study (day 120). At the end of the study, serum was obtained and anti-HBV sAg IgG titers were measured via ELISA to determine endogenous antibody responses. As shown in FIG.23, 10 out of 12 mice treated with an anti- HBV sAg-TLR7 ADC elicited HBV sAg IgG titers above background while none of the PBS mice showed HBV sAg IgG titers which is characteristic of the AAV-HBV mouse model. [0786] One of the hallmarks of chronic hepatitis B infection is tolerogenic immune response to the HBV proteins. This is most notable for immunity to HBV sAg in which there is a minimal T cell response and no HBV sAg IgG responses despite HBV sAg being abundantly expressed. These data suggest that an anti-HBV sAg-TLR7 conjugated mAb (such as mAb3 conjugated to LP6A) can break B cell tolerance to HBV sAg and elicit IgG titers against this viral protein. Table 15
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001

Claims

WHAT IS CLAIMED IS: 1. A compound, wherein the compound is of Formula I:
Figure imgf000269_0001
or is a pharmaceutically acceptable salt thereof, wherein: R1 is H, halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is H, halo, or alkoxy; R3 is -CO2R23, -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or - alkylene-PEG-Y; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; R23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, -NR5R6 or -COZ; Z is -OH, alkoxy or -NR7R8; R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8 are each independently H or alkyl, or, together with the N to which they are attached, form a heterocyclic ring; with the proviso that the compound is not a compound of the formula:
Figure imgf000269_0002
2. The compound of claim 1, wherein: R1 is halo, -NHR4, -OR4, -NH-OR4 or -R4; R2 is halo or alkoxy;
R3 is -CONHR23, -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, heteroalkylene- arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y; R23 is H, alkyl or aryl; R4 is alkyl optionally substituted with alkoxy or heteroalkyl; X is CH or N; Y is -OH, Gly, -NR5R6 or -COZ; Z is -OH or -NR7R8; R5 and R6 are selected from (i), (ii), and (iii): (i) R5 and R6 each H; (ii) R5 is H and R6 is alkyl; (iii) R5 and R6, together with the N to which they are attached, form a heterocyclic ring; and R7 and R8, together with the N to which they are attached, form a heterocyclic ring.
3. The compound of claim 1 or 2 selected with the proviso that R4 is not substituted with hydroxyl.
4. The compound of any one of claims 1-3 selected with the proviso that the alkylene and heteroalkylene portions of R3 are not substituted with oxo.
5. The compound of any one of claims 1-4 selected with the proviso that the compound is not 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine- 2,4-diamine or (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3- methoxyphenyl)methanol.
6. The compound of any one of claims 1-5, wherein R1 is halo, -NHR4, -OR4, -NH-OR4 or -R4, and is a straight chain of 6 atoms in length.
7. The compound of any one of claims 1-6, wherein R1 is halo.
8. The compound of any one of claims 1-6, wherein R1 is -NHR4.
9. The compound of any one of claims 1-6, wherein R1 is -OR4.
10. The compound of any one of claims 1-6, wherein R1 is -NH-OR4.
11. The compound of any one of claims 1-6, wherein R1 is -R4.
12. The compound of any one of claims 1-5, wherein R1 is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt.
13. The compound of any one of claims 1-5, wherein R1 is -NH-n-pentyl.
14. The compound of any one of claims 1-5, wherein R1 is -NH-O-n-butyl.
15. The compound of any one of claims 1-5, wherein R1 is -O-n-pentyl.
16. The compound of any one of claims 1-5, wherein R1 is -n-hexyl.
17. The compound of any one of claims 1-5, wherein R1 is -NH-CH2CH2-OEt.
18. The compound of any one of claims 1-16, wherein R2 is alkoxy.
19. The compound of any one of claims 1-17, wherein R2 is methoxy.
20. The compound of any one of claims 1-16, wherein R2 is H.
21. The compound of any one of claims 1-16, wherein R2 is halo.
22. The compound of any one of claims 1-21, wherein R3 is -CONHR23, -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y.
23. The compound of any one of claims 1-22, wherein R3 is CONHR23.
24. The compound of any one of claims 1-22, wherein R3 is -alkylene-Y.
25. The compound of any one of claims 1-22, wherein R3 is -heteroalkylene-Y.
26. The compound of any one of claims 1-22, wherein R3 is -heteroalkylene-arylene-Y.
27. The compound of any one of claims 1-22, wherein R3 is -(hydroxy)heteroalkylene-Y.
28. The compound of any one of claims 1-22, wherein R3 is -(amino)heteroalkylene-Y.
29. The compound of any one of claims 1-22, wherein R3 is -alkylene-PEG-Y.
30. The compound of any one of claims 1-22, wherein R3 is -CONH2, -CO2H, -CH2-Y, - CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y.
31. The compound of any one of claims 1-22, wherein R3 is -CH2-Y, -CH2-O- heteroalkylene-Y, or -CH2-O-alkylene-Y.
32. The compound of any one of claims 1-22, wherein R3 is -C(Me)2OH, -CO2H -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, - CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, - CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, - CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH - CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, -CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2, or -CH2-1-piperazinyl.
33. The compound of any one of claims 1-22, wherein R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, - CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH - CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, - CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl.
34. The compound of any one of claims 1-33, wherein R4 is n-butyl, n-pentyl, n-hexyl or ethoxyethyl.
35. The compound of any one of claims 1-33, wherein R4 is n-butyl.
36. The compound of any one of claims 1-33, wherein R4 is n-pentyl.
37. The compound of any one of claims 1-33, wherein R4 is n-hexyl.
38. The compound of any one of claims 1-33, wherein R4 is ethoxyethyl.
39. The compound of any one of claims 1-38, wherein R5 and R6 are each independently H or alkyl, or, together with the N to which they are attached, form a piperazinyl ring.
40. The compound of any one of claims 1-38, wherein R5 and R6 are each H.
41. The compound of any one of claims 1-38, wherein R5 is H and R6 is alkyl.
42. The compound of any one of claims 1-38, wherein R5 and R6, together with the N to which they are attached, form 1-piperazinyl.
43. The compound of any one of claims 1-42, wherein Y is OH.
44. The compound of any one of claims 1-42, wherein Y is Gly.
45. The compound of any one of claims 1-42, wherein Y is -NR5R6.
46. The compound of any one of claims 1-42, wherein Y is -COZ.
47. The compound of any one of claims 1-42, wherein Y is -OH, -NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2 or -CO-1-piperazinyl.
48. The compound of any one of claims 1-47, wherein Z is -OH.
49. The compound of any one of claims 1-47, wherein Z is alkoxy.
50. The compound of any one of claims 1-47, wherein Z is -NR7R8.
51. The compound of any one of claims 1-47, wherein Z is -OH, ethoxy, -N-n-Pr2 or 1- piperazinyl.
52. The compound of any one of claims 1-47, wherein Z is -OH or 1-piperazinyl.
53. The compound of any one of claims 1-52, wherein R7 and R8 are each independently H or n-propyl, or, together with the N to which they are attached, form 1-piperazinyl.
54. The compound of any one of claims 1-52, wherein R7 and R8, together with the N to which they are attached, form 1-piperazinyl.
55. A compound selected from:
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0003
and pharmaceutically acceptable salts thereof.
56. A compound, wherein the compound is of Formula II:
Figure imgf000278_0001
or is a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined for Formula I in claim 1; R9 is a divalent group formed by removal of a terminal hydrogen (i.e., a hydrogen distal from the phenyl group to which R9 is attached) from an R3 group, as defined in claim 1; and L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; with the proviso that the compound is not a compound of the formula:
Figure imgf000278_0002
Figure imgf000279_0001
57. The compound of claim 56, wherein R9 is - alkylene-Y1-, -heteroalkylene-Y1-, - heteroalkylene-arylene-Y1-, -(hydroxy)heteroalkylene-Y1, -(amino)heteroalkylene-Y1, or - alkylene-PEG-Y1.
58. The compound of claim 56 or 57, wherein R9 is -alkylene-Y1-.
59. The compound of claim 56 or 57, wherein R9 is -heteroalkylene-Y1-.
60. The compound of claim 56 or 57, wherein R9 is -heteroalkylene-arylene-Y1-.
61. The compound of claim 56 or 57, wherein R9 is -(hydroxy)heteroalkylene-Y1-.
62. The compound of claim 56 or 57, wherein R9 is -(amino)heteroalkylene-Y1.
63. The compound of claim 56 or 57, wherein R9 is -alkylene-PEG-Y1.
64. The compound of claim 56 or 57, wherein R9 is -CH2-Y1-, -CH2-O-heteroalkylene-Y1-, or -CH2-O-alkylene-Y1-.
65. The compound of claim 56 or 57, wherein R9 is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH- , -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CO-, - CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, - CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH-, or -CH2-1-piperazin-4-yl.
66. The compound of any one of claims 56-65, wherein Y1 is -O-.
67. The compound of any one of claims 56-65, wherein Y1 is Gly.
68. The compound of any one of claims 56-65, wherein Y1 is -NR5-.
69. The compound of any one of claims 56-65, wherein Y1 is -COZ1.
70. The compound of any one of claims 56-65, wherein Y1 is -O-, -NH-, 1-piperazin-4-yl, -COO-, or -CO-1-piperazin-4-yl.
71. The compound of claim 69, wherein Z1 is -O-.
72. The compound of claim 69, wherein Z1 is -NR7-.
73. The compound of claim 69, wherein Z1 is -O- or 1-piperazin-4-yl.
74. The compound of claim 68, wherein R5 is H.
75. The compound of claim 68, wherein R5 is alkyl.
76. The compound of any one of claims 55-75, wherein L is non-cleavable under physiological conditions.
77. The compound of any one of claims 55-75, wherein L is cleavable under physiological conditions.
78. The compound of claim 77, wherein L is an acid-labile linker, a hydrolysis-labile linker, an enzymatically cleavable linker, a reduction-labile linkers or a self-immolative linker.
79. The compound of any one of claims 55-78, wherein L is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citruline unit, or a para-aminobenzyl (PAB) unit.
80. The compound of any one of claims 55-79, wherein L comprises one or more amino acids.
81. The compound of any one of claims 55-80, wherein L comprises a self-immolative group.
82. The compound of any one of claims 55-81, wherein L comprises p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC).
83. The compound of any one of claims 55-82, wherein L comprises a maleimido, an N- hydroxysuccinimido ester or cyclooctynyl group.
84. The compound of any one of claims 55-83, wherein L is a group selected from 2- maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl,
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
.
85. A compound selected from:
Figure imgf000283_0002
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0002
and pharmaceutically acceptable salts thereof.
86. A compound, wherein the compound is of Formula III:
Figure imgf000287_0001
or is a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined in claim 1; L is any group or moiety that links, connects, or bonds to an antigen-binding domain ABD; R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form the 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene or heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six.
87. The compound of claim 86, wherein R11 and R12 are, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, wherein when R11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6- membered heterocyclyl; R14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, wherein when R14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R13 to form a 4-, 5-, or 6-membered heterocyclyl; R13 is hydrogen, alkyl, alkylene, or heteroalkylene, wherein when R13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is further bonded to R11 or R14 to form a 4-, 5-, or 6-membered heterocyclyl; R15 is hydrogen or alkyl; R16 is alkylene alkylene-arylene, heteroalkylene, heteroalkylene-arylene, - (hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is zero, one, two, three, four, five, or six.
88. The compound of claim 86 or claim 87, wherein the TLR7 agonist used in preparing the compound is P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, P39, P41, P42 or P43.
89. An antibody-drug-conjugate (ADC), comprising the compound of any one of claims 1- 88 or compounds of the formulae:
Figure imgf000288_0001
90. The ADC of claim 89, wherein the ADC is of Formula IV:
Figure imgf000288_0002
or is a pharmaceutically acceptable salt thereof, wherein: R1, R2, R9 and X are as defined in claim 1 for Formula I and claim 56 for Formula II; L1 is a divalent linker; ABD is an antigen-binding domain; and k is an integer from one to thirty.
91. The ADC of claim 89 or 90 that is ABD-LP1, ABD-LP6A, ABD-LP7A, ABD-LP8A, ABD-LP10A or ABD-LP11A.
92. The ADC of claim 89, wherein the ADC is of Formula V:
Figure imgf000289_0001
or is a pharmaceutically acceptable salt thereof, wherein: R1, R2 and X are as defined in claim 1 for Formula I; R10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene- arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG- NH-; ABD is an antibody that contains a Q295 residue, an N297Q mutation, and/or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14) or LLQLLQGA (SEQ ID NO: 15) sites; and k is an integer from one to thirty.
93. The ADC of claim 92, wherein R10 is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-.
94. The ADC of claim 92 or claim 93, wherein R10 is -alkylene-NH-.
95. The ADC of claim 92 or claim 93, wherein R10 is -heteroalkylene-NH-.
96. The ADC of claim 92 or claim 93, wherein R10 is -heteroalkylene-arylene-NH-.
97. The ADC of claim 92 or claim 93, wherein R10 is -(hydroxy)heteroalkylene-NH-.
98. The ADC of claim 92 or claim 93, wherein R10 is -(amino)heteroalkylene-NH-.
99. The ADC of claim 92 or claim 93, wherein R10 is wherein R10 is -alkylene-PEG-NH-.
100. The ADC of claim 92 or claim 93, wherein R10 is -CH2-NH-, -CH2-O-heteroalkylene- NH-, or -CH2-O-alkylene-NH-.
101. The ADC of claim 92 or claim 93, wherein R10 is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4- NH-1-phenyl), -CH2OCH(NH-)CH2OH or -CH2NH-.
102. The ADC of claim 92 or claim 93, wherein R10 is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4- NH-1-phenyl) or -CH2NH-.
103. The ADC of any one of claims 89-102, having the formula ABD-P4, ABD-P5, ABD- P7, ABD-P9, ABD-P10, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, ABD-P34 or ABD-P41, wherein ABD is attached to the payload (i.e., TLR7 agonist) on the amino group of R3.
104. The ADC of claim 89, wherein the ADC is of Formula VI:
Figure imgf000290_0001
or is a pharmaceutically acceptable salt thereof, wherein: L1 is a divalent linker; R1, R2, R16, R11, R12, R13, R14, R15, X, and x are as defined for Formula III in claim 86; and k is an integer from one to thirty.
105. The ADC of claim 104 having the formula ABD-L1-P2, ABD-L1-P6, ABD-L1-P8, ABD- L1-P17, ABD-L1-P18, ABD-L1-P19, ABD-L1-P20, ABD-L1-P23, ABD-L1-P27, ABD-L1-P29, ABD-L1-P32, ABD-L1-P33, ABD-L1-P37, ABD-L1-P39 or ABD-L1-P42, where ABD-L1 is attached to the payload (i.e., TLR7 agonist) on the alcohol group of R3.
106. The ADC of any one of claims 89-105, wherein ABD has binding specificity for a transmembrane molecule (e.g., receptor) expressed on a tumor.
107. A pharmaceutical composition, comprising a compound of any one of claims 1-88 or an ADC of any one of claims 89-106, and a pharmaceutically acceptable carrier.
108. A method of treating or diagnosing disease, comprising administering to a subject a compound of any one of claims 1-88 or an ADC of any one of claims 89-106 or a pharmaceutical composition of claim 107.
109. The method of claim 108, wherein the method treats a disease.
110. The method of claim 108 or 109, wherein the disease is cancer.
111. The method of any one of claims 108-110, wherein the disease is acute myelogenous leukemia, adult T-cell leukemia, astrocytomas, bladder cancer, breast cancer, PRLR positive (PRLR+) breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastomata, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), Kaposi's sarcoma, kidney cancer, leiomyosarcomas, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC)), lymphomas, malignant gliomas, malignant mesothelioma, melanoma, mesothelioma, malignant mesothelioma, MFH/fibrosarcoma, multiple myeloma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic carcinoma, prostate cancer, castrate-resistant prostate cancer, renal cell carcinoma, residual cancer, rhabdomyosarcoma, stomach cancer, synovial sarcoma, thyroid cancer, uterine cancer and Wilms' tumor.
112. The method of any one of claims 108-111, wherein the disease is breast cancer.
113. The method of any one of claims 108-111, wherein the disease is prostate cancer.
114. An antibody-drug-conjugate (ADC), comprising a. an antigen-binding domain (ABD) having binding specificity to a hepatitis B virus surface antigen (HBV sAg); and b. a Toll-like receptor 7 (TLR7) agonist that links the ABD with a divalent linker.
115. The ADC of claim 114, wherein the TLR7 agonist is any one of P2-P39 and P41-P48.
116. The ADC of claim 114, wherein the TLR7 agonist with a divalent linker is any one of LP1-5, LP6A-6B, LP7A-7E, LP8A-8B, LP9, LP10A-10B, LP11A-11D, and LP12-15.
117. The ADC of claim 114, wherein the ABD is an antibody against an HBV sAg or a fragment thereof.
118. The ADC of claim 114, wherein the ABD is a human antibody or a humanized antibody.
119. The ADC of claim 114, wherein the ABD comprises a scFv having binding specificity to a HBV sAg.
120. The ADC of claim 114, wherein the ABD comprises VH chain and VL chain of an antibody against a HBV sAg.
121. The ADC of claim 114, wherein the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of an antibody against a HBV sAg.
122. The ADC of claim 114, wherein the ABD comprises an Fc region.
123. The ADC of claim 120, wherein the Fc region comprises a modification for enhanced binding to FcγR.
124. The ADC of any one of claims 114-123, wherein said ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 25, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 29.
125. The ADC of claim 124, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 30, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32.
126. The ADC of claim 124 or claim 125, wherein said HCVR comprises the amino acid sequence of SEQ ID NO: 25.
127. The ADC of claim 126, wherein said HCVR is a component of a heavy chain comprising the amino acid sequence of SEQ ID NO: 33.
128. The ADC of claim 124 or claim 125, wherein said LCVR comprises the amino acid sequence of SEQ ID NO: 29.
129. The ADC of claim 126, wherein said LCVR is a component of a light chain comprising the amino acid sequence of SEQ ID NO: 34.
130. The ADC of any one of claims 124-129, wherein said ABD is a component of an antibody or antigen-binding fragment thereof.
131. A method of treatment, comprising administering to a subject in need thereof an effective amount of the ADC of any one of claim 114-130.
132. The method of claim 131, wherein the subject has chronic Hepatitis B.
133. The method of claim 131, wherein said Hepatitis B is chronic Hepatitis B.
134. The method of claims 131-133, wherein the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or the pharmaceutical composition.
135. The method of any one of claims 131-133, further comprising, before the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject.
136. The method of any one of claims 131-135, further comprising, after the administering, measuring circulating HBV DNA or HBV sAg in serum of the subject to assess therapeutic efficacy of the ADC or the pharmaceutical composition.
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