TLR7 agonists and their antibody-drug conjugates

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

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 429,096, filed November 30, 2022, and U.S. Provisional Application No. 63 / 578,109, filed August 22, 2023, both of which are incorporated by reference as if fully set forth.

[0002] (Incorporated by reference to the sequence listing)

[0001] This application contains a Sequence Listing, which has been submitted electronically as an XML file entitled "2387951.xml". The Sequence Listing is 30,417 bytes in size and was created on November 27, 2023. It is incorporated herein by reference in its entirety.

[0003] (Field) Provided herein are TLR7 agonists and antibody drug conjugates (ADCs) thereof. [Background technology]

[0004] (background) Toll-like receptors (TLRs) are a subset of pattern recognition receptors (PRRs) that play an important role in innate immune responses. TLRs are classified into two groups based on their subcellular localization, and endosomal TLRs have attracted pharmaceutical attention. Among these endosomal TLRs, TLR7 has been widely 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 note herein, TLR7 agonists are being investigated as cancer immunotherapeutics. One TLR7 agonist, Aldara® (imiquimod), is approved by the US FDA and is indicated for the treatment of actinic keratosis, superficial basal cell carcinoma, and external genital warts.

[0005] ADCs combine the specificity of antibodies with the ability to site-specifically target specific cell types or tissues using a payload. Research in this area has attracted significant interest and led to the sale of pharmaceutical products, including ADCETRIS® (brentuximab vedotin) and KADCYLA™ (ado-trastuzumab emtansine). ADCs with TLR7 agonist payloads have been reported. See, for example, U.S. Patent Nos. 10,472,420, 10,780,180, 10,548,985, 10,722,591, and 10,675,358; PCT Publication WO 2020 / 181050. However, such ADCs have not been approved for human use.

[0006] Thus, there is a continuing need for TLR agonists and their ADCs for the treatment of various diseases, including cancer and chronic hepatitis B. Summary of the Invention

[0007] (overview) In one aspect, the present disclosure provides a TLR7 agonist of Formula I, or a pharmaceutically acceptable salt thereof, for use in the compositions and methods provided herein: [ka] (In the formula: R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is H, halo, or alkoxy; R 3 -CO2R 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, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and R 7 and R 8 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 disclosure provides a TLR7 agonist-linker of Formula II, or a pharmaceutically acceptable salt thereof, for use in preparing an ADC provided herein: [ka] (In the formula: R 1 , R 2 and X is as defined for formula I; R 9 is defined as R 3 The terminal hydrogen from the group (i.e., R 9 is a divalent radical formed by removal of a hydrogen atom distal to a phenyl group to which is attached L is any group or moiety that links, connects or binds to the antigen binding domain ABD).

[0009] In certain embodiments, the ADCs provided herein are useful in methods of treatment, imaging, or diagnosis.

[0010] Also provided are antibody-drug conjugates (ADCs) comprising an antigen-binding domain (ABD) specific for an antigen and a Toll-like receptor 7 (TLR7) agonist, and uses of the ADCs to treat diseases. In some embodiments, the antigen is 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) with binding specificity for 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 bivalent linker linking the ABD to the TLR7 agonist.

[0013] In some embodiments, the ADC is according to Formula IV: [ka] (In the formula: L 1 is a bivalent linker; R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is H, halo, or alkoxy; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 9 is R 3 is a divalent radical formed by removal of hydrogen from 3 is R 9 is a group attached to the phenyl group at position R 3 -CO2H, -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; X is CH or N; Y is -OH, -Gly, -NR 5 R 6 , or -COZ; Z is -OH, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; R 7 and R 8are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and and k is an integer from 1 to 30.

[0014] In some embodiments, the ADC comprises an ABD linked to a compound of Formula III: [ka] (In the formula: R 1 , R 2 and X is 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, where R 11 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 11 or R 14 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6).

[0015] In some embodiments, 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. 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: [ka] (In the formula: R 1 , R 2 and X is 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-; The ABD may contain the Q295 residue, the N297Q mutation, and / or one or more modified [ka] an antibody containing the and k is an integer from 1 to 30.

[0017] In some embodiments, the ADC is according to Formula VI: [ka] (In the formula: L 1 is a bivalent 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 and k is an integer from 1 to 30.

[0018] In some embodiments, the ADC comprises an ABD-L linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39. 1 Includes:

[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 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 in the ABD.

[0021] In some embodiments, the ABD is an antibody or fragment thereof against HBV sAg. 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 an scFv with binding specificity for HBV sAg. In some embodiments, the ABD is a V of an antibody against HBV sAg. H Chain and V L Contains chains.

[0022] In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody against 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 disclosure provides pharmaceutical compositions comprising an ADC disclosed herein and one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0024] In yet another aspect, the disclosure provides methods of treatment comprising administering to a subject in need thereof an effective amount of an ADC or 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 their serum prior to administration of the ADC or pharmaceutical composition.

[0026] In some embodiments, the method further comprises measuring circulating HBV DNA or HBV sAg in the subject's serum prior to administration. In some embodiments, the method further comprises measuring circulating HBV DNA or HBV sAg in the subject's serum after administration to assess the therapeutic efficacy of the ADC or pharmaceutical composition.

[0027] In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated two, three, or more times. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated at least one week apart, two weeks apart, three weeks apart, or four weeks apart. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated one week apart, two weeks apart, three weeks apart, or four weeks apart. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated one month apart, two months apart, or three months apart.

[0028] In some embodiments, the ADC or pharmaceutical composition is administered orally, intravenously, intraperitoneally, by inhalation, intranasally, intramuscularly, or subcutaneously.

[0029] One aspect of the disclosure provides an ADC or pharmaceutical composition for use in therapy, hi some embodiments, the ADC or pharmaceutical composition is for use in treating chronic hepatitis B in a subject in need thereof.

[0030] Another aspect of the disclosure provides an ADC or pharmaceutical composition for the manufacture of a medicament. In some embodiments, the medicament is for the treatment of chronic hepatitis B in a subject in need thereof. [Brief explanation of the drawings]

[0031] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows the in vitro plasma stability of the anti-HER2 Ab-LP1 ADC (Example 68).

[0032] [Figure 2] FIG. 2 shows the in vitro plasma stability of the anti-HER2 Ab-LP6A ADC (Example 68).

[0033] [Figure 3] Figure 3 shows the in vitro plasma stability of the anti-HER2 Ab-LP11A ADC (Example 68).

[0034] [Figure 4] Figure 4 shows the in vitro plasma stability of the anti-HER2 Ab-LP7A ADC (Example 68).

[0035] [Figure 5] FIG. 5 shows a conjugation scheme for conjugating antibodies with the linker payloads provided herein (Examples 63, 64, and 69).

[0036] [Figure 6] FIG. 6 is a preparative SEC chromatogram of the conjugation mixture showing clear separation of ADC monomers from aggregates (“HMW”) and unconjugated linker payload (“free drug”).

[0037] [Figure 7] FIG. 7 is an analytical SEC chromatogram of the SEC-purified antibody-TLR7 conjugate showing 99.7% monomeric purity.

[0038] [Figure 8] Figure 8 shows the LC-ESI-MS spectrum of the deglycosylated and reduced ADC sample. The calculated average DAR value was 1.84. The deconvoluted mass spectrum showed light chain species (LC, LCl) and heavy chain species (HC, HC1, HC2, etc.). The average DAR can be calculated from the LC and HC drug loadings.

[0039] [Figure 9] Figure 9 shows the HIC chromatogram of the antibody and its LP11A conjugate, revealing a mixture of three species: two DARs (51%), four DARs (28%), and unconjugated antibody (21%). The ADC has an average DAR of 2.1.

[0040] [Figure 10] Figure 10 shows results after a single treatment of anti-HER2 Ab-LP6A ADC (Table 3) in the N87 xenograft tumor model. Dosing occurred on day 0. Tumor regression was observed after treatment with 5 mg / kg (gray circles) of anti-HER2 Ab-LP6A ADC, whereas treatment with 1 mg / kg (gray squares) of anti-HER2 Ab-LP6A ADC resulted in tumor stasis when compared with saline-treated animals (open circles). No regression of N87 gastric tumors was observed in N87 xenograft mice treated with 5 mg / kg of the isotype control Ab-LP6A ADC (Table 3) (black circles), 0.5 mg / kg (gray triangles), or 0.1 mg / kg (gray diamonds) of anti-HER2 Ab-LP6A ADC when compared with saline-treated animals (open circles). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0041] [Figure 11] Figure 11 shows the results after 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 occurred on day 0. Tumor regression was observed after treatment with 5 mg / kg of anti-HER2 Ab-LP6A ADC (gray circles), 5 mg / kg of anti-HER2 Ab-LP11A ADC (gray squares), or 5 mg / kg of anti-HER2 Ab-LP7A ADC (gray triangles) when compared to saline-treated animals (white circles). No regression of N87 gastric tumors was observed in N87 xenografted mice treated with 5 mg / kg of the isotype control Ab-LP6A ADC (black circles) (Table 3), the isotype control Ab-LP11A ADC (black squares) (Table 3), or the isotype control Ab-LP7A ADC (black triangles) (Table 3) when compared with saline-treated animals (white circles). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0042] [Figure 12] Figure 12 shows the results after treatment of trastuzumab-resistant HER2 medium human JIMT-1 xenograft tumors. Dosing began on day 0 and every 7 days thereafter with a total of four doses of anti-HER2 Ab-LP6A ADC (Table 3) or in combination with pertuzumab. Tumor regression was observed after treatment with 5 mg / kg of anti-HER2 Ab-LP6A ADC in combination with 5 mg / kg of pertuzumab (gray squares), while treatment with 5 mg / kg of anti-HER2 Ab-LP6A ADC alone (gray circles) resulted in 45 days of tumor stasis when compared to animals treated with 5 mg / kg of unconjugated mAb2 alone (white circles). No regression of JIMT-1 breast tumors was observed following treatment with 5 mg / kg of the isotype control Ab-LP6A ADC (black circles) (Table 3), the isotype control Ab-LP6A ADC in combination with 5 mg / kg of pertuzumab (black squares), or 5 mg / kg of unconjugated mAb2 in combination with 5 mg / kg of pertuzumab (white squares) when compared to animals treated with 5 mg / kg of unconjugated mAb2 alone (white circles). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0043] [Figure 13]Figure 13 shows the results after treatment of MC38 tumors modified to express human CD20. Beginning on day 0, tumor-bearing mice were treated with a total of three 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 squares), compared with animals treated with saline (white circles). MC38hCD20 tumor regression was not observed in MC38hCD20 syngeneic mice treated with 5 mg / kg of anti-mIgG2a Ctrl Ab-LP1 ADC (black circles) (Table 3) or 5 mg / kg of unconjugated anti-CD20 Ab (white squares), compared with animals treated with 5 mg / kg of saline (white circles). Data represent the mean tumor volume (mean + / - SEM) over time (post-dose).

[0044] [Figure 14] Figure 14 shows HBV sAg levels measured in a mouse model of chronic hepatitis B (CHB) disease after subcutaneous (SC) treatment with anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3 + LP1 or mAb4 + LP1), TLR7 agonist (LP1), or PBS three times at 2-week intervals (Example 51; Experiment 1).

[0045] [Figure 15] Figure 15 shows HBV sAg levels measured in a mouse model of chronic hepatitis B (CHB) disease after subcutaneous (SC) treatment with anti-sAg mAb (mAb3), anti-sAg mAb-TLR7 agonist (mAb3 + LP1 or mAb4 + LP1), TLR7 agonist (LP1), or PBS three times at 2-week intervals (Example 51; Experiment 2).

[0046] [Figure 16]Figure 16 shows the results after parental MC38 tumor cell rechallenge in mice that had first cleared MC38.hTAAPos-implanted tumors (the same as those used in Figure 13). Sixty days after MC38.hTAAPos tumor cell inoculation, tumor-free mice (black squares) were rechallenged with parental MC38 cells that do not overexpress human TAAs. Compared to control, untreated mice (white circles), mice previously treated with anti-CD20-LP6A conjugates are protected from tumor rechallenge. Data represent the mean tumor volume (mean + / - SEM) over time (after rechallenge).

[0047] [Figure 17] Figure 17 shows the results of treatment of mice inoculated with MC38.hTAAPos tumor cells with three doses of anti-CD20-LP11A conjugate every 7 days in wild-type mice (open symbols with solid lines) and humanized IFNAR mice (open symbols with dashed lines), which lack the ability to respond to murine type I IFN. Tumor regression was observed after treatment with 5 mg / kg of anti-CD20-LP11A conjugate (open triangles) compared with animals treated with saline (closed circles) and an isotype control antibody conjugate (closed squares). MC38.hTAAPos tumor regression was not observed in humanized IFNAR mice treated with 5 mg / kg of anti-CD20-LP11A conjugate (open triangles); an isotype control antibody conjugate (open squares); or saline-treated animals (open circles). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0048] [Figure 18]Figure 18 shows results in mice humanized for TAA and human CD3 after treatment of mice inoculated with MC38.hTAAPos tumor cells with 3 doses of anti-CD20-LP6A conjugate every 7 days, with or without 5 doses of anti-CD20 x anti-hCD3 bispecific antibody every 4 days. Tumor regression was observed after treatment with 2.5 mg / kg of anti-CD20-LP6A conjugate in combination with 2.5 mg / kg of anti-CD20×anti-hCD3 bispecific antibody (black triangles), whereas treatment with 2.5 mg / kg of anti-CD20 in combination with 2.5 mg / kg of bispecific antibody isotype control (white triangles), 2.5 mg / kg of anti-CD20×anti-hCD3 bispecific antibody alone (black circles); 2.5 mg / kg of isotype control antibody-(NC-1) in combination with 2.5 mg / kg of anti-CD20×anti-hCD3 bispecific antibody (black squares) resulted in tumor growth delay when compared to mice treated with 2.5 mg / kg of bispecific antibody isotype control (white circles) or 2.5 mg / kg of isotype control antibody in combination with 2.5 mg / kg of bispecific antibody isotype control (white squares). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0049] [Figure 19] Figure 19 shows the opening of the imide bond of an antibody-drug conjugate by conjugation of a cysteine ​​thiol with a maleimide of the linker-payload. The opening of the imide bond under physiological conditions gives two positional isomers, one with the thiol attached to the α-carbon and the other with the thiol attached to the β-carbon relative to the carboxylic acid group.

[0050] [Figure 20] FIG. 20 is a scheme showing one possible metabolic pathway for the compound referred to herein as Qo-LP11A.

[0051] [Figure 21]FIG. 21 is a scheme showing another possible metabolic pathway for the compound referred to herein as Qo-LP11A.

[0052] [Figure 22] Figure 22 shows hepatitis B virus surface antigen (HBV sAg) levels measured in a mouse model of chronic hepatitis B (CHB) disease after five weekly subcutaneous treatments with anti-sAg monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate-buffered saline (PBS). mAb3+LP6A was effective in reducing HBV sAg levels compared to the PBS control.

[0053] [Figure 23] Figure 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) after initial treatment with anti-surface antigen (sAg) monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate-buffered saline (PBS) subcutaneously five times at weekly intervals. Mice treated with mAb3+LP6A had higher titers compared to control mice treated with PBS. DETAILED DESCRIPTION OF THE INVENTION

[0054] (Detailed explanation) (I. Definition) To facilitate understanding of the disclosure set forth herein, several terms are defined below.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used 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 multiple definitions for terms herein, those in this section prevail unless stated otherwise.

[0056] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0057] As used herein, "subject" refers to an animal, e.g., a mammal, including a human, e.g., a patient.

[0058] As used herein, biological activity refers to the in vivo activity of a compound or the physiological response that results from in vivo administration of a compound, composition, or other mixture. Thus, biological activity encompasses the therapeutic effects and pharmacokinetic behavior of such compounds, compositions, or mixtures. Biological activity can be observed in in vitro systems designed to test for such activity.

[0059] As used herein, "antigen-binding domain" or "ABD" refers to any peptide, polypeptide, nucleic acid molecule, scaffold-based molecule, peptide display molecule, or polypeptide-containing construct capable of specifically binding to a particular antigen of interest. As used herein, "antigen-binding domain" includes antibodies and antigen-binding fragments of antibodies. All references herein to proteins, polypeptides, and protein fragments 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 phrases "specifically bind" or "specifically binds to" mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as binding to an antigen with a specific binding affinity of at least about 1 x 10 -8 can be characterized by an equilibrium dissociation constant less than or equal to M (e.g., a smaller K D(Indicates 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 be identified, for example, by real-time label-free biolayer interference assays on an Octet® HTX biosensor that specifically bind to a target antigen. Furthermore, multispecific antibodies that bind to one domain in a target antigen and one or more additional antigens or bispecifics that bind to two different regions of a target antigen are still considered to be "specifically binding" antibodies as used herein. In addition to neutralizing antibodies, antibodies that specifically bind to a target antigen but are non-neutralizing can also be used within the scope of the present disclosure to create antibody-drug conjugates. Such antibodies can function, for example, to deliver a payload to cells expressing the target antigen.

[0061] The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 as measured by a real-time label-free biolayer interference assay, e.g., Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution affinity ELISA. -8 M; preferably 10 -9 M; more preferably, 10 -10 M, and even more preferably 10 -11 M, and even more preferably 10 -12 M's, K D The term "mAb" refers to a mAb that has binding affinity for its target antigen, expressed as

[0062] "Slow dissociation rate", "K off " or "k d " refers to a concentration of 1 x 10 or more as determined by real-time label-free biolayer interference assay, e.g., Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™. -3 s -1 Less than 1 x 10, preferably -4 s -1 This refers to an antibody that dissociates from a target antigen with the following rate constant:

[0063] As used herein, an "unrelated antigen" is a protein, peptide, or polypeptide that has less than 95% amino acid identity with one another.

[0064] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM) that contain four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V). H The heavy chain constant region is C H 1. C H 2, and C H Each light chain contains three domains, designated as V and V. Each light chain contains a light chain variable region (herein referred to as LCVR or V). L The light chain constant region comprises one domain (C L 1) V H and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). 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. H The three CDRs are called HCDR1, HCDR2, and HCDR3, and V L The three CDRs 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 derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0066] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may nevertheless include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences. 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, e.g., a mouse, have been grafted onto human framework sequences.

[0067] As used herein, the term "humanized antibody" refers to a chimeric antibody containing minimal sequence derived from a non-human antibody. Humanized antibodies are typically human antibodies (recipient antibodies) in which residues from one or more CDRs have been replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, e.g., a mouse, rat, rabbit, chicken, or non-human primate antibody, that has the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced with corresponding framework region residues from the donor antibody. Humanized antibodies may also contain 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 any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., 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 involving splicing of human immunoglobulin gene sequences into other DNA sequences.

[0069] The terms "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, e.g., FASTA, BLAST, or GAP, such as those discussed in WO 2016 / 100807 or US 2016 / 0176953 A1, each of which is incorporated herein by reference in its entirety. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0070] As used herein in the context of amino acid sequences, the phrase "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity, when optimally aligned, e.g., by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that 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 detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0072] As used herein, "K D The term "" refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise indicated, the KD K values ​​are determined at 25 °C by surface plasmon resonance assay. D Points to a value.

[0073] As used herein, pharmaceutically acceptable salts include 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 alkaline 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, disodium hydrogen phosphate and disodium phosphate; and salts of mineral acids, such as, but not limited to, hydrochloride and sulfate; and salts of organic acids, such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, mesylate, and fumarate.

[0074] As used herein, the terms "treat," "treating," or "treatment" refer to a reduction in the severity or amelioration of at least one symptom or sign of a disease, e.g., cancer or hepatitis B infection, by administration of a therapeutic agent, such as the disclosed antibodies, to a subject in need thereof. These terms include inhibition of disease progression or worsening of infection. These terms also include a positive prognosis of the disease, e.g., the subject may be free of infection, the subject may have reduced or no viral titers, the subject may have tumor regression, and the like, by administration of a therapeutic agent, such as the disclosed antibodies or antibody-drug conjugates.

[0075] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the development of any symptom or sign of a disease (e.g., cancer or hepatitis B infection) by administration of a disclosed antibody or antibody-drug conjugate. This term includes preventing the spread of infection in a subject who has been exposed to the virus or is at risk of having a hepatitis B infection.

[0076] The phrase "therapeutically effective amount" refers to the 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, e.g., Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

[0077] As used herein, amelioration of symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any relief, whether permanent or temporary, persistent or transient, that can result from or be associated with administration of the compound or pharmaceutical composition.

[0078] As used herein, IC 50 refers to the amount, concentration, or dosage of a particular test compound that achieves 50% inhibition of a maximal response in an assay that measures such response.

[0079] Where moieties are designated by their conventional chemical formula and written from left to right, they equally encompass the chemically identical moiety obtained by writing the structure from right to left, e.g., -CHO- is equivalent to -OCH-.

[0080] The term "alkyl," by itself or as part of another substituent, means, unless otherwise specified, a straight-chain (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 alkyl. Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, and includes groups having 10 or fewer carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 6 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 specified, a straight-chain (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 alkenyl. Typically, an alkenyl (or alkenylene) group has from 1 to 24 carbon atoms, including groups having 10 or fewer carbon atoms. A "lower alkenyl" or "lower alkenylene" is a shorter chain alkenyl or alkenylene group, typically 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 higher homologs and isomers.

[0082] The term “alkynyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical having one or more carbon-carbon triple bonds, and which may include divalent and multivalent radicals having the specified number of carbon atoms (i.e., C1-C2). 10means 1 to 10 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 to refer to an alkyl group attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.

[0084] The term "heteroalkyl," alone or in combination with another term, means, unless otherwise specified, a straight or branched chain hydrocarbon radical containing at least one heteroatom selected from O, N, P, Si, and S in the chain, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen atom may have an alkyl substituent to satisfy valences and / or may be optionally quaternized. The heteroatoms 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)-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 to, -CH-O-CH-CH-, -CH-CH-O-CH-CH-, -CH-O-CH-CH-NH-CH-, -CH-CH-S-CH-CH-, and -CH-S-CH-CH-NH-CH-. 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)R'- represents both -C(O)R'- and -R'C(O)-.

[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, and include 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, mean 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," unless otherwise specified, means a polyvalent unsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (in some embodiments, 1 to 3 rings) that are fused or covalently linked together. The term "heteroaryl" refers to an aryl group containing 1 to 4 heteroatoms selected from N, O, and S in the ring, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. The terms "arylene" and "heteroarylene," alone or as part of another substituent, mean 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, Examples include 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 having a positive charge on one or more heteroatoms.

[0087] Each of the above terms is intended 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 portions of alkyl, heteroalkyl, alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups may, in some embodiments, be selected from deuterium, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, halo, —SiR′R″R′″ in a number ranging from 0 to the number of hydrogen atoms in such radical. , —OC(O)R′, —C(O)R′, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)R′, —NR—C(NR′R″R′′)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSO R′, —CN, and —NO. In some embodiments, the substituent moieties of 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"" are each, in some embodiments, independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy group, or arylalkyl group. When the compounds provided herein include multiple R groups, for example, each of the R groups is independently selected, as is each R', R", R'", and R"" group when multiple of these groups are present. When R' and R" are attached to the same nitrogen atom, they can combine 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 intended to include groups that contain carbon atoms bonded 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)CHOCH3, etc.).

[0089] Substituent moieties of aryl and heteroaryl groups, in some embodiments, include deuterium, halo, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl, in a number ranging from 0 to the total number of hydrogens on the aromatic ring system, -OR', -NR'R'', -SR', -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)R', -NR-C(NR'R''R'')=NR'''', -NR-C(NR' R") = NR"', -S(O)R', -S(O)R', -S(O)NR'R", -NRSOR', -CN, and -NO, -R', -N, -CH(Ph), fluoro(C-C)alkoxy, and fluoro(C-C)alkyl; 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 the compounds provided herein include multiple R groups, for example, each of the R groups is independently selected, as is each R', R", R'", and R"" group when multiple of these groups are present.

[0090] Two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -Q'-C(O)-(CRR') qAlternatively, two of the substituent moieties on adjacent atoms of the aryl or heteroaryl ring can optionally form a ring 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 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 - (wherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-) substituents. 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 term "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 is not 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 intended 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 this disclosure. The ADCs provided herein do not include those known in the art to be too unstable to synthesize and / or isolate.

[0095] (II. TLR7 Agonists) In one aspect, provided herein are TLR7 agonists for use in the compositions and methods provided herein. In some embodiments, the TLR7 agonist is a compound of Formula I: [ka] (In the formula: R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is H, halo, or alkoxy; R 3 -CO2R 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, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and R 7 and R 8 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 Formula (I) is a compound of the formula: [ka] isn't it.

[0096] In some embodiments, in the TLR7 agonist according to Formula I, R 5 and R 6 are (i), (ii), and (iii): (i)R 5 and R 6 are each H; (ii)R 5 is H and R 6 is alkyl; (iii)R 5 and R 6 form a heterocyclic ring together with the N to which they are attached; is selected from R 7 and R 8 together with the N to which they are attached form a heterocyclic ring.

[0097] In some embodiments, the TLR7 agonist is 4 is selected with the proviso that it is not substituted with a hydroxyl.

[0098] In some embodiments, the TLR7 agonist is 3 are selected with the proviso that the alkylene and heteroalkylene moieties are not substituted with oxo.

[0099] In some embodiments, the TLR7 agonist is 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.

[0100] In some embodiments, R 1 is H. In some embodiments, R 1 , halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and is a straight chain six atoms long. In some embodiments, R 1 is halo. In some embodiments, R 1 -NHR 4 In some embodiments, R 1 -OR 4 In some embodiments, R 1 -NH-OR 4 In some embodiments, R 1 -R 4 In some embodiments, R 1 is -NH-n-pentyl, -NH-On-butyl, -On-pentyl, -n-hexyl, or -NH-CHCH-OEt. 1 is -NH-n-pentyl. In some embodiments, R 1 is -NH-On-butyl. In some embodiments, R 1 is -On-pentyl. In some embodiments, R1 is -n-hexyl. In some embodiments, R 1 is -NH-CH2CH2-OEt.

[0101] 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.

[0102] In some embodiments, R 3 -CO2R 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 -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. In some embodiments, 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, -COOH, -CH-Y, -CH-O-heteroalkylene-Y, or -CH-O-alkylene-Y. In some embodiments, R 3 is —CH—Y, —CH—O-heteroalkylene-Y, or —CH—O-alkylene-Y. In some embodiments, R 3-C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2 OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-N H-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -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. 3is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2 CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O) CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl.

[0103] In some embodiments, R 4 is n-butyl, n-pentyl, n-hexyl, or ethoxyethyl. 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.

[0104] 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. 5 and R 6 are each H. In some embodiments, R 5 is H and R 6is alkyl. In some embodiments, R 5 and R 6 together with the N to which they are attached to form 1-piperazinyl.

[0105] In some embodiments, Y is OH. In some embodiments, Y is a divalent glycine group of formula -NHCHC(O)-. In some embodiments, Y is -NR 5 R 6 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.

[0106] In some embodiments, Z is -OH. In some embodiments, Z is alkoxy. In some embodiments, Z is -NR 7 R 8 In some embodiments, Z is -OH, ethoxy, -Nn-Pr2, or 1-piperazinyl. In some embodiments, Z is -OH or 1-piperazinyl.

[0107] In some embodiments, 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. In some embodiments, R 7 and R 8 together with the N to which they are attached to form 1-piperazinyl.

[0108] 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: [Table 1] TIFF2025540064000012.tif243170TIFF2025540064000013.tif223170TIFF2025540064000014.tif237170TIFF2025540064000015.tif225170.

[0109] In some embodiments, the TLR7 agonist can be a known TLR7 agonist, such as 852A, imiquimod, resiquimod, galdiquimod, loxoribine, bropirimine, 3M-011, 3M-052, DSR-6434, DSR-29133, SC1, SZU-101, SM-360320, and SM-276001. Exemplary TLR7 agonists are described, for example, in Chi et al., Front. Pharmacol. 8:34, 31 May 2017, which is incorporated herein by reference in its entirety.

[0110] III. Synthesis of TLR7 Agonists The TLR7 agonists of the present disclosure can be synthesized in any suitable manner. Non-limiting examples of synthetic schemes for synthesizing the TLR7 agonists of the present disclosure are provided in Schemes 1-7 herein.

[0111] Scheme 1. Synthesis of intermediate Aa from compound 1 [ka]

[0112] Scheme 2. Synthesis of intermediates from compound 5 [ka]

[0113] Scheme 3. Synthesis of payloads P1, P2, P20, P23, P27, P29, P32, P33, P37, and P39 [ka]

[0114] Scheme 4. Synthesis of payloads P3, P26, P28, P36, and P38 [ka]

[0115] Scheme 5. Synthesis of payloads P22, P25, P31, P35, P21, P24, P30, and P34 [ka]

[0116] Scheme 6. Synthesis of payloads P4, P5, P6, P8, P9, P10, P11, P12, P17, P18, and P19 [ka]

[0117] Scheme 7. Synthesis of payloads P7, P13, P14, P15, and P16 [ka]

[0118] IV. Linker-TLR7-Agonist (Linker-Payload) In one aspect, provided herein are TLR7 agonist-linkers for use in preparing ADCs. In some embodiments, the ADC comprises an ABD linked to a linker-TLR7 agonist according to Formula II: [ka] (In the formula: R 1 , R 2 and X is as defined elsewhere for formula I; R 9 is R 3is a divalent radical formed by removal of hydrogen from 3 is R 9 is a group attached to the phenyl group at position L is any group or moiety that links, connects, or bonds an antigen binding domain (ABD), as defined elsewhere herein, to a compound of formula I. In some embodiments, the compound of Formula (II) comprises a compound of the formula: [ka] isn't it.

[0119] In some embodiments, 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 In some embodiments, 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 -alkylene-PEG-Y 1 In another embodiment, R 9 is -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1In another embodiment, R 9 -C(Me)2O-, -CO-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2 CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -C H2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CO-, -CH2OCH2CO-1-piperazin-4-yl, -(R)-CHOCH(OH)CHO-, -(S)-CHOCH(OH)CHO-, -CHOCH(NH2)CHO-, -CHO-, -CH2NH-, or -CH2-1-piperazin-4-yl.

[0120] In one embodiment, Y 1 is -O-. In some embodiments, Y 1 is a divalent glycine group of formula -NHCHC(O)-. In some embodiments, Y 1 is -NR 5 In some embodiments, Y 1 -COZ 1 (where Z 1 -O-, -NR 7 In some embodiments, Y is -, -O-alkylene-, or 1-piperazin-4-yl. 1 is —O—, —NH—, 1-piperazin-4-yl, —COO—, or —CO-1-piperazin-4-yl.

[0121] In some embodiments, Z 1 is —O—. In some embodiments, Z 1 is -NR7 In some embodiments, R 7 is H. In some embodiments, R 7 is alkyl.

[0122] 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.

[0123] In some embodiments, the linker L for use herein can be found, for example, in Antibody-Drug Conjugates and Immunotoxins, edited by Phillips, G.L.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, edited by Ducrry, L.; Humana Press, 2013; Antibody-Drug Conjugates, edited by Wang, J., Shen, W.-C., and Zaro, J.L.; Springer International Publishing, 2015. In some embodiments, the L group of the ADCs provided herein is sufficiently stable to take advantage of the circulating half-life of the antigen-binding domain and, at the same time, can release its payload after antigen-mediated internalization of the ADC. The linker L can be cleavable or non-cleavable. Cleavable linkers for use as L herein include linkers that are cleaved by intracellular metabolism after internalization, such as cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers for use as L herein include linkers that release the attached payload via lysosomal degradation of the antigen-binding domain after internalization. Suitable L linkers include, but are not limited to, acid-labile linkers, hydrolytically labile linkers, enzyme-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 include peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, maltocaproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.

[0124] The terms "PEG" or "polyethylene glycol", alone or in combination with another term, refer to, unless otherwise specified, the group -(OCH2CH2O) n- (where n is an integer from about 1 to about 100, e.g., from about 1 to about 10, from about 2 to about 8, from about 4 to about 20, from about 4 to about 12, and from about 12 to about 30). Examples of PEG groups include, but are not limited to, the following [ChemDraw structure in inset]: The PEG group can have any suitable molecular weight, for example, 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.

[0125] Any linker molecule or linker technology known in the art can be used as L to create or construct the ADCs 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, for example, linkers comprising or consisting of MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide moiety in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide moiety in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof. Further examples of L linkers that can be used in the ADCs provided herein are disclosed, for example, in U.S. Pat. No. 7,754,681 and Ducry, Bioconjugate Chem., 2010, 21:5-13, and references cited therein.

[0126] In some embodiments, the L linker is stable under physiological conditions. In some embodiments, the L linker is cleavable, for example, in the presence of an enzyme or at a specific pH range or value, and can release at least the payload portion. In some embodiments, the 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.

[0127] In some embodiments, the L linker comprises a non-cleavable moiety.

[0128] 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, derivatives thereof, or combinations thereof. In some embodiments, one or more side chains of the amino acids are linked to the side chain groups 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.

[0129] In some embodiments, the L linker comprises a self-immolative group. The self-immolative group can be any such group known to those skilled in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-immolative group can undergo a chemical reaction that releases the remaining atoms of the L linker from the payload.

[0130] In other embodiments, the L group can be modified with one or more enhancing groups. In some embodiments, the enhancing group can be linked to the side chain of any amino acid in L. In one embodiment, amino acids for linking an enhancing group include lysine, asparagine, aspartate, glutamine, glutamate, and citrulline. Linkage to the enhancing group can be a direct bond to the amino acid side chain, or the linkage can be indirect via a spacer and / or a reactive group. In one embodiment, spacers and reactive groups include any of those described herein. In some embodiments, the enhancing group can be any group that imparts a beneficial effect to the payload, linker payload, or ADC, including, but not limited to, a biological effect, a biochemical effect, a synthetic effect, a solubilizing effect, an imaging effect, a detection effect, and a reactivity effect. In some embodiments, the enhancing group is a hydrophilic group. In some embodiments, the enhancing group is a cyclodextrin. In some embodiments, the enhancing group is an alkyl sulfonic acid, a heteroalkyl sulfonic acid, an alkenyl sulfonic acid, a heteroalkenyl sulfonic acid, a heteroalkenyl taurine, a heteroalkenyl phosphoric acid or phosphate, a heteroalkenyl amine (e.g., a quaternary amine), or a heteroalkenyl sugar. In some embodiments, the sugar includes, but is not limited to, a monosaccharide, a disaccharide, and a polysaccharide. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like. In some embodiments, the sugar includes a sugar acid such as glucuronic acid, further including conjugated forms (i.e., by glucuronidation) such as glucuronides. 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 the art.In some embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In some embodiments, the cyclodextrin is α-cyclodextrin. In some embodiments, the cyclodextrin is β-cyclodextrin. In some embodiments, the cyclodextrin is γ-cyclodextrin. In some embodiments, the enhancing group can improve the solubility of the remainder of the ADC. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or unsubstituted. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH). 1-5 SO3H, -(CH2) n -NH-(CH2) 1-5 SO3H, -(CH2) n -C(O)NH-(CH2) 1-5 SO3H, -(CH2CH2O) m -C(O)NH-(CH2) 1-5 SO3H, -(CH2) n -N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, -(CH2) n -C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, or -(CH2CH2O) m -C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where 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) 1-5 In some embodiments, the heteroalkyl or heteroalkenyl sulfonic acid is —(CH) n -NH-(CH2) 1-5SO3H, where 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) 1-5 SO3H, where 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) 1-5 SO3H, where m is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2) n -N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where 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) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where 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) 1-5 C(O)NH(CH2) 1-5 SO3H2, where m is 1, 2, 3, 4, or 5.

[0131] In some embodiments, L comprises a maleimide (for conjugation with a thiol, e.g., cysteine, of the antigen-binding domain), an N-hydroxysuccinimide ester (for conjugation with an amine, e.g., lysine, of the antigen-binding domain), or a 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.

[0132] In some embodiments, L contains a maleimide group. In such embodiments, the maleimide group in L reacts with a cysteine ​​residue on the antigen-binding domain to form a carbon-sulfur bond.

[0133] In some embodiments, L contains an N-hydroxysuccinimide ester group. In such embodiments, the N-hydroxysuccinimide ester group reacts with a lysine residue on the antigen-binding domain to form an amide bond.

[0134] In some embodiments, L contains an alkyne that can react with an azide via click chemistry, e.g., to form a click chemistry reaction product. In some embodiments, the alkyne group reacts with an azide on the modified antigen-binding domain. In some embodiments, L contains a functional group or moiety that can undergo click chemistry (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 can undergo a 1,3-cycloaddition reaction with an azide. In such embodiments, alkynes that can be used include strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, such as cyclooctynes, benz-annulated alkynes, and alkynes that can undergo 1,3-cycloaddition reactions with alkynes in the absence of a copper catalyst. In such embodiments, alkynes that can be used also include, but are not limited to, dibenzoazacyclooctynes, dibenzocyclooctynes, biarylazacyclooctynones, difluorinated cyclooctynes, substituted (e.g., fluorinated) alkynes, azacycloalkynes, and bicyclo[6.1.0]nonynes. In other embodiments, alkynes are useful for conjugating antibodies functionalized with azide groups. Such functionalized antibodies include antibodies functionalized with azide-polyethylene glycol groups. In some embodiments, such functionalized antibodies are obtained 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.

[0135] In some embodiments, L is selected from 2-maleimido-1-ethyl, 2-maleimidoacetyl, and 3-maleimidopropanoyl. [ka] Selected from: TIFF2025540064000026.tif249170TIFF2025540064000027.tif221170.

[0136] In some embodiments, L is 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, [ka] is a group selected from

[0137] In some embodiments, the linker-TLR7 agonist is selected from those in Table 2 and pharmaceutically acceptable salts of any of these: [Table 2] TIFF2025540064000030.tif228170TIFF2025540064000031.tif239170TIFF2025540064000032.tif219170TIFF2025540064000033.tif191170.

[0138] In some embodiments, when the payload (i.e., TLR7 agonist) has an alcohol group (i.e., -OH), the payload can be converted to a prodrug prior to attachment to the linking group and formation of the ADC. See, e.g., WO 2020 / 146541. In this embodiment, the payload of Formula I can be converted to a linker-TLR7 agonist of Formula III: [ka] (In the formula: L is a linker as defined elsewhere herein; R 1 , R 2 and X is as defined elsewhere for formula I; R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 11 or R 14 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6).

[0139] In some embodiments, the linker-TLR7 agonist has Formula III, wherein R 11 and R 12are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 11 or R 14 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, R 16 is the R of formula I that connects the phenyl ring to the oxygen atom of the alcohol. 3 This is the part.

[0140] In some embodiments, the TLR7 agonist used in preparing a linker-TLR7 agonist according to Formula III is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, or P39.

[0141] V. TLR7 Agonist-Linker Synthesis Scheme 8. Synthesis of Linker-Payloads LP1, LP2, LP3, and LP4 [ka]

[0142] Scheme 9. Synthesis of Linker-Payload LP5 [ka]

[0143] Scheme 10. Synthesis of Linker-Payload LP6A [ka]

[0144] Scheme 11. Synthesis of Linker-Payload LP7A and LP10A [ka]

[0145] Scheme 12. Synthesis of Linker-Payload LP8A [ka]

[0146] Scheme 13. Synthesis of Linker-Payload LP9 and LP12 [ka]

[0147] Scheme 14. Synthesis of linker-payloads LP6A, LP6B, LP7A, LP7B, LP7C, LP7D, LP10A, LP10B, LP11A, LP11B, LP11C, LP11D, LP12, and LP14 [ka] TIFF2025540064000042.tif240170TIFF2025540064000043.tif51170

[0148] Scheme 14A. Alternating synthesis of linker-payload LP11A [ka] [Table 3]

[0149] Scheme 15. Synthesis of Linker-Payload LP8B2 [ka]

[0150] Scheme 16. Synthesis of Linker-Payload LP13 [ka]

[0151] Scheme 16A: Linker-Payload Q c -LP7A, Q c -LP11A, Q o -LP7A and Q o Synthesis of -LP11-A [ka]

[0152] VI. ADCs for Use in Compositions and Methods 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 bivalent linker linking the ABD to the TLR7 agonist. The ABD can be attached to the TLR7 agonist at any position along the ABD, with or without a linker, so long as the ABD is capable of binding to its target.

[0153] In some embodiments, the ADC is according to Formula IV: [ka] (In the formula: L 1 is a bivalent linker; R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is H, halo, or alkoxy; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 9 is R 3 is a divalent radical formed by removal of hydrogen from 3 is R 9 is a group attached to the phenyl group at position R 3 -CO2H, -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; X is CH or N; Y is -OH, -Gly, -NR 5 R 6 , or -COZ; Z is -OH, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; R 7 and R 8are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and and k is an integer from 1 to 30.

[0154] In some embodiments, the ABC comprises one TLR7 agonist molecule conjugated to an ABD having binding specificity for the target antigen. In some embodiments, the ADC comprises multiple TLR7 agonist molecules per 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 greater.

[0155] 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.

[0156] In some embodiments, R 1 , halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and has a linear chain length of 6 atoms. 1 is halo. In some embodiments, R 1 -NHR 4 In some embodiments, R 1 -OR 4 In some embodiments, R 1 -NH-OR 4In some embodiments, R 1 -R 4 In some embodiments, R 1 is -NH-n-pentyl, -NH-On-butyl, -On-pentyl, -n-hexyl, or -NH-CHCH-OEt. 1 is -NH-n-pentyl. In some embodiments, R 1 is -NH-On-butyl. In some embodiments, R 1 is -On-pentyl. In some embodiments, R 1 is -n-hexyl. In some embodiments, R 1 is -NH-CH2CH2-OEt.

[0157] 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.

[0158] In some embodiments, R 3 -CO2H, -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. In some embodiments, R 3 is -(hydroxy)heteroalkylene-Y. In some embodiments, R3 is -(amino)heteroalkylene-Y. In some embodiments, R 3 is alkylene-PEG-Y. In some embodiments, R 3 is -CONH, -CH-Y, -CH-O-heteroalkylene-Y, or -CH-O-alkylene-Y. In some embodiments, R 3 is —CH—Y, —CH—O-heteroalkylene-Y, or —CH—O-alkylene-Y. In some embodiments, R 3 -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2 OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-N H-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -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. 3is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2 CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O) CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, or -CH2-1-piperazinyl.

[0159] In some embodiments, R 4 is n-butyl, n-pentyl, n-hexyl, or ethoxyethyl. 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.

[0160] 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. 5 and R 6 are each H. In some embodiments, R 5 is H and R 6is alkyl. In some embodiments, R 5 and R 6 together with the N to which they are attached to form 1-piperazinyl.

[0161] In some embodiments, Y is OH. In some embodiments, Y is a divalent glycine group of formula -NHCHC(O)-. In some embodiments, Y is -NR 5 R 6 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.

[0162] In some embodiments, Z is -OH. In some embodiments, Z is alkoxy. In some embodiments, Z is -NR 7 R 8 In some embodiments, Z is -OH, ethoxy, -Nn-Pr2, or 1-piperazinyl. In some embodiments, Z is -OH or 1-piperazinyl.

[0163] In some embodiments, 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. In some embodiments, R 7 and R 8 together with the N to which they are attached to form 1-piperazinyl.

[0164] In some embodiments, ABD-L 1 is R of a compound selected from P1 to P43 3 is linked to the compound by removal of a hydrogen from the group at the position corresponding to

[0165] In some embodiments, 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 In some embodiments, 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 -alkylene-PEG-Y 1 In some embodiments, R 9 is -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 In some embodiments, R 9-C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH 2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, - CHOCH-((4-NH-)-1-phenyl), -CHOCHCOO-, -CHOCHCHOCHCO-, -CHOCHCHOCHCHOCHCO-, -CHOCHCHOCHCHOCH2CHOCH2CO-, -CHOCHCO-1-piperazin-4-yl, -(R)-CHOCH(OH)CHO-, -(S)-CHOCH(OH)CHO-, -CHOCH(NH)CHO-, -CHO-, -CHNH-, or -CH-1-piperazin-4-yl.

[0166] In some embodiments, Y 1 is -O-. In some embodiments, Y 1 is a divalent glycine group of formula -NHCHC(O)-. In some embodiments, Y 1 is -NR 5 In some embodiments, Y 1 -COZ 1 (where Z 1 -O-, -NR 7 -, -O-alkylene-, or 1-piperazin-4-yl). 1 is —O—, —NH—, 1-piperazin-4-yl, —COO—, or —CO-1-piperazin-4-yl.

[0167] In some embodiments, Z 1 is —O—. In some embodiments, Z 1 is -NR 7 In some embodiments, R7 is H. In some embodiments, R 7 is alkyl.

[0168] 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.

[0169] (A.L. 1 (divalent group) In some embodiments, the ADC of the disclosure comprises a TLR7 indirectly linked to an ABD via a linker. In some embodiments, the linker is a bivalent linker (L) that links the ABD to the TLR7 agonist according to Formula IV. 1 In some embodiments, when the ADC is indirectly conjugated to the TLR7 agonist via a linker, the bivalent linker (L 1 ) is generated by reaction between a linker (L) and an ADC for conjugation.

[0170] Linkers (L 1 ) can be found, for example, in Antibody-Drug Conjugates and Immunotoxins, edited by Phillips, G.L.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, edited by Ducrry, L.; Humana Press, 2013; Antibody-Drug Conjugates, edited by Wang, J., Shen, W.-C., and Zaro, J.L.; Springer International Publishing, 2015. In certain embodiments, the L 1The group is sufficiently stable to take advantage of the circulating half-life of the antigen-binding domain, and at the same time is capable of releasing its payload after antigen-mediated internalization of the ADC. 1 As used herein, L can be cleavable or non-cleavable. 1 Cleavable linkers for use as include linkers that are cleaved by intracellular metabolism after internalization, for example, by hydrolysis, reduction, or cleavage via an enzymatic reaction. 1 Non-cleavable linkers for use as L include linkers that release the attached payload via lysosomal degradation of the antigen-binding domain after internalization. 1 Linkers include, but are not limited to, acid-labile linkers, hydrolytically labile linkers, enzyme-cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers. 1 Linkers also include, but are not limited to, those that are or include peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, malcaproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.

[0171] Any linker molecule or linker technology known in the art may be used. 1 can be used to create or construct the ADCs provided herein. In certain embodiments, L 1 The linker is a cleavable linker. 1 The linker is a non-cleavable linker. In certain embodiments, the L that can be used in the ADCs provided herein 1Linkers include, for example, linkers comprising or consisting of MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide moiety in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide moiety in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof. 1 Further examples of linkers are disclosed, for example, in U.S. Pat. No. 7,754,681 and Ducry, Bioconjugate Chem., 2010, 21:5-13, and references cited therein.

[0172] In one embodiment, L 1 The linker is stable under physiological conditions. 1 The linker is cleavable, e.g., capable of releasing at least the payload portion in the presence of an enzyme or at a particular pH range or value. 1 The linker comprises an enzyme-cleavable moiety. In some embodiments, the enzyme-cleavable L 1 Linkers include, but are not limited to, peptide bonds, ester linkages, and hydrazones. 1 The linker comprises a cathepsin-cleavable linker.

[0173] In some embodiments, L 1 The linker comprises a non-cleavable moiety.

[0174] In some embodiments, L 1 The 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, L 1The 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, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of the amino acids are linked to the side chain groups described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, L 1 Linkers include lysine, valine, and citrulline. 1 The linker comprises lysine, valine, and alanine. 1 The linker comprises valine and alanine.

[0175] In some embodiments, L 1 The linker comprises a self-immolative group. The self-immolative group can be any such group known to those of skill in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-immolative group is L 1 It will be appreciated that a chemical reaction can be carried out that releases the remaining atoms of the linker from the payload.

[0176] In other embodiments, L 1 The group can be modified with one or more enhancing groups. In certain embodiments, the enhancing group is L 1The enhancing group can be linked to the side chain of any amino acid in the nucleotide sequence. In some embodiments, amino acids for linking the enhancing group include lysine, asparagine, aspartate, glutamine, glutamate, and citrulline. Linkage to the enhancing group can be a direct bond to the amino acid side chain, or the linkage can be indirect via a spacer and / or a reactive group. In some embodiments, spacers and reactive groups include any of those described herein. In certain embodiments, the enhancing group can be any group that imparts a beneficial effect to the payload, linker payload, or ADC, including, but not limited to, a biological effect, a biochemical effect, a synthetic effect, a solubilization effect, an imaging effect, a detection effect, and a reactivity effect. In certain embodiments, the enhancing group is a hydrophilic group. In certain embodiments, the enhancing group is a cyclodextrin. In certain embodiments, the enhancing group is an alkyl, heteroalkyl, alkenyl, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, a heteroalkenyl amine (e.g., a quaternary amine), or a heteroalkenyl sugar. In some embodiments, sugars include, but are not limited to, 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 (i.e., by glucuronidation) such as glucuronides. 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 skilled in the art. In some embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In some embodiments, the cyclodextrin is α-cyclodextrin.In some embodiments, the cyclodextrin is β-cyclodextrin. In some embodiments, the cyclodextrin is γ-cyclodextrin. In some embodiments, the enhancing group can improve the solubility of the remainder of the ADC. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or unsubstituted. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH). 1-5 SO3H, -(CH2) n -NH-(CH2) 1-5 SO3H, -(CH2) n -C(O)NH-(CH2) 1-5 SO3H, -(CH2CH2O) m -C(O)NH-(CH2) 1-5 SO3H, -(CH2) n -N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, -(CH2) n -C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, or -(CH2CH2O) m -C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H2, where 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) 1-5 In another embodiment, the heteroalkyl or heteroalkenyl sulfonic acid is —(CH) n -NH-(CH2) 1-5 SO3H, where 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, where 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) 1-5 SO3H, where m is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2) n -N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where 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) 1-5 C(O)NH(CH2) 1-5 SO3H)2, where 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) 1-5 C(O)NH(CH2) 1-5 SO3H2, where m is 1, 2, 3, 4, or 5.

[0177] In another embodiment, L 1 contains a 3-thiosuccinimide group (arising from conjugation of a maleimide group with a thiol, e.g., cysteine, of the antigen-binding domain), an amide group (arising from conjugation of an N-hydroxysuccinimide ester with an amine, e.g., lysine, of the antigen-binding domain), or a triazolocyclooctyl group (arising from conjugation of a cyclooctynyl group with an antigen-binding domain modified to contain an azide group using click chemistry). See, e.g., WO 2020 / 132658; Chio et al., Methods Mol. Biol. 2020, 2078:83-87.

[0178] In one embodiment, L1 contains 3-thiosuccinimide. In such an embodiment, L 1 The 3-thiosuccinimide group in results from the reaction of a cysteine ​​residue on the antigen-binding domain with the maleimide group of L to form a carbon-sulfur bond.

[0179] In one embodiment, L 1 is derived from L, which contains a maleimide group. In such embodiments, the maleimide group in L reacts with a cysteine ​​residue on the antigen-binding domain to form a carbon-sulfur bond.

[0180] In some embodiments, L 1 is derived from L, which contains an N-hydroxysuccinimide ester group. In such embodiments, the N-hydroxysuccinimide ester group reacts with a lysine residue on the antigen-binding domain to form an amide bond.

[0181] In other embodiments, L 1is derived from L, which contains an alkyne that can react with an azide via click chemistry, e.g., to form a click chemistry reaction product. In some embodiments, the alkyne group reacts with an azide on the modified antigen-binding domain. In certain embodiments, L contains a functional group or moiety that can undergo click chemistry (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 can undergo a 1,3-cycloaddition reaction with an azide. In such embodiments, alkynes that can be used include strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, such as cyclooctynes, benz-annulated alkynes, and alkynes that can undergo 1,3-cycloaddition reactions with alkynes in the absence of a copper catalyst. In such embodiments, alkynes that can be used also include, but are not limited to, dibenzoazacyclooctynes, dibenzocyclooctynes, biarylazacyclooctynones, difluorinated cyclooctynes, substituted (e.g., fluorinated) alkynes, azacycloalkynes, and bicyclo[6.1.0]nonynes. In other embodiments, alkynes are useful for conjugating antibodies functionalized with azide groups. Such functionalized antibodies include antibodies functionalized with azide-polyethylene glycol groups. In one embodiment, such a functionalized antibody is obtained 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.

[0182] In some embodiments, L 1 contains an amide group. In such an embodiment, L 1The amide group in results from the reaction of the N-hydroxysuccinimide ester group of L with a lysine residue on the antigen-binding domain to form an amide bond.

[0183] In other embodiments, L 1contains a cyclic group resulting from the reaction of an alkyne with an azide via click chemistry, for example, to form a click chemistry reaction product. In some embodiments, the alkyne group reacts with an azide on the modified antigen-binding domain. In some embodiments, the 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 capable of undergoing a 1,3-cycloaddition reaction with an azide. In such embodiments, alkynes that can be used include strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes, such as cyclooctynes, benz-annulated alkynes, and alkynes that can undergo 1,3-cycloaddition reactions with alkynes in the absence of a copper catalyst. In such embodiments, alkynes that can be used also include, but are not limited to, dibenzoazacyclooctynes, dibenzocyclooctynes, biarylazacyclooctynones, difluorinated cyclooctynes, substituted (e.g., fluorinated) alkynes, azacycloalkynes, and bicyclo[6.1.0]nonynes. In other embodiments, alkynes are useful for conjugating antibodies functionalized with azide groups. Such functionalized antibodies include antibodies functionalized with azide-polyethylene glycol groups. In one embodiment, such a functionalized antibody is obtained 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.

[0184] In another embodiment, L 1 are 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, [ka] is a group obtained from

[0185] In one embodiment, L 1 teeth, [ka] is or contains a divalent group selected from:

[0186] In another embodiment, L 1 teeth, [ka] TIFF2025540064000053.tif206170TIFF2025540064000054.tif65170.

[0187] In some embodiments, L 1 is non-cleavable under physiological conditions. 1 is cleavable under physiological conditions. 1 is an acid labile linker, a hydrolytically labile linker, an enzyme-cleavable linker, a reduction-labile linker, or a self-immolative linker. 1 is or includes a peptide, carbohydrate, glucuronide, polyethylene glycol (PEG) unit, hydrazone, mal-caproyl unit, dipeptide unit, valine-citrulline unit, or para-aminobenzyl (PAB) unit. 1 In some embodiments, L 1 In some embodiments, L 1 includes p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC). In some embodiments, L 1comprises a maleimide, an N-hydroxysuccinimide ester, or a cyclooctynyl group. 1 are 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, [ka] The base is obtained from TIFF2025540064000056.tif182170TIFF2025540064000057.tif178170.

[0188] In some embodiments, the ADC comprises an ABD linked to LP1-LP15.

[0189] B. Antigen Binding Domain (ABD) In one embodiment, the antigen-binding domain, i.e., ABD, in Formula IV for use in the ADCs provided herein includes any molecule that specifically interacts with a particular antigen.

[0190] In certain embodiments, the ABD is an antibody or an antigen-binding fragment of an antibody. In certain embodiments, the ABD is an antibody.

[0191] In some embodiments, the ABD is an antibody comprising an Fc region modified to enhance binding affinity to FcγR. In some embodiments, the ABD is an antibody having one or more mutations selected from F243L, R292P, Y300L, V305I, and P396L. In some embodiments, the ABD is an antibody having one or more mutations selected from S239D and I332E. In some embodiments, the ABD is an antibody having one or more mutations selected from S239D, I332E, and A330L. In some embodiments, the ABD is an antibody having one or more mutations selected from S298A, E333A, and K334A. In some embodiments, the ABD is an antibody having one or more mutations selected from L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, the ABD is an antibody having one or more mutations selected from D270E, K326D, A330M, and K334E. In some embodiments, the ABD is an antibody having L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in one heavy chain and D270E, K326D, A330M, and K334E in the opposite heavy chain. In some embodiments, the ABD is an antibody having one or more mutations selected from G236A, S239D, and I332E. In some embodiments, the ABD is an antibody having one or more mutations selected from M252Y, S254T, and T256E. In some embodiments, the ABD is an antibody having one or more mutations selected from M428L and N434S. In some embodiments, the ABD is an antibody with one or more mutations selected from S267E and L328F. In some embodiments, the ABD is an antibody with one or more mutations selected from N325S and L328F.

[0192] In some embodiments, the ABD is an antibody comprising a glutamine residue. Antibodies comprising glutamine residues can be isolated from natural sources or modified to comprise one or more glutamine residues. Techniques for artificially creating glutamine residues in antibody polypeptide chains (glutaminyl-modified antibodies) are within the capabilities of those skilled in the art. In other embodiments, the ABD is an N297Q mutant antibody. In further embodiments, Z comprises one or more modified [ka] Antibodies having a nucleotide sequence similar to that of the nucleotide sequence of the present invention are antibodies having a nucleotide sequence similar to that of the nucleotide sequence of the present invention. See, e.g., U.S. Patent No. 9,676,871 and U.S. Patent Application Publication No. 2003 / 0138785.

[0193] In certain embodiments, the antibody is aglycosylated. In some embodiments, the antibody is glycosylated.

[0194] In certain embodiments, the ABD is an antibody that is a monoclonal antibody, a human antibody, a humanized antibody, a camelized antibody, or a chimeric antibody. In other embodiments, the 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, the ABD has a molecular weight of at least 500, 600, 700, 800, 900, 1,000, 10,000, 50,000, or 100,000 daltons.

[0195] 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 the ligand-binding portion of a receptor that specifically binds to a particular antigen, antigen-binding scaffolds (e.g., scaffolds based on DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and natural repeat proteins (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, the ADC comprises an scFv that has binding specificity for a target antigen.

[0196] Methods for determining whether two molecules specifically bind to one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, an antigen-binding domain as used herein has a K 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 by a surface plasmon resonance assay. D The present invention includes a polypeptide that binds to a target antigen or a portion thereof.

[0197] In certain embodiments, the framework regions (FRs) of antibodies or antigen-binding fragments thereof for use in the ADCs provided herein may be identical to human germline sequences or may be naturally or artificially modified. Amino acid consensus sequences can be defined based on comparative analysis of two or more CDRs.

[0198] 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 CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of 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 antibodies.

[0199] Antigen-binding domains for use in the ADCs provided herein may comprise or consist of antigen-binding fragments of intact antibody molecules. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules using any suitable standard technique, 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 readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0200] 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 amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. In other embodiments, antigen-binding fragments of antibodies include 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.

[0201] In certain embodiments, an antigen-binding fragment of an antibody comprises at least one variable domain. A variable domain may be of any size or amino acid composition and typically comprises at least one CDR adjacent to or in-frame with one or more framework sequences. H Domain is V L In the antigen-binding fragment associated with the domain, V H Domains and V L The domains may be in any suitable arrangement relative to each other. For example, the variable region may be a dimer and the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0202] 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 arrangements 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 LIn any arrangement of variable and constant domains, including any of the exemplary arrangements above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that create a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. In further embodiments, the antigen-binding fragments may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the above arrangements of variable and constant domains non-covalently associated (e.g., by disulfide bonds) with the domains.

[0203] In another embodiment, the antigen-binding domain used in the ADCs provided herein may comprise or consist of a human antibody and / or recombinant human antibody, or an antigen-binding fragment thereof.

[0204] In another embodiment, the antigen-binding domain used in the ADCs provided herein may comprise or consist of a recombinant human antibody or antigen-binding fragment thereof. In some embodiments, such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when using animals transgenic for human Ig sequences) and thus the V and V of the recombinant antibody have been modified. H and V L The amino acid sequence of the region is human germline V H and V L A sequence that is derived from and related to a sequence, but that may not naturally occur within the human antibody germline repertoire in vivo.

[0205] In another embodiment, the antigen-binding domain used in the ADCs provided herein also includes bispecific antigen-binding molecules, such as bispecific antibodies. Methods for making bispecific antibodies are known in the art and can 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, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2Bispecific formats include those described herein (see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and the references cited therein, for a review of such formats). See also, for example, US2018 / 0134794, which discloses bispecific antigen-binding molecules. Briefly, a bispecific antigen-binding molecule 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"). Simultaneous binding of two distinct epitopes by a bispecific antigen-binding molecule results in effective ligand blockade with minimal activation of target signaling. In certain embodiments, the D1 and D2 domains of a bispecific antibody are non-competitive with each other. Non-competition between D1 and D2 means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to the target. Exemplary antigen-binding protein competition assays are known in the art. In some embodiments, D1 and D2 bind to different (e.g., non-overlapping or partially overlapping) epitopes on the target. Bispecific antigen-binding molecules can be constructed using the antigen-binding domains of two separate monospecific antibodies. For example, a collection of monoclonal monospecific antibodies can be produced using standard methods known in the art. The individual antibodies thus produced can be tested against each other for cross-competition with the target protein. If two different antibodies can simultaneously bind to the target (i.e., do not compete with each other), the antigen-binding domain from a first antibody and the antigen-binding domain from a second, non-competing 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 each other. Any antigen-binding construct capable of simultaneously binding to two separate, non-identical epitopes on a target molecule is considered a bispecific antigen-binding molecule. The bispecific antigen-binding molecule or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) as known to those skilled in the art.In another embodiment, bispecific antibodies are also provided in which 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, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), 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 aforementioned formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to create site-specific antibody-oligonucleotide conjugates that subsequently self-assemble into multimeric complexes with defined composition, valency, and shape (see, e.g., Kazane et al., J. Am. Chem. Soc. (Epub: Dec. 4, 2012)).

[0206] In another embodiment, the antigen-binding domain for use in the ADCs provided herein also includes antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences known in the art. In some embodiments, variants also include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences known in the art with one or more conservative substitutions. For example, the antigen-binding domain includes an antibody or antigen-binding fragment thereof having an HCVR, LCVR, and / or CDR amino acid sequence with 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences known in the art. In another embodiment, the antigen-binding domain includes an antibody or antigen-binding fragment thereof that also includes variants with substantial sequence identity to any of the HCVR, LCVR, and / or CDR amino acid sequences known in the art. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0207] 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 example, GCG software includes 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, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of maximum overlap between the query and search sequences (Pearson, 2000, supra). Another preferred algorithm for comparing the sequences provided herein to a database containing a large number of sequences from various organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with 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.

[0208] Antigen-binding domains for use in the ADCs provided herein include proteins having an amino acid sequence that differs from that of the described antibodies but retains the ability to bind to a target protein. Such variant antigen-binding domains contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies.

[0209] Two antigen-binding domains are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antigen-binding domains are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their rate is not; however, such differences in absorption rate may be considered bioequivalent because they are intentional, reflected in the labeling, are not essential for achieving effective body drug concentrations during chronic use, and are not considered medically significant for the particular drug being studied.

[0210] In some embodiments, two antigen-binding domains are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0211] In some embodiments, two antigen-binding domains are bioequivalent if a patient can switch between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuous therapy without such switching.

[0212] In some embodiments, two antigen-binding domains are biologically equivalent if they both act by a common mechanism(s) of action for the condition(s) of use, to the extent such mechanisms are known.

[0213] Bioequivalence can be demonstrated by in vivo and in vitro methods. Methods for assessing bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antigen-binding domain or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with or reasonably predict in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding domain.

[0214] Biologically equivalent variants of antigen-binding domains for use in the ADCs provided herein can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antigen-binding domains can include variants containing amino acid changes that modify the glycosylation characteristics of the antigen-binding domain, for example, mutations that ablate or eliminate glycosylation.

[0215] In certain embodiments, the antigen-binding domain for use in the ADCs provided herein binds to a human target protein but does not bind to target proteins from other species. In other embodiments, the antigen-binding domain for use in the ADCs provided herein binds to a human target protein and to target proteins from one or more non-human species. For example, the antigen-binding domain for use in the ADCs provided herein can bind to a human target protein and, optionally, may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee target proteins. In some embodiments, the antigen-binding domain specifically binds to a human target protein and a cynomolgus monkey (e.g., Macaca fascicularis) target protein. In other embodiments, the antigen-binding domain for use herein binds to a human target protein but does not bind, or only weakly binds, to a cynomolgus monkey target protein.

[0216] (1. ABD sequence) In some embodiments, the ABD comprises the heavy and light chains of an antibody.

[0217] In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0218] (2.Connection part) The ABD can be linked to the TLR7 agonist directly through attachment at a specific amino acid within the ABD or indirectly via a linker. Exemplary amino acid additions that can be used in connection with this embodiment of the disclosure include, for example, 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 Nos. 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), formylglycine (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), unnatural amino acids (see, e.g., WO Examples of suitable linkers include amino acids, amino acids, and amino acids (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).

[0219] In some examples, the ABD is bound to the linker via a lysine residue. In some embodiments, the antibody or antigen-binding molecule is bound to the linker via a cysteine ​​residue, a lysine residue, or a glutamine residue. In some embodiments, the ABD is bound to the linker via a cysteine ​​residue. In some embodiments, the maleimide moiety of the linker binds to a cysteine ​​residue of the antibody. In some embodiments, the ABD is bound to the linker via a lysine residue. In some embodiments, the N-hydroxysuccinimide moiety of the linker binds to a lysine residue of the antibody to form an amide linkage.

[0220] In some embodiments, the ABD is attached to the linker via 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 containing glutamine residues can be isolated from natural sources or modified to contain one or more glutamine residues. In some embodiments, the antibody or antigen-binding molecule is modified by mutation, e.g., insertion or deletion, to facilitate reaction with transglutaminase. In some embodiments, the antibody or antigen-binding molecule is modified to remove one or more glycosylation sites. In some embodiments, the antibody or antigen-binding molecule is modified to add one or more glutamine residues. In some embodiments, the glutamine residues are added within the TGase recognition tag described herein. Techniques for artificially incorporating glutamine residues into antibody polypeptide chains (glutaminyl-modified antibodies or antigen-binding molecules) are within the capabilities of one of ordinary skill in the art. In certain embodiments, the antibody is aglycosylated.

[0221] In certain embodiments, the ABD comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the ABD comprises two heavy chain polypeptides, each having one Gln295 or Q295 residue. In further embodiments, the ABD comprises one or more glutamine residues at a site other than 295 in the heavy chain. Included herein are antibodies of this section that carry the N297Q mutation described herein. In certain embodiments, a glutamine residue is added to the C-terminus of the heavy chain.

[0222] In some embodiments, glutamine is a polypeptide into which a glutamine-containing tag (e.g., a glutamine-containing peptide tag, a Q-tag, or a TGase recognition tag) has been artificially incorporated. The term "TGase recognition tag" or "Q-tag" refers to a sequence of amino acids containing a glutamine residue that, when incorporated (e.g., added) into a polypeptide sequence under suitable conditions, is recognized by transglutaminase ("TGase") and results in cross-linking by TGase through a reaction between an amino acid side chain and a reactive group within the amino acid sequence. The recognition tag may be a peptide sequence that does not naturally occur in a polypeptide. In some embodiments, the TGase recognition tag contains at least one glutamine. In some embodiments, the TGase recognition tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., the conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, He, Met, Pro, Thr, Lys, or Trp, or an unconventional amino acid). [ka] and (iii) a sequence of amino acids selected from the group consisting of: See, e.g., WO2012059882, the contents of which are incorporated herein in their entirety.

[0223] In some embodiments, the ABD comprises an antibody heavy chain and further comprises a TGase recognition tag at the C-terminus of the antibody heavy chain. In some embodiments, the ABD comprises an antibody heavy chain and further comprises a TGase recognition tag at the C-terminus of the antibody heavy chain, wherein the TGase recognition tag has the pentapeptide sequence [ka] In some embodiments, the ABD comprises two antibody heavy chains and further comprises a TGase recognition tag at the C-terminus of each antibody heavy chain. In some embodiments, the ABD comprises two antibody heavy chains and further comprises a TGase recognition tag at the C-terminus of each antibody heavy chain, wherein the TGase recognition tag has the pentapeptide sequence [ka] is.

[0224] ABDs can also be modified with 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 that can react with a reactive group on a linker-payload. In some embodiments, the primary amine compound provides a diene or dienophile. In some 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 some embodiments, the primary amine compound provides an azide group. In some embodiments, the primary amine compound provides an azide group and the linker-payload provides a complementary alkyne for conjugation via a Click reaction.

[0225] In some embodiments, the ABD comprises a heavy chain, and the heavy chain is linked to the ABD directly or indirectly via a linker. In some embodiments, the ABD comprises a light chain, and the light chain is linked to the ABD directly or indirectly via a linker.

[0226] In some embodiments, the ABD comprises a heavy chain, and the C-terminus of the heavy chain is linked to the ABD, either 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 the ABD, either directly or indirectly via a linker.

[0227] In some embodiments, the ABD comprises two heavy chains, each of which is linked to the ABD directly or indirectly via a linker. In some embodiments, the ABD comprises two light chains, each of which is linked to the ABD directly or indirectly via a linker.

[0228] In some embodiments, the ABD comprises two heavy chains, and the C-terminus of each of the two heavy chains is linked to the ABD, either directly or indirectly via a linker. In some embodiments, the ABD comprises two light chains, and the C-terminus of each of the two light chains is linked to the ABD, either directly or indirectly via a linker.

[0229] 3. Epitope Mapping and Related Techniques The epitope to which the antigen-binding domain binds may consist of a single contiguous sequence of three 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 the target protein. Alternatively, the relevant epitope may consist of multiple 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 the binding domain of the target protein.

[0230] Various techniques known to those skilled in the art can be used to determine the epitope with which the antigen-binding domain used in the ADCs provided herein interacts. Exemplary techniques that can be used to determine the epitope or binding domain of a particular antigen-binding domain include, for example, point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be utilized (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide with which an antigen-binding domain interacts is hydrogen / deuterium exchange, detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterium-labeling a protein of interest and then binding the antigen-binding domain to the deuterium-labeled protein. The protein / antigen-binding domain complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antigen-binding domain (which remains deuterium-labeled). After dissociation of the antigen-binding domain, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing deuterium-labeled residues corresponding to the specific amino acids with which the antigen-binding domain interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography can also be used to identify the amino acids within a polypeptide with which the antigen-binding domain interacts.

[0231] (4. Synthesis of ABD) In one embodiment, the antibody for use in ADC provided herein is a fully human antibody.The method for producing monoclonal antibodies, including fully human monoclonal antibodies, is known in the art.Any such known method can be used in the context of the present disclosure to produce human antibodies that specifically bind to human protein targets.

[0232] For example, using VELOCIMMUNE™ technology or any other similar known method for generating fully human monoclonal antibodies, high-affinity chimeric antibodies against human protein targets are first isolated, having human variable regions and mouse constant regions. The antibodies are characterized and selected for desired characteristics, including affinity, ligand-blocking activity, selectivity, epitope, etc. If necessary, the mouse constant region is replaced with a desired human constant region, such as wild-type or modified IgG1 or IgG4, to generate a fully human antibody. The constant region selected can vary depending on the specific application, but the high-affinity antigen binding and target specificity characteristics reside in the variable region. In some cases, fully human antibodies are isolated directly from antigen-positive B cells.

[0233] Monoclonal antibodies can be produced by any technique familiar to those skilled in the art. Such methods include, but are not limited to, transformation of human peripheral blood cells (e.g., containing B lymphocytes) with Epstein-Barr virus (EBV), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V-region phage library, or other procedures 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, in 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. NY Acad. Sci. 764:525-35. In this technique, elements of the human heavy and light chain loci are introduced into mouse strains derived from embryonic stem cell lines containing targeted disruptions of the endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes can be minigene constructs or transgene loci on yeast artificial chromosomes, which undergo B cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissues. Fully human monoclonal antibodies can be obtained by immunizing transgenic mice that can then produce human antibodies specific to target antigens. Lymphoid cells from immunized transgenic mice can be used to produce hybridomas that secrete human antibodies according to the methods described herein.Polyclonal sera containing fully human antibodies can also be obtained from the blood of the immunized animals.

[0234] Another method for producing the human antibodies of the present disclosure involves immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. Such immortalized B cell lines (or lymphoblastoid cell lines) producing monoclonal antibodies that specifically bind to a target antigen can be identified by the immunodetection methods provided herein, such as ELISA, and then isolated by standard cloning techniques. The stability of lymphoblastoid cell lines producing antibodies against a target antigen can be improved by fusing the transformed cell line with a mouse myeloma to generate a mouse-human hybrid cell line according to methods known in the art (see, for example, Glasky et al., Hybridoma 8:377-89 (1989)). Another method for producing human monoclonal antibodies is in vitro immunization, which involves priming human splenic B cells with the target antigen and then fusing the primed B cells with a heterohybrid fusion partner. See, for example, Boerner et al., 1991 J. Immunol. 147:86-95.

[0235] In one embodiment, B cells producing antibodies against the target antigen are selected, and the light and heavy chain variable regions are cloned from the B cells according to molecular biology techniques known in the art (WO 92 / 02551; U.S. Patent No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B cells from immunized animals can be isolated from the spleen, lymph nodes, or peripheral blood samples by selecting cells producing antibodies that specifically bind to the target antigen. B cells can also be isolated from humans, for example, from peripheral blood samples.

[0236] Methods for detecting single B cells producing antibodies with desired specificity are well known in the art, such as by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibodies, etc. Methods for selecting B cells producing specific antibodies include, for example, preparing a single-cell suspension of B cells in soft agar containing the target antigen. Binding of the specific antibody produced by the B cells to the antigen results in the formation of a complex that is visible as an immunoprecipitate.

[0237] Methods for obtaining antibodies of the present disclosure can also employ various phage display techniques 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. Combinatorial libraries of human or mouse immunoglobulin variable region genes can be generated in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to a target antigen or variants or fragments thereof. See, e.g., U.S. Pat. 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 can be inserted in frame with a sequence encoding a phage coat protein into the genome of a filamentous bacteriophage, such as M13 or a mutant thereof. The fusion protein can be a fusion of the coat protein with a light chain variable region domain and / or a heavy chain variable region domain. In some embodiments, immunoglobulin Fab fragments can also be displayed on phage particles (see, e.g., U.S. Patent No. 5,698,426).

[0238] Antigen-bearing phage can also be enriched using antibody fragments fused to another protein, e.g., a minor coat protein. Rearranged heavy chains (V) from mice immunized with the target antigen (e.g., HBV sAg, tumor-specific antigen) are then cloned into the phage. H ) and light chain (V L Random combinatorial libraries of antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG1, IgG1, IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG1, IgG1, IgG2, IgG4, IgG4, IgG5, IgG6, IgG1, IgG1, IgG2, IgG4, IgG4, IgG5, IgG6, IgG1, IgG1, IgG1, IgG2, IgG4, IgG4, IgG5, IgG6, IgG1, IgG1, IgG2, IgG4 ...

[0239] Heavy and light chain immunoglobulin cDNA expression libraries can also be prepared in lambda phage using, for example, λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B cell population and used to generate heavy and light chain immunoglobulin cDNA expression libraries in λImmunoZap(H) and λImmunoZap(L) vectors. These vectors can be screened individually or coexpressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can then be converted to non-lytic plasmids that allow high-level expression of monoclonal antibody fragments from E. coli.

[0240] In some embodiments, the variable regions of the genes expressing the monoclonal antibody of interest in the hybridoma are amplified using nucleotide primers. These primers may be synthesized by one skilled in the art or purchased from commercial sources (e.g., V Ha , V Hb , V Hc , V Hd , C H1 , V L, and C L (See Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including primers for the V region. These primers can be used to amplify the heavy or light chain variable region, which can then be inserted into a vector such as ImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively. These vectors can then be introduced into E. coli, yeast, or mammalian-based systems for expression. H Domains and V L Large amounts of single-chain proteins containing fusions of the domains can be produced using these methods (see Bird et al., Science 242:423-426, 1988).

[0241] Once cells producing an antibody according to the present disclosure have been obtained using any of the above immunization and other techniques, the specific antibody gene can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures described herein, the antibody produced therefrom can be sequenced, the CDRs identified, and the DNA encoding the CDRs manipulated as previously described to generate other antibodies according to the present disclosure.

[0242] A binding agent of the present disclosure preferably modulates the activity of the target antigen in a cell-based assay described herein and / or an in vivo assay described herein, and / or binds to one or more of the domains described herein, and / or cross-blocks the binding of one of the antibodies described in the present application, and / or is cross-blocked from binding to the target antigen by one of the antibodies described in the present application. Accordingly, such binding agents can be identified using the assays described herein.

[0243] In some 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 herein and / or whose binding to a target antigen is cross-blocked by one of the antibodies described herein. The CDR regions from these antibodies are then used to insert into a suitable biocompatible framework to generate a binding agent for 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 for characterization of the binding agent. Preferably, the binding agent of the present disclosure is an antibody as defined herein.

[0244] Other antibodies according to the present disclosure can be obtained by conventional immunization and cell fusion procedures described herein and known in the art.

[0245] Molecular evolution of the complementarity-determining region (CDR) at the center of the antibody binding site has also been used to isolate antibodies with increased affinity, for example, antibodies with increased affinity for c-erbB-2, as described in Schier et al., 1996, J. Mol. Biol. 263:551. Therefore, such techniques are useful for preparing antibodies against target antigens. Antigen-binding proteins against target antigens can be used, for example, in assays to detect the presence of the target antigen either in vitro or in vivo. Antigen-binding proteins can also be used to purify the target antigen by immunoaffinity chromatography.

[0246] While human, partially human, or humanized antibodies are preferred for many uses, particularly those involving administration of antibodies to human subjects, other types of antigen-binding proteins are preferred for certain uses. Non-human antibodies of the present disclosure can be obtained, for example, from any antibody-producing animal, such as a mouse, rat, rabbit, goat, donkey, or non-human primate (e.g., a monkey (e.g., a cynomolgus or rhesus monkey) or an ape (e.g., a chimpanzee)). Antibodies from a particular species can be produced, for example, by immunizing an animal of that species with a desired immunogen (e.g., HBV sAg, tumor-specific antigen), or by using artificial systems to produce antibodies of that species (e.g., bacterial or phage display-based systems to produce antibodies of a particular species), or by converting an antibody from one species to an antibody from another species, for example, by replacing 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 to more closely resemble the sequence of an antibody from another species. In some embodiments, the antibody is a chimeric antibody comprising amino acid sequences derived from antibodies from two or more different species.

[0247] Antigen binding proteins can be prepared by any of several conventional techniques and screened for desired properties. Some of these techniques involve isolating a nucleic acid encoding the 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 by recombinant DNA technology. The nucleic acid can be fused to another nucleic acid of interest or modified (e.g., by mutagenesis or other conventional techniques) to, for example, add, delete, or substitute one or more amino acid residues. Furthermore, antigen binding proteins can be isolated from cells that naturally express them (e.g., antibodies can be isolated from the hybridoma that produces them) or can be produced in recombinant expression systems using any technique known in the art. See, e.g., 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).

[0248] Any expression system known in the art can be used to produce the recombinant polypeptides of the present disclosure. Expression systems have been comprehensively detailed above. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Among the host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia 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 (ATCC CCL 70), which is derived from the African green monkey kidney cell line CVI described in McMahan et al., 1991, EMBO J. 10:2821. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0249] It will be understood that the antibodies of the present disclosure can have at least one amino acid substitution, provided that the antibody retains its binding specificity. Therefore, modifications to the antibody structure are encompassed within the scope of the present disclosure. These can include amino acid substitutions, which can be conservative or non-conservative, that do not destroy the antibody's ability to bind to its target. Conservative amino acid substitutions can include non-natural amino acid residues, typically incorporated by chemical peptide synthesis rather than by synthesis in a biological system. These include peptidomimetics and other reversed or inverted amino acid moieties. Conservative amino acid substitutions can also include substitutions of natural amino acid residues with standard residues that have little or no effect on the polarity or charge of the amino acid residue at that position.

[0250] Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid mimetics for a member of another class having different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues can be introduced into regions of the human antibody that are homologous with the non-human antibody, or into the non-homologous regions of the molecule.

[0251] Furthermore, those skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is discovered that a change to a particular amino acid residue results in destroyed, unnecessarily reduced, or inappropriate activity, variants with such changes can be avoided. That is, based on the information collected from such routine experiments, those skilled in the art can easily determine amino acids for which further substitutions should be avoided, either alone or in combination with other mutations.

[0252] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. In some embodiments, those skilled in the art can identify suitable regions of the molecule that can be changed without destroying activity by targeting regions that are not considered important for activity. In some embodiments, they can identify residues and portions of the molecule that are conserved between similar polypeptides. In some embodiments, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitution without destroying biological activity or adversely affecting polypeptide structure.

[0253] Additionally, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues in the similar protein that are important for activity or structure. One skilled in the art can select chemically similar amino acid substitutions for such important amino acid residues.

[0254] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence with respect to its structure in similar polypeptides. Given such information, one skilled in the art can 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 can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, because such residues may be involved in important interactions with other molecules.

[0255] In some embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In some embodiments, variants contain more or fewer N-linked glycosylation sites than the native protein. N-linked glycosylation sites are characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitution to eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided are rearrangements of N-linked carbohydrate chains in which one or more N-linked glycosylation sites (usually those present naturally) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or substituted with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be useful when antibodies must be refolded into a biologically active conformation, for example, after isolation of insoluble inclusion bodies. Cysteine ​​variants typically have fewer cysteine ​​residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0256] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art when such substitutions are desired. In some embodiments, preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. In some embodiments, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) can be made in the naturally occurring sequence (in some embodiments, in portions of the polypeptide outside the domains that form intermolecular contacts). In some embodiments, conservative amino acid substitutions typically do not substantially alter the structural features of the parent sequence (e.g., the replacement amino acid should not tend to disrupt helices occurring in the parent sequence or other types of secondary structure that characterize 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, NY (1991)); and Thornton et al., Nature 354:105 (1991), each of which is incorporated herein by reference.

[0257] In certain embodiments, the antibodies of the present disclosure may be chemically conjugated to a polymer, lipid, or other moiety.

[0258] The binding agent can 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 sufficient to form a conformationally stable structural support, or framework, or scaffold, capable of presenting one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that bind to an antigen within a localized surface region. Such structures can be naturally occurring polypeptides or polypeptide "folds" (structural motifs) or can have one or more modifications, e.g., additions, deletions, or substitutions, of amino acids compared to naturally occurring polypeptides or folds. These scaffolds can be derived from polypeptides of any species (or multiple species), e.g., humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0259] Typically, biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains, such as those based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and tendamistat domain (see, e.g., Nygren and Uhlen, 1997, Curr. Opin. in Struct. Biol., 7, 463-469).

[0260] 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).

[0261] Furthermore, those skilled in the art will recognize that suitable binding agents comprise the following portions of these antibodies: LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3. The non-CDR portion of an antibody may be a non-protein molecule that cross-blocks the binding of an antibody disclosed herein to a target antigen. The non-CDR portion of an antibody may be a non-protein molecule that exhibits a binding pattern with a target antigen similar to that exhibited by at least one of the antibodies disclosed herein in a competitive binding assay. The non-CDR portion of an antibody may be a recombinant binding protein or synthetic peptide, and the recombinant binding protein may be composed of amino acids that cross-block the binding of an antibody disclosed herein to a target antigen and / or neutralize the target antigen. The non-CDR portion of an antibody may be a recombinant antibody, and the recombinant antibody may be composed of amino acids that exhibit a binding pattern with a target antigen similar to that exhibited by at least one of the antibodies disclosed herein in a target epitope competitive binding assay (described below) and / or neutralize the target antigen.

[0262] When an antibody comprises one or more of the above-described HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, it can be obtained by expression from a host cell containing DNA encoding these sequences. DNA encoding each CDR sequence can be determined based on the amino acid sequence of the CDR and, if necessary, synthesized together with any desired antibody variable region framework and constant region DNA sequences using oligonucleotide synthesis, site-directed mutagenesis, and polymerase chain reaction (PCR). DNA encoding variable region frameworks and constant regions is widely available to those skilled in the art from gene sequence databases such as GenBank®. In some embodiments, the heavy and light chains of an antibody are expressed from a single DNA construct. In some embodiments, the heavy and light chains of an antibody are expressed from two or more separate DNA constructs.

[0263] Once synthesized, DNA encoding the antibodies or fragments thereof of the present disclosure can be propagated and expressed using any number of known expression vectors according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection. Thus, in certain embodiments, expression of antibody fragments may be preferred in prokaryotic hosts such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol. 178:497-515). In certain other embodiments, expression of antibodies or fragments thereof may be preferred in eukaryotic host cells, 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 (e.g., murine NSO strains), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells.

[0264] One or more replicable expression vectors containing DNA encoding antibody variable and / or constant regions can be prepared and used to transform an appropriate cell line, such as a non-producing myeloma cell line such as the murine NSO strain, or bacteria such as Escherichia coli, in which antibody production will occur. To ensure efficient transcription and translation, the DNA sequence in each vector should contain appropriate regulatory sequences, particularly a promoter and leader sequence, operably linked to the variable domain sequence. Specific methods for producing antibodies in this manner are generally well known and routinely used. For example, basic molecular biology procedures are described in 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).Furthermore, numerous publications describe suitable techniques for manipulating DNA, creating expression vectors, and preparing antibodies by transforming and culturing appropriate cells (Mountain A and Adair, JR in Biotechnology and Genetic Engineering Reviews (Tombs, MP ed.), 10, Chapter 1, 1992, Intercept, Andover, UK); Current Protocols in Molecular Biology, 1999, FM Ausubel (ed.), Wiley Interscience, New York).

[0265] If it is desired to improve the affinity of an antibody according to the present disclosure containing one or more of the above-described CDRs, it can be improved by maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), using mutant 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, These affinity maturation methods are discussed in detail in Vaughan et al. (Nature Biotech., 16, 535-539, 1998).

[0266] Those skilled in the art will appreciate that some proteins, such as antibodies, can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications can include glycosylation variations, methionine oxidation, diketopiperidine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of a carboxy-terminal basic residue (e.g., lysine or arginine) by the action of carboxypeptidase (discussed in Harris, RJ Journal of Chromatography 705:129-134, 1995).

[0267] (5.Biological equivalents) Antigen-binding domains for use in the ADCs provided herein include proteins having amino acid sequences that differ from those of the described antibodies but that retain the ability to bind to a target protein. Such variant antigen-binding domains contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies.

[0268] Two antigen-binding domains are considered bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antigen-binding domains are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their rate is not; however, such differences in absorption rate may be considered bioequivalent because they are intentional, reflected in the labeling, are not essential for achieving effective body drug concentrations during chronic use, and are not considered medically significant for the particular drug being studied.

[0269] In one embodiment, two antigen-binding domains are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0270] In one embodiment, two antigen-binding domains are bioequivalent if a patient can switch between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuous therapy without such switching.

[0271] In one embodiment, two antigen-binding domains are biologically equivalent if they both act by a common mechanism(s) of action for the condition(s) of use, to the extent such mechanisms are known.

[0272] Bioequivalence can be demonstrated by in vivo and in vitro methods. Methods for assessing bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of the antigen-binding domain or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with or reasonably predict in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding domain.

[0273] Biologically equivalent variants of antigen-binding domains for use in the ADCs provided herein can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antigen-binding domains can include variants containing amino acid changes that modify the glycosylation characteristics of the antigen-binding domain, for example, mutations that ablate or eliminate glycosylation.

[0274] 6. Species Selectivity and Species Cross-Reactivity In certain embodiments, the antigen-binding domain for use in the ADCs provided herein binds to a human target protein but not to target proteins from other species. In other embodiments, the antigen-binding domain for use in the ADCs provided herein binds to a human target protein and to target proteins from one or more non-human species. For example, the antigen-binding domain for use in the ADCs provided herein can bind to a human target protein and, optionally, may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee target proteins. In one embodiment, the antigen-binding domain specifically binds to a human target protein and a cynomolgus monkey (e.g., Macaca fascicularis) target protein. In other embodiments, the antigen-binding domain for use herein binds to a human target protein but does not bind, or only weakly binds, to a cynomolgus monkey target protein.

[0275] 7. Exemplary Antibodies and Antigen Targets Antigen-binding domains (ABDs) for use in the ADCs provided herein can have binding specificity for any antigen (target protein) deemed suitable by one of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., a receptor) or a surface protein.

[0276] (ABD for Hepatitis B Virus (HBV) antigen) Chronic hepatitis B infection is usually associated with increased circulating HBV DNA and HBV surface antigen (HBV sAg) in serum. Sustained loss of circulating HBV sAg and HBV DNA is a hallmark of successful infection control or "functional cure." Current therapies using nucleoside analogs reduce HBV load in plasma but are rarely accompanied by HBV sAg loss. Therefore, nucleoside therapy must be administered lifelong to prevent viral regrowth. Some embodiments of the present disclosure relate to ADCs targeting HBV antigens. The ADCs can be used for the treatment of hepatitis B. In some embodiments, the ADCs comprise an ABD specific for HBV sAg, where the HBV sAg can be associated with non-infectious HBV sAg particles, infectious HBV virions, or cells expressing HBV sAg.

[0277] In some embodiments, the ABD comprises the heavy chain and light chain of an antibody specific for HBV sAg. In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody specific for HBV sAg. In some embodiments, the ABD is an antibody specific for HBV sAg. In some embodiments, the HBV antigen is HBV sAg available from Prospect Bio with catalog number HBS-872.

[0278] (ABD against tumor antigens) Some embodiments of the present disclosure relate to ADCs that target tumor antigens. The ADCs can be used to treat cancer. In some embodiments, the ADCs comprise an ABD specific for the tumor antigen.

[0279] In some embodiments, the antigen is expressed on a tumor. In some embodiments, the binding agent interacts with or binds to a tumor antigen, including an antigen specific to a certain type of tumor or an antigen shared, overexpressed, or modified on a particular type of tumor. In one embodiment, the antigen is expressed on a solid tumor. Exemplary antigens include lipoproteins; alpha 1-antitrypsin; cytotoxic T-lymphocyte-associated antigen (CTLA), such as CTLA-4; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; 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 receptors, FOLRI, mesothelin, Crypto, alpha v beta 6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins, e.g., 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 antibodies that bind 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; bone morphogenetic factors; immunotoxins; bone morphogenetic proteins (BMPs); T cell receptors; surface membrane proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM;Tumor-associated antigens, such as AFP, ALK, B7H4, BAGE protein, 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, and EGFRvI II, endoglin, Epcam, EphA2, ErbB2 / Her2, ErbB3 / Her3, ErbB4 / Her4, ETV6-AML, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, globoH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / EBNA1, HLA / k-ras, HLA / MAGE-A3, hTERT, IGF1R, LGR5, LMP 2, MAGE protein, MART-1, mesothelin, ML-IAP, Muc1, Muc16, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-E SO1, 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 protein, 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 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 members 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), but are not limited to these. 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 protein is selected from MAGE-1, -2, -3, -4, -6, and -12. In some embodiments, the GAGE ​​protein is selected from GAGE-1 and GAGE-2.

[0280] (Antibody scaffold) In some embodiments, the antibody contains 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 Gln295 or Q295. Those skilled in the art will recognize that this is a conserved glutamine residue in the wild-type sequences of many antibodies. In other embodiments, the antibody can be modified to contain a glutamine residue. In some embodiments, the antibody contains one or more N297Q mutations. Techniques for modifying antibody sequences to contain glutamine residues are within the capabilities of those skilled in the art (see, e.g., Ausubel et al., Current Protoc. Mol. Biol. (John Wiley & Sons)).

[0281] The antibody may have a Q295 residue, a N297Q mutation, or one or more altered [ka] In embodiments containing a moiety, the payload of Formula I, where R 3is -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y; where Y is -NR 5 R 6 and R 5 and R 6 and each are H) can be directly conjugated to the antibody to form an ADC of Formula V: [ka] (In the formula: R 1 , R 2 and X is as defined 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-; The ABD may contain the Q295 residue, the N297Q mutation, and / or one or more modified [ka] an antibody containing the and k is an integer from 1 to 30.

[0282] In another embodiment, R 10 is -alkylene-NH-, -heteroalkylene-NH-, or -heteroalkylene-arylene-NH-. In another embodiment, R 10 is -alkylene-NH-. In another embodiment, R 10 is -heteroalkylene-NH-. In another embodiment, R 10 is -heteroalkylene-arylene-NH-. In another embodiment, R 10is —CH—NH—, —CH—O-heteroalkylene-NH—, or —CH—O-alkylene-NH—. In another embodiment, R 10 is —CH2OCH2CH2NH—, —CH2OCH2CH2CH2CH2NH—, —CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2NHC(O)CH2NH—, —CH2OCH2-(4-NH-1-phenyl), —CH2OCH(NH—)CH2OH, or —CH2NH—. 10 is —CH2OCH2CH2NH—, —CH2OCH2CH2CH2CH2NH—, —CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2NHC(O)CH2NH—, —CH2OCH2-(4-NH-1-phenyl), or —CH2NH—.

[0283] 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, where ABD is R 3 The compound has a payload (i.e., a TLR7 agonist) attached to the amino group of the compound.

[0284] In another embodiment, an ADC provided herein for use in the compositions and methods provided herein is prepared from a linker-TLR7 agonist of Formula III and has Formula VI: or a pharmaceutically acceptable salt thereof: [ka] (In the formula: R 1 , R 2 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , L1 , ABD, and x are as defined elsewhere for formula III; and and k is an integer from 1 to 30.

[0285] In another embodiment, 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 (in the formula, ABD-L 1 has a payload (i.e., a TLR7 agonist) attached to it.

[0286] VII. Synthesis of ADCs Also provided herein are methods for synthesizing an ADC comprising an ABD, a linker, and a TLR7 agonist. Each component of the ADC (i.e., the ABD, the linker, and the TLR7 agonist) can be synthesized separately and then linked or conjugated to form the ADC.

[0287] In some embodiments, the method comprises the steps of partially reducing the antigen-binding domain with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of the reduced cysteine ​​residue with a maleimide-functionalized linker-payload (i.e., a TLR7 agonist). In some embodiments, the antigen-binding domain is partially reduced by the addition of a 1.5-3.0-fold molar excess of TCEP and 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS pH 7.4 at 37°C for 2 hours. The reduced antigen-binding domain may be buffer-exchanged into PBS containing 1% w / v polysorbate 20. The linker-payload may be added at a linker-payload / antigen-binding domain molar ratio of 5-10 and allowed to react for an additional 2 hours at 25°C in the presence of 12% v / v dimethyl sulfoxide (DMSO). The mixture may be purified, for example, by size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase) to obtain the ADCs provided herein.

[0288] Alternatively, for ADCs of Formula V, in some embodiments, the reaction of the ABD with the payload (i.e., the TLR7 agonist) is mediated by a transglutaminase enzyme. In another embodiment, the transglutaminase enzyme is mouse transglutaminase enzyme. In another embodiment, when the Gln of the ABD is Q295 of the N297 antibody, the ABD is reacted with a PNGase, such as, but not limited to, PNGase F, to deglycosylate N297 prior to the reaction of the ABD with the payload.

[0289] Chemical site-selective protein modification has become increasingly popular for antibody-based bioconjugates. Among all bioorthogonal reactions developed to date, the inverse electron demand Diels-Alder (IEDDA) reaction, a [4 + 2] cycloaddition of 1,2,4,5-tetrazine (s-tetrazine, Tz) with various dienophiles, satisfies most of the bioorthogonal criteria required for conjugation (e.g., fast, selective, biocompatible, and catalyst-free). In this study, the tetrazine linker was designed to have two functions: (1) as a handle with an additional chemical moiety (e.g., an amine) to which an antibody can be attached, while the tetrazine moiety can react with a linker-payload to generate an ADC; and (2) as a linker-payload linker to which an antibody handle can be attached (Titas Deb et al., Chem. Rev. 2021, 121, 12, 6850-6914; Astrid-Caroline Knall and Christian Slugovc., Chem. Soc. Rev., 2013, 42, 5131).

[0290] In one embodiment, the ADC provided herein is selected from those in Table 3: [Table 4] TIFF2025540064000067.tif235170

[0291] VIII. PHARMACEUTICAL COMPOSITIONS In one aspect, the present disclosure provides a pharmaceutical composition comprising an ADC described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a target antigen (e.g., HBV sAg). In some embodiments, the pharmaceutical composition comprises an immune complex (IC) of the ADC and HBV sAg.

[0292] The TLR7 agonist or ADC can be formulated into a suitable pharmaceutical preparation. Typically, the TLR7 agonist or ADC is formulated into a pharmaceutical composition using techniques and procedures well known in the art (see, for example, Ansel, Introduction to Pharmaceutical Dosage Forms, 7th ed., 1999).

[0293] In the composition, an effective concentration of one or more TLR7 agonists or ADCs or pharmaceutically acceptable salts is mixed with a suitable pharmaceutical carrier. In certain embodiments, the concentration of TLR7 agonist or ADC in the composition is effective to deliver an amount that, upon administration, treats, prevents, or ameliorates one or more of the symptoms and / or progression of a disease or disorder disclosed herein.

[0294] Typically, the compositions are formulated for single-dosage administration. To formulate the compositions, a weight fraction of the TLR7 agonist or ADC is dissolved, suspended, dispersed, or otherwise mixed in the selected carrier at an effective concentration to alleviate or ameliorate the condition being treated. Pharmaceutical carriers suitable for administering 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.

[0295] In some embodiments, the TLR7 agonist or ADC is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects on the treated subject. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo systems described herein and known to those skilled in the art, and then human dosages can be extrapolated therefrom. In some embodiments, the ADC is administered in a manner that achieves a therapeutically effective concentration of the payload. In some embodiments, a companion diagnostic (see, e.g., Olsen D and Jorgensen JT, 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 a particular subject or subject population.

[0296] The concentration of the TLR7 agonist or ADC in the pharmaceutical composition will depend on the absorption, tissue distribution, inactivation, and excretion rates of the TLR7 agonist or ADC, the physicochemical characteristics of the TLR7 agonist or ADC, the dosing schedule, and the amount administered, as well as other factors known to those skilled in the art. For example, the amount delivered will be sufficient to ameliorate one or more symptoms of a disease or disorder disclosed herein.

[0297] The composition may be administered at once, or may be divided into several smaller doses that are administered at intervals.It is understood that the exact dosage and duration of treatment are a function of the disease being treated and can be determined experimentally using known testing protocols or by extrapolation from in vivo or in vitro test data.It should be noted that concentration and dosage values ​​may also vary depending on the severity of the condition to be alleviated.It should further be understood that for any particular subject, specific dosage regimens should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0298] The compositions may include other active compounds to obtain a desired combination of properties. The TLR7 agonists or ADCs provided herein, or pharmaceutically acceptable salts thereof described herein, may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the general art to be useful in treating one or more of the diseases or medical conditions mentioned herein. It should be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

[0299] The pharmaceutical composition may be in any form suitable for human or veterinary medicine, including a liquid, oil, emulsion, gel, colloid, aerosol, or solid.

[0300] The pharmaceutical compositions may be formulated for administration by any route of administration suitable for human or veterinary medicine, including enteral and parenteral routes of administration.

[0301] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.

[0302] (IX. Administration) The compounds and pharmaceutical compositions provided herein can be administered in specific therapeutically or prophylactically effective amounts, at specific time intervals, in specific dosage forms, and by specific dosage administration methods, as described below.

[0303] Although some diseases or disorders are more prevalent in certain age groups, the methods provided herein encompass treating patients regardless of their age.

[0304] The TLR7 agonists or ADCs, or pharmaceutically acceptable salts thereof, provided herein can be administered repeatedly if necessary, for example, until the subject experiences stable or regression of disease, or until the subject experiences disease progression or unacceptable toxicity.

[0305] The TLR7 agonists or ADCs, or pharmaceutically acceptable salts thereof, provided herein can be administered once daily (QD) or divided into multiple daily doses, e.g., twice daily (BID), three times daily (TID), and four times daily (QID). Furthermore, administration can be continuous (i.e., every day for consecutive days, or every day), intermittent, e.g., cyclical (i.e., with drug-free periods of several days, weeks, or months). As used herein, the term "daily" is intended to mean that a therapeutic compound, such as a TLR7 agonist or ADC, or a pharmaceutically acceptable salt thereof, provided herein is administered, for example, once or multiple times daily over a period of time. The term "continuous" is intended to mean that a therapeutic compound, such as a TLR7 agonist or ADC, or a pharmaceutically acceptable salt thereof, provided herein is administered daily without interruption for a period of at least 10 days to 52 weeks. As used herein, the term "intermittent" or "intermittently" is intended to mean stopping and starting at regular or irregular intervals. For example, intermittent administration of a TLR7 agonist or ADC, or a pharmaceutically acceptable salt thereof, provided herein can be administration 1-6 days per week, cyclical administration (e.g., daily administration for 2-8 consecutive weeks, followed by a rest period of up to 1 week), or every other day administration. As used herein, the term "cycling" is intended to mean that a therapeutic compound, such as a TLR7 agonist or ADC, or a pharmaceutically acceptable salt thereof, provided herein is administered daily or continuously, but with a rest period. In some such embodiments, administration is once daily for 2-6 days, followed by a rest period of 5-7 days without administration.

[0306] (X.Treatment method) In some embodiments, methods are provided for treating a subject using a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof. In some embodiments, methods are provided for treating a subject using a pharmaceutical composition comprising a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition comprises any of the TLR7 agonists or ADCs disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0307] 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 the expression, signaling, or activity of the target protein of the antigen-binding domain.

[0308] In certain embodiments, the ADCs provided herein are used to treat primary and / or metastatic tumors arising in the brain and cerebral meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive organs, muscle, bone, skin and appendages, connective tissue, spleen, immune system, hematopoietic 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 the following cancers: acute myeloid leukemia, adult T-cell leukemia, astrocytoma, 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, glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC)), and other cancers. SCLC), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, malignant mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, castration-resistant prostate cancer, renal cell carcinoma, residual cancer (wherein "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject after treatment with an anti-cancer therapy), rhabdomyosarcoma, gastric 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.

[0309] In some embodiments, the subject has chronic hepatitis B. In some embodiments, the ADCs provided herein are used to treat chronic hepatitis B.

[0310] In some embodiments, the subject is diagnosed with chronic hepatitis B. In some embodiments, the subject has elevated circulating HBV DNA or HBV sAg in their serum before administration of the ADC or pharmaceutical composition. In some embodiments, the methods of treatment provided herein further comprise measuring circulating HBV DNA or HBV sAg in the subject's serum before administration of the ADC or pharmaceutical composition. In some embodiments, the methods of treatment provided herein further comprise measuring circulating HBV DNA or HBV sAg in the subject's serum after administration to assess the therapeutic efficacy of the ADC or pharmaceutical composition.

[0311] In the context of the methods of treatment provided herein, the TLR7 agonist or ADC can be administered as 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).

[0312] VII. Combination Therapy with a Second Active Agent Provided herein are compositions comprising any of the TLR7 agonists or ADCs provided herein in combination with one or more additional therapeutically active ingredients, and methods of treatment comprising administering such combinations to a subject.

[0313] The TLR7 agonists or ADCs provided herein can be used in combination with other MET antagonists (e.g., anti-MET antibodies (e.g., onartuzumab, emibetuzumab, and H4H14639D), or small molecule inhibitors of MET), EGFR antagonists (e.g., anti-EGFR antibodies (e.g., cetuximab or panitumumab), or small molecule inhibitors of EGFR (e.g., gefitinib or erlotinib)), antagonists of another EGFR family member, e.g., Her2 / ErbB2, ErbB3, or ErbB4 (e.g., anti-ErbB2 (e.g., trastuzumab or T-DM1 {KADCYLA®}), anti-ErbB3 or anti-ErbB4 antibodies, or ErbB2, ErbB3, or small molecule inhibitors of ErbB4 activity), EGFRvIII antagonists (e.g., anti-EGFRvIII antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies or small molecule kinase inhibitors such as, e.g., imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C, or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., VEGF-Trap, e.g., aflibercept, e.g., U.S. Pat. No. 7,087,087,No. 411 (also referred to herein as "VEGF-inhibitory fusion proteins"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 2009 / 0142354, e.g., REGN421), Ang2 antagonists (e.g., see US 2009 / 0142354, e.g., REGN421), Ang3 antagonists (e.g., see US 2009 / 0142354, e ... anti-Ang2 antibodies disclosed in 2011 / 0027286 (e.g., H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin (e.g., anti-UPK3A) antibodies), MUC16 antagonists (e.g., anti-MUC16 antibodies) , 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), an LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody, e.g., rituximab), a CD20xCD3 bispecific antibody, a PD-1 blocker (e.g., an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab), and the like. Other agents that can be advantageously administered in combination with the antibodies provided herein include, for example, 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, and IL-18, or their respective receptors.

[0314] By way of example, a PD-1 inhibitor, such as an anti-PD-1 antibody, can be combined with a TLR7 agonist or ADC described herein.

[0315] 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; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, and fenesterine. , prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., aclacinomycin (aclacinomycin), actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as florinic acid acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestravsil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (trademark); razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, NJ), and docetaxel (Taxotere™; Aventis Antony, France); chlorambucil; gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogues, such as cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate;Included in this definition are CPT-11, the topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY 117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0316] The TLR7 agonists or ADCs provided herein can also be administered in combination with and / or co-formulated with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotective agents, metal chelators, IFN-γ, and / or NSAIDs.

[0317] The additional therapeutically active ingredient, for example, any of the above-mentioned agents or derivatives thereof, can be administered immediately before, simultaneously with, or shortly after administration of the TLR7 agonists or ADCs provided herein. In some embodiments, provided are pharmaceutical compositions in which the TLR7 agonists or ADCs provided herein are co-formulated with one or more of the additional therapeutically active ingredients described herein.

[0318] As used herein, the term "in combination" includes the use of more than one therapies (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 patient 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 a subject. Triple therapies are also contemplated herein.

[0319] Administration of a compound or derivative thereof provided herein and one or more second active agents to a subject can be simultaneous or sequential, by the same or different routes of administration. The suitability of a particular route of administration for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without being degraded before entering the bloodstream) and the disease or disorder being treated.

[0320] XI. Further Implementations and Provisions The present disclosure is further illustrated by the following non-limiting embodiments. Embodiment 1. A compound of Formula I: or a pharmaceutically acceptable salt thereof [ka] (In the formula: R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2is H, halo, or alkoxy; R 3 -CO2R 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, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and R 7 and R 8 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; provided that the compound is a compound of the following formula: [ka] isn't it). Embodiment 2. A compound according to embodiment 1 or a pharmaceutically acceptable salt thereof. (In the formula: R 1 , halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is halo or alkoxy; R 3 -CONHR23 , -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 and; R 5 and R 6 are (i), (ii), and (iii): (i)R 5 and R 6 are each H; (ii)R 5 is H and R 6 is alkyl; (iii)R 5 and R 6 form a heterocyclic ring together with the N to which they are attached; is selected from R 7 and R 8 together with the N to which they are attached form a heterocyclic ring). Embodiment 3. R 4 The compound of embodiment 1 or 2, selected with the proviso that is not substituted with hydroxyl. Embodiment 4. R 3 The compound of any one of embodiments 1-3, wherein the alkylene and heteroalkylene moieties are selected from the group consisting of: with the proviso that they are not substituted with oxo. Embodiment 5. The compound of any one of embodiments 1 to 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. R 1 But, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and is a linear chain six atoms in length. Embodiment 7. R 1 The compound of any one of embodiments 1-6, wherein is halo. Embodiment 8. R 1 Ga-NHR 4 7. The compound of any one of embodiments 1 to 6, wherein: Embodiment 9. R 1 -OR 4 7. The compound of any one of embodiments 1 to 6, wherein: Embodiment 10. R 1 -NH-OR 4 7. The compound of any one of embodiments 1 to 6, wherein: Embodiment 11. R 1 Ga-R 4 7. The compound of any one of embodiments 1 to 6, wherein: Embodiment 12. R 1 The compound of any one of embodiments 1-5, wherein is -NH-n-pentyl, -NH-On-butyl, -On-pentyl, -n-hexyl, or -NH-CH2CH2-OEt. Embodiment 13. R 1 The compound of any one of embodiments 1-5, wherein is -NH-n-pentyl. Embodiment 14. R 1 The compound of any one of embodiments 1-5, wherein is -NH-On-butyl. Embodiment 15. R1 The compound of any one of embodiments 1-5, wherein is -On-pentyl. Embodiment 16. R 1 The compound of any one of embodiments 1-5, wherein is -n-hexyl. Embodiment 17. R 1 The compound of any one of embodiments 1-5, wherein is —NH—CH 2 CH 2 —OEt. Embodiment 18. R 2 Embodiment 18. The compound of any one of embodiments 1-17, wherein is alkoxy. Embodiment 19. R 2 Embodiment 18. The compound of any one of embodiments 1-17, wherein is methoxy. Embodiment 20. R 2 The compound of any one of embodiments 1-17, wherein is H. Embodiment 21. R 2 Embodiment 18. The compound of any one of embodiments 1-17, wherein is halo. Embodiment 22. R 3 But -CONHR 23 , -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y. Embodiment 23. R 3 CONHR 23 23. The compound of any one of embodiments 1-22, wherein: Embodiment 24. R 3 23. The compound of any one of embodiments 1-22, wherein is -alkylene-Y. Embodiment 25. R 3 23. The compound of any one of embodiments 1-22, wherein is -heteroalkylene-Y. Embodiment 26. R 3 23. The compound of any one of embodiments 1-22, wherein is -heteroalkylene-arylene-Y. Embodiment 27. R 323. The compound of any one of embodiments 1-22, wherein is -(hydroxy)heteroalkylene-Y. Embodiment 28. R 3 23. The compound of any one of embodiments 1-22, wherein is -(amino)heteroalkylene-Y. Embodiment 29. R 3 23. The compound of any one of embodiments 1-22, wherein is -alkylene-PEG-Y. Embodiment 30. R 3 23. The compound of any one of embodiments 1-22, wherein is -CONH2, -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. Embodiment 31. R 3 23. The compound of any one of embodiments 1-22, wherein is —CH 2 —Y, —CH 2 —O-heteroalkylene-Y, or —CH 2 —O-alkylene-Y. Embodiment 32. R 3 is -C(Me)2OH, -CO2H-CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH 2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), - 23. The compound of any one of embodiments 1-22, which is 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. R 3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OC H2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2 23. The compound of any one of embodiments 1-22, which is OCH2-(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. R 4 Embodiment 34. The compound according to any one of embodiments 1-33, wherein is n-butyl, n-pentyl, n-hexyl, or ethoxyethyl. Embodiment 35. R 4 The compound of any one of embodiments 1-33, wherein is n-butyl. Embodiment 36. R 4 Embodiment 34. The compound of any one of embodiments 1-33, wherein is n-pentyl. Embodiment 37. R 4 Embodiment 34. The compound of any one of embodiments 1-33, wherein is n-hexyl. Embodiment 38. R 4 Embodiment 34. The compound according to any one of embodiments 1-33, wherein is ethoxyethyl. Embodiment 39. R 5 and R 6are each independently H or alkyl, or together with the N to which they are attached form a piperazinyl ring. Embodiment 40. R 5 and R 6 and R are each H. Embodiment 41. R 5 is H and R 6 Embodiment 39. The compound of any one of embodiments 1-38, wherein is alkyl. Embodiment 42. R 5 and R 6 taken together with the N to which they are attached to form 1-piperazinyl. Embodiment 43. The compound of any one of Embodiments 1 to 42, wherein Y is OH. Embodiment 44. The compound of any one of Embodiments 1 to 42, wherein Y is Gly. Embodiment 45. Y is -NR 5 R 6 43. The compound of any one of embodiments 1-42, wherein: Embodiment 46. The compound of any one of Embodiments 1 to 42, wherein Y is -COZ. Embodiment 47. The compound of any one of Embodiments 1 to 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 to 47, wherein Z is —OH. Embodiment 49. The compound of any one of Embodiments 1 to 47, wherein Z is alkoxy. Embodiment 50. Z is -NR 7 R 8 48. The compound of any one of embodiments 1-47, wherein: Embodiment 51. The compound of any one of Embodiments 1 to 47, wherein Z is -OH, ethoxy, -Nn-Pr2, or 1-piperazinyl. Embodiment 52. The compound of any one of Embodiments 1 to 47, wherein Z is —OH or 1-piperazinyl. Embodiment 53. R 7 and R 8 is each independently H or n-propyl, or together with the N to which they are attached form 1-piperazinyl. Embodiment 54. R 7 and R 8 taken together with the N to which they are attached to form 1-piperazinyl. Embodiment 55. [Table 5] TIFF2025540064000071.tif240170TIFF2025540064000072.tif248170TIFF2025540064000073.tif239170TIFF2025540064000074.tif249170TIFF2025540064000075.tif59170: and a pharmaceutically acceptable salt of any of these compounds. Embodiment 56. Formula II: or a pharmaceutically acceptable salt thereof [ka] (In the formula: R 1 , R 2 and X is as defined in embodiment 1 for Formula I; R 9 is as defined in embodiment 1, 3 The terminal hydrogen from the group (i.e., R 9 is a divalent radical formed by removal of a hydrogen atom distal to a phenyl group to which is attached L is any group or moiety that links, connects or binds to the antigen binding domain ABD; provided that the compound is a compound of the following formula: [ka] isn't it). Embodiment 57. R 9 But, -alkylene-Y 1 -, -heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 , or -alkylene-PEG-Y 1 57. The compound of embodiment 56, wherein: Embodiment 58. R 9 -Alkylene-Y 1 58. The compound of embodiment 56 or 57, wherein - Embodiment 59. R 9 Ga-heteroalkylene-Y 1 58. The compound of embodiment 56 or 57, wherein - Embodiment 60. R 9 -heteroalkylene-arylene-Y 1 58. The compound of embodiment 56 or 57, wherein - Embodiment 61. R 9 -(hydroxy)heteroalkylene-Y 1 58. The compound of embodiment 56 or 57, wherein Embodiment 62. R 9 -(amino)heteroalkylene-Y 1 58. The compound of embodiment 56 or 57, wherein Embodiment 63. R 9 -Alkylene-PEG-Y 1 58. The compound of embodiment 56 or 57, wherein Embodiment 64. R 9 But -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 58. The compound of embodiment 56 or 57, wherein - Embodiment 65. R 9is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2O CH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-( 58. The compound of embodiment 56 or 57, which is (4-NH-)-1-phenyl), -CHOCHCOO-, -CHOCHCHOCHCO-, -CHOCHCHOCHOCHCO-, -CHOCHCHOCHCHOCHCO-, -CHOCHCO-1-piperazin-4-yl, -(R)-CHOCH(OH)CHO-, -(S)-CHOCH(OH)CHO-, -CHOCH(NH)CHO-, -CHO-, -CHNH-, or -CH-1-piperazin-4-yl. Embodiment 66. Y 1 The compound of any one of embodiments 55-65, wherein is —O—. Embodiment 67. Y 1 The compound of any one of embodiments 55-65, wherein is Gly. Embodiment 68. Y 1 Ga-NR 5 - . Embodiment 69. Y 1 Ga-COZ 1 66. The compound of any one of embodiments 55-65, wherein: Embodiment 70. Y 1 66. The compound of any one of embodiments 55-65, wherein is -O-, glycine, -NH-, 1-piperazin-4-yl, -COO-, or -CO-1-piperazin-4-yl. Embodiment 71. Z 1 70. The compound of embodiment 69, wherein is -O-. Embodiment 72. Z 1 Ga-NR7 70. The compound of embodiment 69, wherein Embodiment 73. Z 1 70. The compound of embodiment 69, wherein is -O- or 1-piperazin-4-yl. Embodiment 74. R 5 is H. Embodiment 75. R 5 69. The compound of embodiment 68, wherein is alkyl. Embodiment 76. The compound of any one of Embodiments 55 to 75, wherein L is non-cleavable under physiological conditions. Embodiment 77. The compound of any one of Embodiments 55 to 75, wherein L is cleavable under physiological conditions. Embodiment 78. The compound of embodiment 77, wherein L is an acid labile linker, a hydrolytically labile linker, an enzyme-cleavable linker, a reduction-labile linker, or a self-immolative linker. Embodiment 79. The compound of any one of embodiments 55 to 78, wherein L is or comprises a peptide, carbohydrate, glucuronide, polyethylene glycol (PEG) unit, hydrazone, malcaproyl unit, dipeptide unit, valine-citrulline unit, or 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 maleimide, an N-hydroxysuccinimide ester, or a cyclooctynyl group. Embodiment 84. L is 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, [ka] TIFF2025540064000079.tif246170TIFF2025540064000080.tif181170. Embodiment 85. [Table 6] TIFF2025540064000082.tif245170TIFF2025540064000083.tif182170: and a pharmaceutically acceptable salt of any of these compounds. Embodiment 86. A compound of Formula III: or a pharmaceutically acceptable salt thereof [ka] (In the formula: R 1 , R 2 and X is as defined in embodiment 1 for Formula I; L is any group or moiety that links, connects or binds to the antigen binding domain ABD; R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 11 or R 14 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylenealkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6). Embodiment 87. R 11 and R 12 is independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 11 or R 14 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16is alkylenealkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6; The compound of embodiment 86. 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. A compound according to any one of embodiments 1 to 88 or a compound of the formula: [ka] An antibody drug conjugate (ADC) comprising: Embodiment 90. An ADC according to embodiment 89 having formula IV: or a pharmaceutically acceptable salt thereof. [ka] (In the formula: R 1 , R 2 , R 9 and X is as defined in embodiment 1 for formula I and in embodiment 56 for formula II; L 1 is a bivalent linker; ABD is the antigen binding domain; and and k is an integer from 1 to 30. Embodiment 91. The ADC of embodiment 89 or 90, which is ABD-LP1, ABD-LP6A, ABD-LP7A, ABD-LP8A, ABD-LP10A, or ABD-LP11A. Embodiment 92. An ADC according to embodiment 89 having formula V: or a pharmaceutically acceptable salt thereof. [ka] (In the formula: R 1 , R 2 and X is as defined in embodiment 1 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-; and The ABD may contain the Q295 residue, the N297Q mutation, and / or one or more modified [ka] an antibody containing the and k is an integer from 1 to 30. Embodiment 93. R 10 93. The ADC of embodiment 92, wherein is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-. Embodiment 94. R 10

[0099] Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is -alkylene-NH-. Embodiment 95. R 10

[0088] Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is -heteroalkylene-NH-. Embodiment 96. R 10

[0097] Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is -heteroalkylene-arylene-NH-. Embodiment 97. R 10 Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is --(hydroxy)heteroalkylene-NH-. Embodiment 98. R 10

[0099] Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is -(amino)heteroalkylene-NH-. Embodiment 99. R 10

[0097] Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is -alkylene-PEG-NH-. Embodiment 100. R 10 Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein is —CH—NH—, —CH—O-heteroalkylene-NH—, or —CH—O-alkylene-NH—. Embodiment 101. R 10 is —CH2OCH2CH2NH—, —CH2OCH2CH2CH2CH2NH—, —CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2NHC(O)CH2NH—, —CH2OCH2-(4-NH-1-phenyl), —CH2OCH(NH—)CH2OH, or —CH2NH—. Embodiment 102. R 10 is —CH2OCH2CH2NH—, —CH2OCH2CH2CH2CH2NH—, —CH2OCH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2CH2OCH2CH2NH—, —CH2OCH2CH2OCH2CH2OCH2CH2NH—, —CH2OCH2NHC(O)CH2NH—, —CH2OCH2-(4-NH-1-phenyl), or —CH2NH—. Embodiment 103. A compound 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 R 3 The ADC of any one of embodiments 89 to 102, wherein the payload (i.e., the TLR7 agonist) is attached to the amino group of the ADC. Embodiment 104. An ADC according to embodiment 89 having formula VI: [ka] (In the formula: L 1 is a bivalent linker; R 1 , R 2 , R 16 , R 11 , R 12, R 13 , R 14 , R 15 , X, and x are as defined in embodiment 86 for Formula III; and and k is an integer from 1 to 30. Embodiment 105. 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, wherein ABD-L 1 R 3 The ADC of embodiment 104, wherein the payload (i.e., TLR7 agonist) is attached on the alcohol group of Embodiment 106. The ADC of any one of embodiments 89 to 105, wherein the ABD has binding specificity for a transmembrane molecule (e.g., a receptor) expressed on a tumor. Embodiment 107. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 88 or an ADC according to any one of embodiments 89 to 106 and a pharmaceutically acceptable carrier. Embodiment 108. A method for treating or diagnosing a disease, comprising administering to a subject a compound according to any one of embodiments 1 to 88, or an ADC according to any one of embodiments 89 to 106, or a pharmaceutical composition according to 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.

[0321] The present disclosure is further described by the following non-limiting clauses. Article 1. a. an antigen-binding domain (ABD) having binding specificity for hepatitis B virus surface antigen (HBV sAg); and b. Toll-like receptor 7 (TLR7) agonists An antibody drug conjugate (ADC) comprising: Clause 2. The ADC of clause 1, further comprising a bivalent linker connecting said ABD to said TLR7 agonist. Clause 3. The ADC of Clause 2, wherein said ADC is according to Formula IV: or a pharmaceutically acceptable salt thereof. [ka] (In the formula: L 1 is a bivalent linker; R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is H, halo, or alkoxy; R 4 is alkyl optionally substituted with alkoxy or heteroalkyl; R 9 is R 3 is a divalent radical formed by removal of hydrogen from 3 is R 9 is a group attached to the phenyl group at position R 3 -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, alkyl, or aryl; X is CH or N; Y is -OH, -Gly, -NR 5 R 6 , or -COZ; Z is -OH, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; R 7 and R 8 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and and k is an integer from 1 to 30. Clause 4. The ADC: 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 3. An ADC as described in clause 3, selected provided that it does not contain: Article 5.R 1 But, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and having a linear chain six atoms in length. Article 6.R 1 is a halo. Article 7.R 1 Ga-NHR 4 6. The ADC according to clause 5, Article 8. R 1 -OR 4 6. The ADC according to clause 5, Article 9.R 1 -NH-OR 4 6. The ADC according to clause 5, Article 10.R1 Ga-R 4 6. The ADC according to clause 5, Article 11.R 1 The ADC of clause 5, wherein is -NH-n-pentyl, -NH-On-butyl, -On-pentyl, -n-hexyl, or -NH-CH2CH2-OEt. Article 12.R 1 The ADC according to clause 5, wherein is -NH-n-pentyl. Article 13.R 1 The ADC according to clause 5, wherein is -NH-On-butyl. Article 14.R 1 The ADC according to clause 5, wherein is -On-pentyl. Article 15.R 1 The ADC according to clause 5, wherein is -n-hexyl. Article 16.R 1 The ADC of clause 5, wherein is -NH-CH2CH2-OEt. Article 17.R 2 17. The ADC of any one of clauses 3 to 16, wherein is alkoxy. Article 18.R 2 17. The ADC of any one of clauses 3 to 16, wherein is methoxy. Article 19.R 2 is H. Article 20.R 2 17. The ADC of any one of clauses 3 to 16, wherein is halo. Article 21.R 3 But -CONHR 23 21. The ADC of any one of clauses 3 to 20, wherein the heteroalkylene-Y is -alkylene-Y, -heteroalkylene-Y or -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y. Article 22.R 3 CONHR 23 22. The ADC of clause 21, Article 23.R 3 22. The ADC of clause 21, wherein is -alkylene-Y. Article 24.R3 22. The ADC of clause 21, wherein is -heteroalkylene-Y. Article 25.R 3 22. The ADC of clause 21, wherein is -heteroalkylene-arylene-Y. Article 26.R 3 22. The ADC of clause 21, wherein is -(hydroxy)heteroalkylene-Y. Article 27.R 3 22. The ADC of clause 21, wherein is -(amino)heteroalkylene-Y. Article 28.R 3 22. The ADC of clause 21, wherein is -alkylene-PEG-Y. Article 29.R 3 21. The ADC of any one of clauses 3 to 20, wherein is -CONH2, -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. Article 30.R 3 21. The ADC of any one of clauses 3 to 20, wherein is —CH 2 —Y, —CH 2 —O-heteroalkylene-Y, or —CH 2 —O-alkylene-Y. Article 31.R 3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OC H2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC 21. The ADC of any one of clauses 3-20, wherein (O) is CH2NH2, -CHOCH2-(4-NH2-1-phenyl), -CHOCH2COOH, -CHOCH2COOEt, -CHOCH2CON(n-Pr)2, -CHOCH2CO-1-piperazinyl, -(R)-CHOCH(OH)CH2OH, -(S)-CHOCH(OH)CH2OH, -CHOCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2, or -CH2-1-piperazinyl. Article 32.R3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OC H2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH 21. The ADC of any one of clauses 3-20, wherein the ADC is 2OCH2-(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. Article 33.R 4 33. The ADC of any one of clauses 3 to 32, wherein is n-butyl, n-pentyl, n-hexyl, or ethoxyethyl. Article 34.R 4 34. The ADC of clause 33, wherein is n-butyl. Article 35.R 4 34. The ADC according to clause 33, wherein is n-pentyl. Article 36.R 4 34. The ADC of clause 33, wherein is n-hexyl. Article 37.R 4 34. The ADC according to clause 33, wherein is ethoxyethyl. Article 38.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. Article 39.R 5 and R 6and each is H. Article 40.R 5 is H and R 6 39. The ADC of clause 38, wherein is alkyl. Article 41.R 5 and R 6 together with the N to which they are attached form 1-piperazinyl. Clause 42. The ADC of any one of clauses 3 to 41, wherein Y is OH. Clause 43. The ADC of any one of clauses 3 to 41, wherein Y is glycine. Article 44. Y is -NR 5 R 6 42. The ADC of any one of clauses 3 to 41, wherein Clause 45. The ADC of any one of clauses 3 to 41, wherein Y is -COZ. Clause 46. The ADC of any one of clauses 3 to 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 to 41, wherein Y is -OH, -NH2, 1-piperazinyl, -COOH, or -CO-1-piperazinyl. Clause 48. The ADC according to clause 45, wherein Z is -OH. Clause 49. The ADC according to Clause 45, wherein Z is alkoxy. Article 50. Z is -NR 7 R 8 46. ​​The ADC according to Clause 45, Clause 51. The ADC of Clause 45, wherein Z is -OH, ethoxy, -Nn-Pr2, or 1-piperazinyl. Clause 52. The ADC according to Clause 45, wherein Z is -OH or 1-piperazinyl. Article 53.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. Article 54.R 7 and R 8 together with the N to which they are attached form 1-piperazinyl. Article 55. ABD-L 1 is R of the compound 3 to a compound selected from P1-P43 and pharmaceutical salts thereof by removal of a hydrogen from the group at the position corresponding to: [Table 7] TIFF2025540064000092.tif249170TIFF2025540064000093.tif246170TIFF2025540064000094.tif249170TIFF2025540064000095.tif133170Article 56. R 9 But, -alkylene-Y 1 -, -heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 , or -alkylene-PEG-Y 1 55. The ADC of any one of clauses 3 to 54, wherein Article 57.R 9 -Alkylene-Y 1 57. An ADC according to clause 56, wherein Article 58.R 9 Ga-heteroalkylene-Y 1 57. An ADC according to clause 56, wherein Article 59.R 9 -heteroalkylene-arylene-Y 1 57. An ADC according to clause 56, wherein Article 60.R 9 -(hydroxy)heteroalkylene-Y 1 57. The ADC according to Clause 56, Article 61.R 9 -(amino)heteroalkylene-Y1 57. The ADC according to Clause 56, Article 62.R 9 -Alkylene-PEG-Y 1 57. An ADC according to clause 56, wherein Article 63.R 9 But -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 57. An ADC according to clause 56, wherein Article 64.R 9 is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH 2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazin-4-yl-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH 57. The ADC of clause 56, which is 2-((4-NH-)-1-phenyl), -CHOCHCOO-, -CHOCHCHOCHCO-, -CHOCHCHOCHOCHCO-, -CHOCHCHOCHCHOCHCO-, -CHOCHCO-1-piperazin-4-yl, -(R)-CHOCH(OH)CHO-, -(S)-CHOCH(OH)CHO-, -CHOCH(NH)CHO-, -CHO-, -CHNH-, or -CH-1-piperazin-4-yl. Article 65. Y 1 65. The ADC of any one of clauses 56-64, wherein is -O-. Article 66. Y 1 65. The ADC of any one of clauses 56 to 64, wherein is Gly. Article 67. Y 1 Ga-NR 5 65. The ADC of any one of clauses 56 to 64, wherein Article 68. Y 1 Ga-COZ1 where Z 1 -O-, -NR 7 65. The ADC of any one of clauses 56 to 64, wherein the ADC is -, -O-alkylene-, or 1-piperazin-4-yl. Article 69. Y 1 65. The ADC of any one of clauses 56-64, wherein is -O-, -NH-, 1-piperazin-4-yl, -COO-, or -CO-1-piperazin-4-yl. Article 70.Z 1 69. The ADC of clause 68, wherein is -O-. Article 71.Z 1 Ga-NR 7 68. An ADC according to clause 68, wherein Article 72.R 7 72. The ADC of clause 71, wherein Article 73.R 7 72. The ADC of clause 71, wherein is alkyl. Article 74.Z 1 69. The ADC of clause 68, wherein is 1-piperazin-4-yl. Article 75. Y 1 70. The ADC of clause 69, wherein is 1-piperazin-4-yl. Article 76. Y 1 70. The ADC of clause 69, wherein is -CO-1-piperazin-4-yl. Article 77. L 1 77. The ADC of any one of clauses 3 to 76, wherein Article 78. L 1 77. The ADC of any one of clauses 3 to 76, wherein is cleavable under physiological conditions. Article 79. L 1 is an acid labile linker, a hydrolytically labile linker, an enzyme-cleavable linker, a reduction-labile linker, or a self-immolative linker. Article 80. L 180. The ADC of any one of clauses 3-79, wherein X is or comprises a peptide, carbohydrate, glucuronide, polyethylene glycol (PEG) unit, hydrazone, mal-caproyl unit, dipeptide unit, valine-citrulline unit, or para-aminobenzyl (PAB) unit. Article 81. L 1 81. The ADC of any one of clauses 3 to 80, wherein Article 82. L 1 79. An ADC according to clause 79, wherein Article 83. L 1 81. The ADC of clause 80, wherein comprises p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC). Article 84. L 1 77. The ADC of any one of clauses 3 to 76, wherein Article 85. L 1 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, [ka] 77. The ADC of any one of clauses 3 to 76, which is a group derived from TIFF2025540064000097.tif215170TIFF2025540064000098.tif141170. Clause 86. The ADC according to Clause 3, comprising an ABD linked to a compound selected from LP1, LP6 to LP12, and pharmaceutically acceptable salts of these compounds, wherein LP1 and LP6 to LP12 have the following structures: [Table 8] TIFF2025540064000100.tif245170TIFF2025540064000101.tif238170TIFF2025540064000102.tif202170. Clause 87. An ADC according to Clause 3 comprising an ABD linked to a compound of Formula III: [ka] (In the ceremony R 1 , R 2 and X is 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, where R 11 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 11 or R 14 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylenealkylene-arylene, heteroalkylene, heteroalkylene-arylene-, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6). Article 88. R 11 and R 12 is independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 11 or R 14 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylenealkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6; ADC as described in Article 87. Article 89. 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. Clause 90. The ADC of clause 89, wherein said ABD is linked to a compound selected from LP1, LP6, LP7, LP8, LP10, and LP11. Clause 91. The ADC of Clause 3, wherein said ADC is according to Formula V: or a pharmaceutically acceptable salt thereof. [ka] (In the formula: R 1 , R 2 and X is 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-; The ABD may contain the Q295 residue, the N297Q mutation, and / or one or more modified [ka] an antibody containing the and k is an integer from 1 to 30. Article 92.R 10 92. The ADC of clause 91, wherein is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-. Article 93.R 10 93. The ADC of clause 92, wherein is -alkylene-NH-. Article 94.R 10 93. The ADC of clause 92, wherein is -heteroalkylene-NH-. Article 95.R 10 93. The ADC of clause 92, wherein is -heteroalkylene-arylene-NH-. Article 96.R 10 93. The ADC of clause 92, wherein is -(hydroxy)heteroalkylene-NH-. Article 97.R 10 93. The ADC of clause 92, wherein is -(amino)heteroalkylene-NH-. Article 98. R 10 93. The ADC of clause 92, wherein is -alkylene-PEG-NH-. Article 99.R 10 93. The ADC of clause 91 or 92, wherein is -CH2-NH-, -CH2-O-heteroalkylene-NH-, or -CH2-O-alkylene-NH-. Article 100.R 10 92. The ADC of clause 91, wherein is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl)-, -CH2OCH(NH-)CH2OH, or -CH2NH-. Article 101. R 10 92. The ADC of clause 91, wherein is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl)-, or -CH2NH-. Article 102.R 3 4. The ADC of clause 3, comprising an ABD linked via an amino group of to a compound selected from P4, P5, P7, P9, P11, P12, P19, P21, P24, P30, and P34. Clause 103. The ADC according to Clause 2, wherein said ADC is according to Formula VI: or a pharmaceutically acceptable salt thereof. [ka] (In the formula: L 1 is a bivalent linker; R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2is 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, where R 11 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 11 or R 14 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylenealkylene-arylene, heteroalkylene, heteroalkylene-arylene-, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; X is CH or N; x is 0, 1, 2, 3, 4, 5, or 6; and and k is an integer from 1 to 30. Clause 104. ABD-L linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39.1 104. The ADC of clause 103, comprising: 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 ABD comprises a heavy chain and the C-terminus of the heavy chain is L 1 104. The ADC of clause 103, conjugated to Clause 108. The ABD comprises two heavy chains, and the C-terminus of each of the two heavy chains is L. 1 104. The ADC of clause 103, conjugated to Article 109. L 1 is linked to a cysteine ​​residue of said ABD. Clause 110. The ADC of any one of clauses 1 to 109, wherein said ABD is an antibody against HBV sAg or a fragment thereof. Clause 111. The ADC of any one of clauses 1 to 110, wherein said ABD is a human antibody or a humanized antibody. Clause 112. The ADC of any one of clauses 1 to 111, wherein said ABD is IgG1 or IgG2a. Clause 113. The ADC of any one of clauses 1 to 110, wherein said ABD comprises an scFv with binding specificity for HBV sAg. Clause 114. The ABD is a V of antibody to HBV sAg H Chain and V L 114. The ADC of any one of clauses 1-113, comprising a chain. Clause 115. The ADC of any one of clauses 1-114, wherein said ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody against HBV sAg. Clause 116. The ADC of any one of clauses 1-102, wherein said ABD comprises an Fc region. Clause 117. The ADC of clause 116, wherein said Fc region comprises a modification for enhanced binding to an FcγR. Clause 118. The ADC of any one of clauses 1 to 114, wherein said ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised 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) comprised 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 to 123, wherein said ABD is a component of an antibody or antigen-binding fragment thereof. Clause 125. The ADC of clause 1, wherein said TLR7 agonist having a bivalent 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 to 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 an ADC according to any one of clauses 1 to 117 or a pharmaceutical composition according to clause 118. Clause 128. The method of clause 127, wherein said 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 said subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of said ADC or said pharmaceutical composition. Clause 131. The method of any one of clauses 127 to 130, further comprising measuring circulating HBV DNA or HBV sAg in the serum of said subject prior to administration. Clause 132. The method of any one of clauses 127 to 131, further comprising measuring circulating HBV DNA or HBV sAg in the serum of said subject after administration to assess the therapeutic efficacy of said ADC or said pharmaceutical composition. Clause 133. The method of any one of clauses 127 to 132, wherein the step of administering said ADC or said pharmaceutical composition is repeated. Clause 134. The method of clause 133, wherein the step of administering said ADC or said pharmaceutical composition is repeated two, three, or more times. Clause 135. The method of clause 133 or 134, wherein the step of administering said ADC or said pharmaceutical composition is repeated at least one week apart, two weeks apart, three weeks apart, or four weeks apart. Clause 136. The method of clause 133 or 134, wherein the step of administering said ADC or said pharmaceutical composition is repeated at 1 week intervals, 2 week intervals, 3 week intervals, or 4 week intervals. Clause 137. The method of clause 133 or 134, wherein the step of administering said ADC or said pharmaceutical composition is repeated at monthly intervals, at bimonthly intervals, or at three-monthly intervals. Clause 138. The method of any one of clauses 127 to 137, wherein said ADC or pharmaceutical composition is administered by intravenous, intraperitoneal, inhalation, intranasal, intramuscular, or subcutaneous administration. Clause 139. An ADC according to any one of clauses 1 to 117 or a pharmaceutical composition according to clause 126 for use in therapy. Clause 140. The ADC of any one of clauses 1 to 117 or the pharmaceutical composition of clause 126 for use in the treatment of chronic hepatitis B in a subject in need thereof. Clause 141. Use of an ADC according to any one of clauses 1 to 117 or a pharmaceutical composition according to clause 126 for the manufacture of a medicament. Clause 142. Use of an ADC according to any one of clauses 1 to 117 or a pharmaceutical composition according to clause 126 for the manufacture of a medicament for the treatment of chronic hepatitis B in a subject in need thereof. [Example]

[0322] XII. Examples The following examples are intended to illustrate certain embodiments provided herein and are not intended to limit the scope of the disclosure.

[0323] Example 1 Synthesis of intermediate Aa (see Scheme 1)

[0324] Methyl 4-{[2-chloro-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (3) [ka]

[0325] To a mixture of compound 1 (0.50 g, 2.7 mmol) in acetonitrile (10 mL) was 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 h, which was monitored by LCMS (ESI m / z: 366.1 (M+H) +). To the resulting mixture, potassium carbonate (0.37 g, 2.7 mmol) and 1-pentanamine (0.70 g, 7.9 mmol) were added, and the reaction mixture was stirred at 85° C. for 5 hours, which was monitored by LCMS. After cooling 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 (×3). The combined organic solution was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 3 (0.23 g, 21% yield) as a pale yellow solid. ESI m / z: 417.2 (M+H) + .

[0326] Methyl 4-{[2-azido-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (4) [ka]

[0327] 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 °C for 6 h, which was monitored by LCMS. After cooling to room temperature, the mixture was diluted with ethyl acetate (40 mL) and saturated aqueous 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 and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound 4 (60 mg, 30% yield) as a pale yellow solid. ESI m / z: 424.2 (M+H). + .

[0328] Methyl 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Aa) (Method 1) [ka]

[0329] To a solution of compound 4 (0.12 g, 0.28 mmol) in acetic anhydride (10 mL) was added zinc dust (0.56 g, 8.5 mmol) at 0 °C under the protection of nitrogen flow. The reaction mixture was then stirred at 85 °C for 4 h. After cooling to room temperature, the mixture was filtered through Celite, and the filtrate was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.05%)) to give intermediate Aa (65 mg, 58% yield) as a pale yellow solid. ESI m / z: 398.3 (M+H) + .

[0330] Example 2 General Procedure I for the Synthesis of Compound 7 [ka]

[0331] To a mixture of compounds 5a, b (1.0 equiv.) in acetonitrile (0.2 M), compounds 6a–c (1.1–1.2 equiv.) and potassium carbonate (2.0 equiv.) were added, and the suspension was stirred at room temperature for 16 h, 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 compounds 7a–d (34–94% yield) as pale yellow solids.

[0332] Methyl 4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzoate (7a) [ka]

[0333] Following general procedure I starting from compounds 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 pale yellow solid after purification by silica gel flash chromatography (50–100% ethyl acetate in petroleum ether over 20 min). [ka]

[0334] Methyl 4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzoate (7b) [ka]

[0335] Following general procedure I starting from compounds 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 pale yellow solid after purification by silica gel flash chromatography (5–10% methanol in DCM over 20 min). [ka]

[0336] tert-Butyl N-{[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)phenyl]methyl}carbamate (7c) [ka]

[0337] Following 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 pale yellow solid after purification by silica gel flash chromatography (20–25% ethyl acetate in petroleum ether over 20 min). [ka]

[0338] 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') [ka]

[0339] Following general procedure I starting from compounds 5b (0.50 g, 2.9 mmol) and 6a (0.84 g, 3.2 mmol), compound 7d (0.35 g, 34% yield) and its isomer 7d′ (0.15 g, 15% yield) were obtained separately as white solids after purification by silica gel flash chromatography (0–5% methanol in DCM).

[0340] 7d: ESI m / z: 348.1(M+H) + , 717.3(2M+Na) + ; LCMS retention time: 1.64 min; [ka]

[0341] 7d': ESI m / z: 348.1(M+H) + ; LCMS retention time: 1.67 min; [ka]

[0342] The structures of 7d and 7d' were determined from the NOE data of 9b and 9b'.

[0343] Example 3 General Procedure II for the Synthesis of Intermediates Aa, Ba, Bb, Bc, and Fa [ka]

[0344] To a suspension of compounds 7a–d (1.0 equiv.) in acetonitrile (40–50 mM), potassium carbonate (4.0 equiv.) and 1-pentanamine 8a (5.0 equiv.) or o-butylhydroxylamine 8b (HCl salt, 2.0 equiv.) were added, and the reaction mixture was stirred at 85°C for 15 h, which was monitored by LCMS. Volatiles were removed in vacuo, and the residue was purified by silica gel flash chromatography (0–10% methanol in DCM) to give intermediates Aa, Ba, Bb, Bc, or Fa (28–98% yields) as solids.

[0345] Methyl 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Aa) (Method 2) [ka]

[0346] Following general procedure II starting from 7a (0.60 g, 1.7 mmol) together 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). + .

[0347] Methyl 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Ba) [ka]

[0348] Following general procedure II starting from 7a (0.45 g, 1.3 mmol) together 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). [ka]

[0349] Methyl 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}benzoate (Bb) [ka]

[0350] Following general procedure II starting from 7b (0.40 g, 1.3 mmol) together 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). [ka]

[0351] tert-Butyl N-[(4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}phenyl)methyl]carbamate (Bc) [ka]

[0352] Following 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) + .

[0353] Methyl 4-{[2-amino-6-(pentylamino)-7H-purin-7-yl]methyl}-3-methoxybenzoate (Fa) [ka]

[0354] Following general procedure II starting from 7d (0.28 g, 0.81 mmol) together 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). [ka]

[0355] Example 4 General Procedure III for the Synthesis of Intermediates Ca, Cb, and Cc [ka]

[0356] To a solution of compounds 7a-c (1.0 equiv.) in 1-butanol (0.10-0.15 M), DIPEA (4 equiv.) and 2-ethoxyethan-1-amine 8c (2.0 equiv.) were added, and the reaction mixture was protected with argon and stirred in a sealed tube at 120 °C for 4-5 h, which was monitored by LCMS. After cooling, the reaction mixture was concentrated in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give intermediates Ca-c (51-61% yield) as yellow solids.

[0357] Methyl 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzoate (Ca) [ka]

[0358] Following general procedure III starting from 7a (0.17 g, 0.49 mmol) along with amine 8c (87 mg, 0.98 mmol), intermediate Ca (0.12 g, 61% yield) was obtained as a yellow solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 400.3 (M+H). + .

[0359] Methyl 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzoate (Cb) [ka]

[0360] Following 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 reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 370.3 (M+H). + .

[0361] tert-Butyl N-{[4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)phenyl]methyl}carbamate (Cc) [ka]

[0362] Following general procedure III starting from 7c (0.18 g, 0.45 mmol) along with amine 8c (80 mg, 0.90 mmol), intermediate Cc (90 mg, 51% yield) was obtained as a yellow solid after purification by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)). ESI m / z: 441.5 (M+H). + .

[0363] Example 5 Methyl 4-{[2-amino-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Da)

[0364] [ka]

[0365] To a solution of compound 7a (0.50 g, 1.4 mmol) in 1-pentanol (5 mL), HCl in dioxane (4 M, 1 mL) was added, and the reaction mixture was sealed and stirred at 120 °C for 5 h, which was monitored by LCMS. After cooling, 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 in the next step without further purification. ESI m / z: 399.2 (M Da +H) + , 385.3(M P35-1 +H) + .

[0366] Example 6 General Procedure IV for the Synthesis of Intermediates 9a and 9b [ka]

[0367] To a solution of compound 7a or 7d (1.0 equiv.) in dioxane and water (v / v = 4, 0.10-0.12 M) was 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 °C for 15 h, 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.

[0368] Methyl 4-[(2-amino-4-(hex-1-en-1-yl)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]-3-methoxybenzoate (9a) [ka]

[0369] Following 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. [ka]

[0370] Methyl 4-{[2-amino-6-(hex-1-en-1-yl)-7H-purin-7-yl]methyl}-3-methoxybenzoate (9b) [ka]

[0371] Following 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. [ka]

[0372] Methyl 4-{[2-amino-6-(hex-1-en-1-yl)-9H-purin-9-yl]methyl}-3-methoxybenzoate (9b') [ka]

[0373] Following general procedure IV starting from 7d' (0.10 g, 0.29 mmol), compound 9b' (60 mg, 53% yield) was obtained as a yellow solid. [ka]

[0374] Comparing the NOE data of 9b and 9b', the NOESY spectrum of 9b shows a signal between benzyl CH2 (5.58 ppm, s, 2H) and CH=CH (6.27 ppm, d, J=15.1 Hz, 1H), whereas the NOESY spectrum of 9b' shows no signal between benzyl CH2 (5.29 ppm, s, 2H) and CH=CH (6.66 ppm, d, J=16.3 Hz, 1H). This result indicates that compounds 7d and 9b are the desired intermediates.

[0375] Example 7 Methyl 4-({2-amino-4-hexyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzoate (Ea) [ka]

[0376] To a solution of compound 9a (0.38 g, 0.96 mmol) in ethyl acetate (30 mL), palladium on carbon (containing 10% palladium, 40 mg) was added 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 Celite, and the filtrate was concentrated in vacuo to give compound Ea (0.38 g, 95% yield) as a white solid. [ka]

[0377] Example 8 Methyl 4-[(2-amino-6-hexyl-7H-purin-7-yl)methyl]-3-methoxybenzoate (Fb) [ka]

[0378] Palladium hydroxide (20 mg, 10 wt%) was added to a solution of compound 9b (0.20 g, 0.51 mmol) in ethanol (15 mL) under nitrogen protection, and the reaction mixture was stirred at 50° C. under a hydrogen balloon (1.1 atmospheres) for 15 hours, which was monitored by LCMS. After cooling, the resulting mixture was filtered through Celite, and the filtrate was concentrated in vacuo to give compound Fb (0.21 g, 95% yield) as a white solid, which was used in the next step without further purification. ESI m / z: 398.3 (M+H) + .

[0379] Example 9 General procedure V for the reduction of methyl benzoate to benzyl alcohol. [ka]

[0380] To a stirred suspension of lithium aluminum tetrahydride (LAH, 2.0 equiv.) in anhydrous THF (0.04–0.20 M), a solution of the ester (1.0 equiv.) in anhydrous THF (0.02–0.20 M) was added dropwise over 5 min at 0 °C under nitrogen protection. The reaction mixture was stirred at 0 °C for 15 min, then at room temperature for 30 min until complete reduction of the ester was confirmed by LCMS. The resulting mixture was cooled to 0 °C and carefully quenched with saturated aqueous sodium bicarbonate (5% vol.) and water (5% vol.). The mixture was filtered, and the filtrate was extracted with ethyl acetate (×3). The combined organic solution was washed with brine and concentrated in vacuo. The crude product was purified by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.05%)) to afford the payload (43% yield, TFA salt) as a white solid.

[0381] Payload P1

[0382] (4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methanol (P1) [ka]

[0383] General procedure V was followed starting from intermediate Aa (65 mg, 0.16 mmol) to afford payload P1 (35 mg, 43% yield, TFA salt) as a white solid. [ka]

[0384] Payload P20

[0385] (4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methanol (P20) [ka]

[0386] General procedure V was followed starting from intermediate Ba (0.15 g, 0.38 mmol) to afford payload P1 (0.13 g, 72% yield, TFA salt) as a white solid. [ka]

[0387] Payload P23

[0388] [4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxyphenyl]methanol (P23) [ka]

[0389] General procedure V was followed starting from intermediate Ca (30 mg, 75 μmol) to afford payload P23 (15 mg, 41% yield, TFA salt) as a white solid. [ka]

[0390] Payload P27

[0391] (4-{[2-amino-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methanol (P27) [ka]

[0392] General procedure V was followed starting from intermediate Da (60 mg, 0.15 mmol) to afford payload P27 (45 mg, 62% yield, TFA salt) as a white solid. [ka]

[0393] Payload P29

[0394] [4-({2-amino-4-hexyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxyphenyl]methanol (P29) [ka]

[0395] General procedure V was followed starting from intermediate Ea (0.38 g, 0.96 mmol) to afford payload P29 (0.30 g, 65% yield, TFA salt) as a white solid. [ka]

[0396] Payload P32

[0397] (4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}phenyl)methanol (P32) [ka]

[0398] General procedure V was followed starting from intermediate Bb (82 mg, 0.22 mmol) to afford payload P32 (70 mg, 69% yield, TFA salt) as a white solid. [ka]

[0399] Payload P33

[0400] [4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)phenyl]methanol (P33) [ka]

[0401] Following 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. [ka]

[0402] Payload P37

[0403] (4-{[2-amino-6-(pentylamino)-7H-purin-7-yl]methyl}-3-methoxyphenyl)methanol (P37) [ka]

[0404] Following 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. [ka]

[0405] Payload P39

[0406] {4-[(2-amino-6-hexyl-7H-purin-7-yl)methyl]-3-methoxyphenyl}methanol (P39) [ka]

[0407] General procedure V was followed starting from intermediate Fb (0.21 g, 0.53 mmol) to afford payload P37 (0.13 g, 51% yield, TFA salt) as a white solid. [ka]

[0408] Example 10 Payload P2

[0409] 2-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)propan-2-ol (P2) [ka]

[0410] To a cooled (-5 to 5°C) solution of intermediate Aa (50 mg, 0.13 mmol) in anhydrous THF (5.0 mL), methylmagnesium bromide (3.0 M in THF, 0.43 mL, 1.3 mmol) was added dropwise under nitrogen protection. The reaction mixture was stirred at 0°C for 30 minutes 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 (x3). The combined organic solution was washed with brine and concentrated in vacuo. The crude product was purified by reverse-phase flash chromatography (5 to 100% acetonitrile in aqueous TFA (0.01%)) to afford payload P2 (10 mg, 15% yield, TFA salt) as a white solid. [ka]

[0411] Example 11 General Procedure VI for the Synthesis of (Piperazine Analogues P3, P26, P28, P36, and P38) [ka]

[0412] To a suspension of alcoholic payload P1, P27, P29, P37, or P39 (1.0 equiv.) in DCM (50 mM) was added dropwise a solution of thionyl chloride (1.2 equiv.) in DCM (0.1 M) at 0°C. The reaction mixture was stirred at room temperature for 5 h. Volatiles were removed in vacuo to give crude products corresponding to chlorides P3-1, P26-1, P28-1, P36-1, or P38-1 as yellow semi-solids, which were dissolved in DMF (25 mg / mL). To this solution were added potassium carbonate (2.0 equiv.) and N-Boc piperazine (1.0 equiv.), and the reaction mixture was stirred at room temperature for 15 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–95% methanol in aqueous TFA (0.01%)) to afford the Boc-payloads P3-2, P26-2, P28-2, P36-2, or P38-2 as white solids, which were dissolved in DCM (25 mg / mL). To this solution, the hydrochloride salts of the Boc-payloads were added in dioxane (4 M, v / v DCM A solution of 2-(4 ...

[0413] Payload P3

[0414] 5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P3) [ka]

[0415] Following general procedure VI starting from P1, payload P3 (0.23 g, 31% yield, TFA salt) was obtained as a white solid. [ka]

[0416] Payload P26

[0417] 5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (P26) [ka]

[0418] Following general procedure VI starting from P27, payload P26 (15 mg, 31% yield, TFA salt) was obtained as a white solid. [ka]

[0419] Payload P28

[0420] 4-Hexyl-5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (P28) [ka]

[0421] Following general procedure VI starting from P29, payload P28 (46 mg, 53% yield, TFA salt) was obtained as a white solid. [ka]

[0422] Payload P36

[0423] 7-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-N 6-Pentyl-7H-purine-2,6-diamine (P36) [ka]

[0424] Following general procedure VI starting from P37, payload P36 (78 mg, 66% yield, TFA salt) was obtained as a white solid. [ka]

[0425] Payload P38

[0426] 6-Hexyl-7-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-7H-purin-2-amine (P38) [ka]

[0427] Following general procedure VI starting from P39, payload P38 (79 mg, 72% yield, TFA salt) was obtained as a white solid. [ka]

[0428] Example 12 General Procedure VII for the Synthesis of Amides P22, P25, P31, and P35 [ka]

[0429] To a solution of the ester Ba, Ca, Bb, or Cb (1.0 equiv.) in isopropanol or methanol (50–100 mM) was added aqueous sodium hydroxide or lithium hydroxide (1 M, 0.5 v of alcohol solvent). The reaction mixture was stirred at room temperature for 1 h and monitored by LCMS. The remaining solution was neutralized to pH 6 with dilute aqueous hydrochloric acid (1 M), and the resulting mixture was concentrated. The residue was purified by reverse-phase flash chromatography (0–95% acetonitrile in aqueous TFA (0.01%)) to afford the acid P22-1, P25-1, P31-1, or P35-1 as a yellow solid, which was dissolved in DMF (50–80 mM). To this solution was 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 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–100% acetonitrile in aqueous TFA (0.01%)) to afford amides P22, P25, P31, and P35 (4–51% yield, TFA salt) as white solids.

[0430] Payload P22

[0431] 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzamide (P22) [ka]

[0432] Following general procedure VII starting from Ba, payload P22 (82 mg, 28% yield, TFA salt) was obtained as a white solid. [ka]

[0433] Payload P25

[0434] 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzamide (P25) [ka]

[0435] Following general procedure VII starting from Ca, payload P25 (60 mg, 31% yield, TFA salt) was obtained as a white solid. [ka]

[0436] Payload P31

[0437] 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}benzamide (P31) [ka]

[0438] Following general procedure VII starting from Bb, payload P31 (5.0 mg, 4% yield, TFA salt) was obtained as a white solid. [ka]

[0439] Payload P35

[0440] 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzamide (P35) [ka]

[0441] Following general procedure VII starting from Cb, payload P35 (50 mg, 51% yield, TFA salt) was obtained as a white solid. [ka]

[0442] 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoic acid (P47) [ka]

[0443] Following general procedure VII starting from Aa without amidation, crude acid P47 was recovered. Further purification of this crude acid by preparative HPLC afforded pure P47 (5 mg, 60% yield) as a pale yellow solid. ESI m / z: 384.2 (M+H). + .

[0444] Example 13 Payload P21

[0445] 5-{[4-(aminomethyl)-2-methoxyphenyl]methyl}-N 4 -Butoxy-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P21) [ka]

[0446] To a stirred suspension of lithium aluminum tetrahydride (39 mg, 1.0 mmol) in anhydrous THF (6 mL), a solution of compound P22 (65 mg, 0.17 mmol) in anhydrous THF (4 mL) was added dropwise over 10 min at 0 °C under nitrogen protection. The reaction mixture was stirred at 65 °C for 4 h and monitored by LCMS. The resulting mixture was cooled to 0 °C 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 reverse-phase flash chromatography (0 to 100% acetonitrile in aqueous TFA (0.01%)) to afford P21 (31 mg, 38% yield, TFA salt) as a pale yellow solid. [ka]

[0447] Example 14 Payload P24

[0448] 5-{[4-(aminomethyl)-2-methoxyphenyl]methyl}-N 4 -(2-ethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P24) [ka]

[0449] To a solution of compound P25 (29 mg, 75 μmol) in anhydrous THF (4 mL), a solution of borane in THF (1 M, 0.23 mL) was added dropwise at 65°C under argon protection. The reaction mixture was refluxed at 65°C for 4 hours, which was monitored by LCMS. After cooling to 0°C, the resulting mixture was carefully quenched with saturated aqueous sodium bicarbonate (0.24 mL) and water (0.24 mL). The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0 to 100% acetonitrile in aqueous TFA (0.01%)) to give P24 (5 mg, 14% yield, TFA salt) as a pale yellow solid. [ka]

[0450] Example 15 Payload P30

[0451] 5-{[4-(aminomethyl)phenyl]methyl}-N 4 -Butoxy-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P30) [ka]

[0452] To a suspension of compound Bc (45 mg, 0.10 mmol) in DCM (1 mL) was added a solution of its hydrochloride salt (4 M in dioxane, 0.5 mL). The reaction mixture was stirred at room temperature for 1 h until the Boc group was completely removed, as monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was purified by reverse-phase flash chromatography (5–95% acetonitrile in aqueous TFA (0.01%)) to afford P30 (45 mg, 97% yield, TFA salt) as a white solid. [ka]

[0453] Example 16 Payload P34

[0454] 5-{[4-(aminomethyl)phenyl]methyl}-N 4 -(2-ethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P34) [ka]

[0455] Following a similar procedure to payload P30, except substituting Cc for Bc, payload P34 (0.10 g, 60% yield, TFA salt) was obtained as a white solid. [ka]

[0456] Example 17 Intermediate P3-1 was synthesized as described in the procedure for payload P3.

[0457] Example 18 5-{[4-(bromomethyl)-2-methoxyphenyl]methyl}-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P4-1) [ka]

[0458] To a solution of P1 (0.30 g, 0.81 mmol) in DCM, phosphorus tribromide (0.38 mL) was added, and the reaction mixture was stirred at room temperature for 3 h, which was monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give P4-1 (0.32 g, 91% yield) as a yellow solid. ESI m / z: 432.1 (M+H). + .

[0459] Example 19 General procedure for the synthesis of ethers by alcohol reaction with benzyl halides. VIII. [ka]

[0460] To a solution of alcohol P#-2 (2.0 equiv.) in anhydrous THF or DMF (40 mM), sodium hydride (60% in mineral oil, 2.5 equiv.) was added in one portion under nitrogen protection at −15 to 0°C. The resulting suspension was stirred for 10 min at −15 to 0°C under nitrogen protection. Tetrabutylammonium iodide (TBAI) (0.05 equiv.) and benzyl halide (1.0 equiv.) were then added to the stirred mixture, and the reaction mixture was stirred at −15 to 0°C for 30 min, which was monitored by LCMS. The resulting mixture was quenched with methanol. The mixture (with Boc or propylidene protection) was used directly in the next step without further purification. Alternatively, the mixture was neutralized with TFA (to pH 6–7) and then concentrated in vacuo. The residual mixture was purified by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.05%)) to provide the desired ester.

[0461] Example 20 General Procedure IX for the Synthesis of P4, P5, P10, P17, P18, P19, P41, and P42 by Acidification with TFA [ka]

[0462] The quenched reaction mixture containing P#-3 was diluted with DCM (in the case of P4, P5, P10, P41, and P42) or THF (in the case of P17, P18, and P19). To this solution, TFA (25% vol.) was added, and the reaction mixture was stirred at room temperature for 30 min, which was monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was purified by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.05%)) to afford payloads P4, P5, P10, P17, P18, P19, P41, or P42 (20% yield) as white solids.

[0463] Example 21 Payload P4

[0464] 5-({4-[(2-aminoethoxy)methyl]-2-methoxyphenyl}methyl)-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P4) [ka]

[0465] Later, following general procedures VIII and IX 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. [ka]

[0466] Example 22 Payload P5

[0467] 5-({4-[(4-aminobutoxy)methyl]-2-methoxyphenyl}methyl)-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P5) [ka]

[0468] Subsequently, following general procedures VIII and IX starting from benzyl bromide P4-1 reacting with N-Boc-aminobutanol P5-2, payload P5 (2 mg, 2% yield, formate salt) was obtained as a pale yellow solid after purification by preparative HPLC (5–95% acetonitrile in aqueous formic acid (0.1%)). [ka]

[0469] Example 23 Payload P6

[0470] 2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]ethoxy}ethan-1-ol (P6) [ka]

[0471] Following 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. [ka]

[0472] Example 24 Payload P8

[0473] 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) [ka]

[0474] Following general procedure VIII starting from benzyl chloride P3-1 reacting with tetraethylene glycol P8-2 (CAS: 112-60-7) afforded payload P8 (36 mg, 6.4% yield, TFA salt) as an off-white solid. [ka]

[0475] Payload P44

[0476] 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) [ka]

[0477] Hydroxy-PEG3-CH2CO2 t Following general procedure VIII starting from benzyl chloride P3-1 reacting with tBu (CAS: 518044-31-0), payload P44 (4.6 mg, 13% yield) was obtained as a white solid (the tBu group disappeared during the reaction). [ka]

[0478] Payload P45

[0479] 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) [ka]

[0480] Hydroxy-PEG2-CH2CO2 t Following general procedure VIII starting from benzyl chloride P3-1 reacting with tBu (CAS: 149299-82-1), payload P45 (3.9 mg, 15% yield) was obtained as a white solid (the tBu group disappeared during the reaction). ESI m / z: 516.3 (M+H) + .

[0481] Payload P46

[0482] 2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]ethoxy}acetic acid (P46) [ka]

[0483] Following 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 (the tBu group disappeared during the reaction). ESI m / z: 472.3 (M+H) + .

[0484] Payload P48

[0485] 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]ethan-1-ol (P48) [ka]

[0486] Following 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 a white solid. ESI m / z: 414.3 (M+H) + .

[0487] Example 25 Payload P9

[0488] 5-{[4-(13-azido-2,5,8,11-tetraoxatridecan-1-yl)-2-methoxyphenyl]methyl}-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P9-3)

[0489] [ka]

[0490] Following 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). + .

[0491] 5-{[4-(13-amino-2,5,8,11-tetraoxatridecan-1-yl)-2-methoxyphenyl]methyl}-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P9)

[0492] [ka]

[0493] To a solution of compound P9-3 (1.7 g, 3.0 mmol) in methanol (50 mL), palladium on carbon (10 wt% palladium, 0.20 g) was added under nitrogen. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours and monitored by LCMS. The resulting suspension was filtered through Celite, and the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.05%)) to give P9 (0.40 g, 20% yield, di-TFA salt) as a pale yellow solid. [ka]

[0494] Example 26 (Payloads P10 and P43)

[0495] 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.

[0496] 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) [ka]

[0497] Following 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). + .

[0498] 5-({2-methoxy-4-[13-(piperazin-1-yl)-2,5,8,11-tetraoxatridecan-1-yl]phenyl}methyl)-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P10) [ka]

[0499] Following general procedure IX starting from P43, payload P10 (22 mg, 16% yield, di-TFA salt) was obtained as a yellow solid. [ka]

[0500] Example 27 Payload P17

[0501] (2R)-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]propane-1,2-diol (P17) [ka]

[0502] Later, following general procedures VIII and IX 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 pale yellow solid. [ka]

[0503] Example 28 Payload P18

[0504] (2S)-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]propane-1,2-diol (P18) [ka]

[0505] Later, following general procedures VIII and IX 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 pale yellow solid. [ka]

[0506] Example 29 Payload P19

[0507] 2-Amino-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]propan-1-ol (P19) [ka]

[0508] Later, following general procedures VIII and IX 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. [ka]

[0509] Example 30 Payload P41

[0510] 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) [ka]

[0511] Following general procedure VIII starting from benzyl bromide P4-1 reacting with N-Boc-PEG-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). + .

[0512] 5-{[4-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)-2-methoxyphenyl]methyl}-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P41) [ka]

[0513] Following general procedure 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). + . [ka]

[0514] Example 31 Payload P42

[0515] 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() [ka]

[0516] Following general procedure VIII starting from benzyl bromide P4-1 reacting with N-Boc-PEG-OH (P41-2), except using 4 equivalents of sodium hydride (60% in mineral oil) instead of 2.5 equivalents, compound P41-3 (80 mg, 36% yield) and compound P42-3 (20 mg, 9% yield) were obtained separately as white solids after purification by preparative HPLC (5-95% acetonitrile in aqueous ammonium bicarbonate (10 mM)).

[0517] For 41-3: Rt in LC: 2.05 min; ESI m / z: 601.3 (M+H) + .

[0518] For 42-3: Rt in LCMS: 1.84 min, ESI m / z: 601.3 (M+H) + .

[0519] 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) [ka]

[0520] Following general procedure 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). + . [ka]

[0521] Example 32 Payload P11

[0522] 2-Amino-N-{[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]methyl}acetamide (P11) [ka]

[0523] To a solution of P1 (0.30 g, 0.81 mmol) in anhydrous THF (5 mL), potassium tert-butoxide (0.18 g, 1.6 mmol) and compound P11-1 (CAS: 1599440-06-8) (0.45 g, 1.2 mmol) were added under nitrogen at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, which was monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% methanol in aqueous TFA (0.01%)) to give compound P11-2 (0.15 g, ESI m / z: 678.5 (M+H)). +) was obtained as a yellow solid.

[0524] 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 h until complete removal of Fmoc by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (5 mg, 8% yield from P1, TFA salt) as a yellow solid. [ka]

[0525] Example 33 Payload P12

[0526] 5-[(4-{[(4-aminophenyl)methoxy]methyl}-2-methoxyphenyl)methyl]-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P12) [ka]

[0527] To a solution of P1 (20 mg, 54 μmol) in DMF (3 mL) was 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 °C for 18 h, which was monitored by LCMS. The mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound P12-2 (5 mg, ESI m / z: 575.5 (M+H)). +) was obtained as a yellow solid, which was dissolved in DCM (2.5 mL). To this solution, TFA (0.5 mL) was added, and the reaction mixture was stirred at room temperature for 3 h until complete removal of Boc by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (5–95% acetonitrile in aqueous TFA (0.01%)) to afford P12 (2 mg, 6% yield from P1, TFA salt) as a white solid. [ka]

[0528] Example 34 Payload P7

[0529] Methyl 4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3-methoxybenzoate (P7-2) [ka]

[0530] To a solution of compound P7-1 (CAS: 79236-96-7) (0.10 g, 0.51 mmol) in pyridine (5 mL), tert-butylchlorodiphenylsilane (TBDPS-Cl) (0.14 g, 0.51 mmol) and DMAP (4 mg, 33 μmol) were added, and the reaction mixture was stirred at room temperature for 16 h, which was monitored by LCMS. The resulting mixture was extracted twice with ethyl acetate, 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). + .

[0531] (4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3-methoxyphenyl)methanol (P7-3) [ka]

[0532] To a stirred suspension of lithium aluminum tetrahydride (LAH, 8.7 mg, 0.23 mmol) in anhydrous THF (4 mL), a solution of ester P7-2 (0.10 g, 0.23 mmol) in anhydrous THF (1 mL) was added dropwise under nitrogen protection at 0 °C over 5 min. The reaction mixture was stirred at 0 °C for 15 min, then at room temperature for 20 min until complete reduction of the ester was confirmed by LCMS. The resulting mixture was cooled to 0 °C and carefully quenched with two drops of saturated aqueous sodium bicarbonate and two drops of water. The mixture was filtered, and the filtrate was extracted with ethyl acetate (×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 a colorless oil. ESI m / z: 429.3 (M+Na). + .

[0533] {[4-(bromomethyl)-2-methoxyphenyl]methoxy}(tert-butyl)diphenylsilane (P7-4) [ka]

[0534] To a cooled solution of compound P7-3 (2.0 g, 4.9 mmol) in diethyl ether (30 mL), phosphorus tribromide (0.67 g, 2.5 mmol) was added under argon protection, and the reaction mixture was stirred at 0 °C for 3 h, which was monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-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). + .

[0535] tert-Butyl N-(2-{2-[(4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3-methoxyphenyl)methoxy]ethoxy}ethyl)carbamate (P7-6) [ka]

[0536] To a cooled (0 °C) solution of 2-(2-Boc-aminoethoxy)ethanol P7-5 (CAS: 139115-91-6) (34 mg, 0.16 mmol) in anhydrous THF (4 mL), sodium hydride (60% in mineral oil, 9 mg, 0.22 mmol) was added in two portions under an argon atmosphere. The resulting suspension was stirred at 0 °C for 40 min under argon. To the mixture was then added a solution of TBAI (4.0 mg, 11 μmol) and P7-4 (50 mg, 0.11 mmol) in THF (1 mL), and the reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 6 h, which was monitored by LCMS. The resulting mixture was carefully quenched with water. The volatiles were removed in vacuo. The residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in water) to give P7-6 (19 mg, 30% yield) as a colorless oil. ESI m / z: 616.3 (M+H) + .

[0537] tert-Butyl N-[2-(2-{[4-(hydroxymethyl)-3-methoxyphenyl]methoxy}ethoxy)ethyl]carbamate (P7-7) [ka]

[0538] To a solution of P7-6 (0.20 g, 0.34 mmol) in anhydrous THF (5 mL), a solution of TBAF (1 M in THF, 0.68 mL, 0.68 mmol) was added, and the reaction mixture was stirred at room temperature for 3 h, which was monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.1%)) to give P7-7 (80 mg, 66% yield) as a colorless oil. ESI m / z: 378.3 (M+Na). + .

[0539] tert-Butyl N-[2-(2-{[4-(bromomethyl)-3-methoxyphenyl]methoxy}ethoxy)ethyl]carbamate (P7-8) [ka]

[0540] To a cooled (0 °C) 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 °C for 1 h. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in water) to give P7-8 (50 mg, 52% yield) as an oil. ESI m / z: 440.1 (M+Na). + .

[0541] 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) [ka]

[0542] To a mixture of P7-8 (50 mg, 0.12 mmol) in DMF (4 mL), intermediate 6a (20 mg, 0.12 mmol), potassium carbonate (30 mg, 0.24 mmol), and potassium iodide (5 mg, 30 μmol) were added, and the suspension was stirred at room temperature for 18 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.1%)) to give P7-9 (30 mg, 49% yield) as a white solid. ESI m / z: 506.2 (M+H). + .

[0543] 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) [ka]

[0544] 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 °C for 8 h and monitored by LCMS. After cooling, the mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.1%)) to give P7-10 (30 mg, 45% yield) as a white solid. ESI m / z: 557.4 (M+H). + .

[0545] 5-[(4-{[2-(2-aminoethoxy)ethoxy]methyl}-2-methoxyphenyl)methyl]-N 4 -Pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P7) [ka]

[0546] 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 h until the Boc group was completely removed in vacuo by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0–100% acetonitrile in aqueous TFA (0.01%)) to afford P7 (10 mg, 49% yield, di-TFA salt) as a white solid. [ka]

[0547] Example 35 Payload P14

[0548] Ethyl 2-[(4-{[(tert-butyldiphenylsilyl)oxy]methyl}-3-methoxyphenyl)methoxy]acetate (P14-6) [ka]

[0549] To a cooled (0 °C) solution of compound P7-3 (0.10 g, 0.23 mmol) in anhydrous DMF (4 mL), sodium hydride (60% in mineral oil, 26 mg, 0.39 mmol) was added under argon protection. The suspension was stirred at 0 °C for 30 min, followed by 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 °C for 25 min and then at room temperature for 16 h, which was monitored by LCMS. The resulting mixture was carefully quenched with water at 0 °C and extracted twice with ethyl acetate. 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 a colorless oil. ESI m / z: 515.2 (M+Na). + .

[0550] Ethyl 2-{[4-(hydroxymethyl)-3-methoxyphenyl]methoxy}acetate (P14-7) [ka]

[0551] Following a similar procedure to P7-7, except that P14-6 (0.20 g, 0.40 mmol) was substituted for P7-6, compound P14-7 (60 mg, 59% yield) was obtained as a colorless oil. [ka]

[0552] Ethyl 2-{[4-(bromomethyl)-3-methoxyphenyl]methoxy}acetate (P14-8) [ka]

[0553] Following the same procedure as for P7-8, except that P14-7 (30 mg, 0.12 mmol) was substituted for P7-7, compound P14-8 (20 mg, 53% yield) was obtained as a white solid. ESI m / z: 340.3 (M+Na). + .

[0554] Ethyl 2-{[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxyphenyl]methoxy}acetate (P14-9) [ka]

[0555] Following the same procedure as for P7-9, except that P14-8 (50 mg, 0.16 mmol) was substituted for P7-8, compound P14-9 (30 mg, 47% yield) was obtained as a white solid. ESI m / z: 405.2 (M+H). + .

[0556] Ethyl 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]acetate (P14) [ka]

[0557] Following a similar procedure to P7-10, except substituting P14-9 (0.10 g, 0.25 mmol) for P7-9, the payload P14 (60 mg, 42% yield, TFA salt) was obtained as a white solid. [ka]

[0558] Example 36 Payload P13

[0559] 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]acetic acid (P13) [ka]

[0560] To a solution of P14 (20 mg, 44 μmol) in isopropanol (3 mL) was added aqueous sodium hydroxide (1 N, 60 μL). The reaction mixture was stirred at room temperature for 2 h and monitored by LCMS. The resulting mixture was neutralized with TFA to pH < 7. The mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.1%)) to afford P13 (10 mg, 42% yield, TFA salt) as a white solid. [ka]

[0561] Example 37 Payload P15

[0562] 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]-N,N-dipropylacetamide (P15) [ka]

[0563] 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). The reaction mixture was stirred at room temperature for 3 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–100% acetonitrile in aqueous TFA (0.01%)) to afford P15 (2 mg, 58% yield, TFA salt) as a white solid. [ka]

[0564] Example 38 Payload P16

[0565] 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) [ka]

[0566] Following the same procedure as for P15, except substituting N-Boc-piperazine for the dipropylamine reaction with P13 (30 mg, 35 μmol), compound P16-1 (20 mg, 48% yield, ESI m / z: 596.2 (M+H)) was obtained. +) was obtained as a white solid, which was dissolved in DCM (6 mL). To this solution, TFA (1 mL) was added, and the reaction mixture was stirred at room temperature for 4 h until complete removal of Boc by LCMS. The volatiles were removed in vacuo, and the residue was purified by preparative HPLC (5–95% acetonitrile in aqueous TFA (0.01%)) to afford P16 (10 mg, 22% yield from P13, di-TFA salt) as a white solid. [ka]

[0567] Example 39 Linker-Payload LP1

[0568] 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) [ka]

[0569] To a stirred solution of payload P3 (0.10 g, 0.23 mmol) in methanol (5 mL) was 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. The reaction mixture was stirred at room temperature for 1 h and monitored by LCMS. The resulting solution was directly purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.05%)) to afford LP1 (10 mg, 6% yield, TFA salt) as a white solid. [ka]

[0570] Example 40 Linker-Payload LP2

[0571] 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) [ka]

[0572] To a solution of payload P3 (0.10 g, 0.23 mmol) in DMF (5 mL), N-succinimidyl 3-maleimidopropionate (BMPS, CAS: 55750-62-4) (0.12 g, 0.46 mmol) and DIPEA (0.15 g, 1.2 mmol) were added, and the reaction mixture was stirred at room temperature for 3 h, which was monitored by LCMS. The resulting solution was directly purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.05%)) to give LP2 (20 mg, 15% yield, TFA salt). [ka]

[0573] Example 41 Linker-Payload LP3

[0574] (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) [ka]

[0575] To a stirred solution of payload P3 (43 mg, 98 μmol) in methanol (5 mL), N-Boc-2-aminoacetaldehyde (LP3-1, CAS: 89711-08-0) (48 mg, 0.30 mmol) and sodium cyanoborohydride (63 mg, 1.0 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was directly purified by preparative HPLC (5-95% acetonitrile in aqueous TFA (0.05%)) to give Boc-LP3-2 (45 mg, ESI m / z: 581.5 (M+H)). + ) was obtained as a pale yellow solid, which was dissolved in DCM (5 mL). To this solution, hydrochloride salt in dioxane (4N, 5 mL) was added, and the solution was stirred at room temperature for 1 h until the Boc was completely removed by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give LP3-2 (35 mg, 57% yield, tetrahydrochloride salt (presumed)) as a yellow solid. ESI m / z: 481.4 (M+H) + .

[0576] 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) [ka]

[0577] To a solution of LP3-2 (35 mg, HCl salt, 56 μmol (calculated)) in DMF (1 mL) was 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 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to afford LP3 (25 mg, 34% yield from P3, TFA salt) as a white solid. [ka]

[0578] Example 42 Linker-Payload LP4

[0579] (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) [ka]

[0580] To a solution of cysteine ​​(41 mg, 0.34 mmol) in aqueous hydrochloride (2 N, 0.50 mL) was added saturated aqueous sodium bicarbonate until pH = 7.0. To this aqueous solution was added a solution of LP1 (20 mg, 36 μmol) in acetonitrile (3 mL), and the reaction mixture was stirred at room temperature for 3 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0 to 100% acetonitrile in aqueous TFA (0.01%)) to afford LP4-1 (15 mg, 61% yield, TFA salt) as a white solid. [ka]

[0581] 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) [ka]

[0582] 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 aqueous sodium hydroxide (1 M, 0.5 mL) was added to the solution. The reaction mixture was then stirred at room temperature for 3 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0–100% acetonitrile in aqueous ammonium bicarbonate (10 mM)) to give LP4-2 (5 mg, 38% yield) as a white solid. ESI m / z: 700.2 (M+H). + .

[0583] 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) [ka]

[0584] To a solution of LP4-2 (5 mg, 7.2 μmol) in DMF (1.0 mL), cyclooctyne-O-NHS ester (S1d, CAS: 1425803-45-7) (2.5 mg, 8.6 μmol) and DIPEA (1.5 mg, 12 μmol) were added, and the mixture was stirred at room temperature for 3 h, which was monitored by LCMS. The resulting mixture was directly purified by preparative HPLC (5-95% acetonitrile in aqueous ammonium bicarbonate (10 mM)) to give LP4 (5 mg, 80% yield) as a white solid. ESI m / z: 864.2 (M+H). + .

[0585] Example 43 Linker-Payload LP5

[0586] 2-Amino-N-{[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]methyl}acetamide (LP5-2) [ka]

[0587] To a solution of compound P1 (0.30 g, 0.81 mmol) in anhydrous THF (5 mL), compound LP5-1 (CAS: 1599440-06-8) (0.45 g, 1.2 mmol) and potassium tert-butoxide (0.18 g, 1.6 mmol) were added under nitrogen protection at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% methanol in aqueous formic acid (0.01%)) to give Fmoc-LP5-2 (0.15 g, ESI m / z: 678.4 (M+H)). +) was obtained as a yellow solid, which was dissolved in DMF. To this solution, piperidine (94 mg, 1.1 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h, which was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous 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) + .

[0588] 2-[2-(2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}acetamido)acetamido]acetic acid (LP5-4) [ka]

[0589] 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 h and monitored by LCMS. The resulting mixture was purified by reverse-phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.01%)) to give compound LP5-4 (0.25 g, 59% yield) as a yellow solid. ESI m / z: 601.4 (M+H). + .

[0590] 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}carbamoyl)methyl]-1-[2-(cyclooct-2-yn-1-...

Claims

1. The compound is of Formula I: or a pharmaceutically acceptable salt thereof 【Chemistry 1】 (In the formula: R 1 H, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is H, halo, or alkoxy; R 3 -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, alkoxy, or -NR 7 R 8 and; R 5 and R 6 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; and R 7 and R 8 are each independently H or alkyl, or together with the N to which they are attached form a heterocyclic ring; provided that the compound is a compound of the following formula: 【Chemistry 2】 isn't it).

2. R 1 But, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and; R 2 is halo or alkoxy; R 3 But -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, or -NR 5 R 6 , or -COZ; Z is -OH or -NR 7 R 8 and; R 5 and R 6 However, (i), (ii), and (iii): (i)R 5 and R 6 are each H; (ii)R 5 is H and R 6 is alkyl; (iii)R 5 and R 6 form a heterocyclic ring together with the N to which they are attached; is selected from R 7 and R 8 together with the N to which they are attached form a heterocyclic ring, The compound of claim 1.

3. R 4 3. The compound of claim 1 or 2, wherein is selected with the proviso that is is not substituted with hydroxyl.

4. R 3 The compound of any one of claims 1 to 3, wherein the alkylene and heteroalkylene moieties are selected from the group consisting of the formula:

5. The compound of any one of claims 1 to 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. R 1 But, halo, -NHR 4 , -OR 4 , -NH-OR 4 , or -R 4 and is a linear chain six atoms in length.

7. R 1 The compound of any one of claims 1 to 6, wherein is halo.

8. R 1 Ga-NHR 4 7. The compound of any one of claims 1 to 6, wherein

9. R 1 -OR 4 7. The compound of any one of claims 1 to 6, wherein

10. R 1 -NH-OR 4 7. The compound of any one of claims 1 to 6, wherein

11. R 1 Ga-R 4 7. The compound of any one of claims 1 to 6, wherein

12. R 1 is -NH-n-pentyl, -NH-On-butyl, -On-pentyl, -n-hexyl, or -NH-CH 2 CH 2 6. The compound of any one of claims 1 to 5, wherein:

13. R 1 6. The compound of any one of claims 1 to 5, wherein is -NH-n-pentyl.

14. R 1 6. The compound of any one of claims 1 to 5, wherein is -NH-On-butyl.

15. R 1 6. The compound of any one of claims 1 to 5, wherein is -On-pentyl.

16. R 1 6. The compound of any one of claims 1 to 5, wherein is -n-hexyl.

17. R 1 -NH-CH 2 CH 2 6. The compound of any one of claims 1 to 5, wherein:

18. R 2 17. The compound of any one of claims 1 to 16, wherein is alkoxy.

19. R 2 18. The compound of any one of claims 1-17, wherein is methoxy.

20. R 2 17. The compound of any one of claims 1 to 16, wherein is H.

21. R 2 17. The compound of any one of claims 1-16, wherein is halo.

22. R 3 But -CONHR 23 22. The compound of any one of claims 1 to 21, wherein said heteroalkylene-Y is -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y.

23. R 3 CONHR 23 23. The compound of any one of claims 1 to 22, wherein

24. R 3 23. The compound of any one of claims 1-22, wherein is -alkylene-Y.

25. R 3 23. The compound of any one of claims 1-22, wherein is -heteroalkylene-Y.

26. R 3 23. The compound of any one of claims 1-22, wherein is -heteroalkylene-arylene-Y.

27. R 3 23. The compound of any one of claims 1-22, wherein is -(hydroxy)heteroalkylene-Y.

28. R 3 23. The compound of any one of claims 1-22, wherein is -(amino)heteroalkylene-Y.

29. R 3 23. The compound of any one of claims 1-22, wherein is -alkylene-PEG-Y.

30. R 3 But -CONH 2 , -CO 2 H, -CH 2 -Y, -CH 2 -O-heteroalkylene-Y or -CH 2 23. The compound of any one of claims 1 to 22, wherein Y is -O-alkylene-Y.

31. R 3 But -CH 2 -Y, -CH 2 -O-heteroalkylene-Y or -CH 2 23. The compound of any one of claims 1 to 22, wherein Y is -O-alkylene-Y.

32. R 3 が、-C(Me) 2 OH、-CO 2 H,-CH 2 AND 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 CH 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 OH、-CH 2 AND 2 CH 2 AND 2 CH 2 OH、-CH 2 AND 2 CH 2 AND 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 OH、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 -1-ピペラジニル、-CH 2 AND 2 NHC(O)CH 2 NH 2 、-CH 2 AND 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 COOH, -CH 2 OCH 2 COOEt, -CH 2 OCH 2 CON(n-Pr) 2 , -CH 2 OCH 2 CO-1-piperazinyl, -(R)-CH 2 OCH(OH)CH 2 OH, -(S)-CH 2 OCH(OH)CH 2 OH, -CH 2 OCH(NH 2 )CH 2 OH, -CH 2 OH, -CH 2 NH 2 , -CONH 2 , or -CH 2 23. The compound of any one of claims 1 to 22, which is -1-piperazinyl.

33. R 3 が、-C(Me) 2 OH、-CO 2 H、-CH 2 AND 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 CH 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 OH、-CH 2 AND 2 CH 2 AND 2 CH 2 OH、-CH 2 AND 2 CH 2 AND 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 OH、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 NH 2 、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 -1-ピペラジニル、-CH 2 AND 2 NHC(O)CH 2 NH 2 、-CH 2 AND 2 -(4-NH 2 -1-phenyl), -CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 COOH, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 COOH, -CH 2 OCH 2 CO-1-piperazinyl, -(R)-CH 2 OCH(OH)CH 2 OH, -(S)-CH 2 OCH(OH)CH 2 OH, -CH 2 OCH(NH 2 )CH 2 OH, -CH 2 OH, -CH 2 NH 2 , or -CH 2 23. The compound of any one of claims 1 to 22, which is -1-piperazinyl.

34. R 4 34. The compound of any one of claims 1-33, wherein is n-butyl, n-pentyl, n-hexyl, or ethoxyethyl.

35. R 4 34. The compound of any one of claims 1-33, wherein is n-butyl.

36. R 4 34. The compound of any one of claims 1-33, wherein is n-pentyl.

37. R 4 34. The compound of any one of claims 1-33, wherein is n-hexyl.

38. R 4 34. The compound of any one of claims 1-33, wherein is ethoxyethyl.

39. R 5 and R 6 is each independently H or alkyl, or together with the N to which they are attached form a piperazinyl ring.

40. R 5 and R 6 and each is H.

41. R 5 is H and R 6 39. The compound of any one of claims 1-38, wherein is alkyl.

42. R 5 and R 6 together with the N to which they are attached to form 1-piperazinyl.

43. 43. The compound of any one of claims 1-42, wherein Y is OH.

44. 43. The compound of any one of claims 1 to 42, wherein Y is Gly.

45. Y is -NR 5 R 6 43. The compound of any one of claims 1 to 42, wherein

46. 43. The compound of any one of claims 1-42, wherein Y is -COZ.

47. Y is -OH, -NH 2 , 1-piperazinyl, -COOH, -COOEt, -CONPr 2 or -CO-1-piperazinyl.

48. 48. The compound of any one of claims 1-47, wherein Z is -OH.

49. 48. The compound of any one of claims 1-47, wherein Z is alkoxy.

50. Z is -NR 7 R 8 48. The compound of any one of claims 1 to 47, wherein

51. Z is -OH, ethoxy, -Nn-Pr 2 or 1-piperazinyl.

52. 48. The compound of any one of claims 1-47, wherein Z is -OH or 1-piperazinyl.

53. R 7 and R 8 is each independently H or n-propyl, or together with the N to which they are attached form 1-piperazinyl.

54. R 7 and R 8 together with the N to which they are attached to form 1-piperazinyl. 【Request Item 55】 【Table 1】 and pharmaceutically acceptable salts thereof.

56. The compound is of formula II: or a pharmaceutically acceptable salt thereof. 【Transformation 3】 (In the formula: R 1 , R 2 and X are as defined in claim 1 for formula I; R 9 is defined as R 3 The terminal hydrogen from the group (i.e., R 9 is a divalent radical formed by removal of a hydrogen atom distal to a phenyl group to which is attached L is any group or moiety that links, connects or binds to the antigen binding domain ABD; provided that the compound is a compound of the following formula: 【Chemistry 4】 isn't it).

57. R 9 But, -alkylene-Y 1 -, -heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 , or -alkylene-PEG-Y 1 57. The compound of claim 56, wherein:

58. R 9 -Alkylene-Y 1 58. The compound of claim 56 or 57, wherein:

59. R 9 Ga-heteroalkylene-Y 1 58. The compound of claim 56 or 57, wherein:

60. R 9 -heteroalkylene-arylene-Y 1 58. The compound of claim 56 or 57, wherein:

61. R 9 -(hydroxy)heteroalkylene-Y 1 58. The compound of claim 56 or 57, wherein:

62. R 9 -(amino)heteroalkylene-Y 1 58. The compound of claim 56 or 57, wherein:

63. R 9 -Alkylene-PEG-Y 1 58. The compound of claim 56 or 57, wherein:

64. R 9 But -CH 2 -Y 1 -, -CH 2 -O-heteroalkylene-Y 1 - or -CH 2 -O-alkylene-Y 1 58. The compound of claim 56 or 57, wherein:

65. R 9 が、-C(Me) 2 O-、C(O)-、-CH 2 AND 2 CH 2 NH-、-CH 2 AND 2 CH 2 CH 2 CH 2 NH-、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 AND 2 CH 2 AND 2 CH 2 NH-、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 NH-、-CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 AND 2 CH 2 -1-ピペラジン-4-yl-、-CH 2 AND 2 NHC(O)CH 2 NH-、-CH 2 AND 2 -((4-NH-)-1-phenyl)、-CH 2 AND 2 COO-、-CH 2 AND 2 CH 2 AND 2 CO-、-CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CO-, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 CO-, -CH 2 OCH 2 CO-1-piperazin-4-yl, -(R)-CH 2 OCH(OH)CH 2 O-, -(S)-CH 2 OCH(OH)CH 2 O-, -CH 2 OCH(NH 2 )CH 2 O-, -CH 2 O-, -CH 2 NH- or -CH 2 58. The compound of claim 56 or 57, which is -1-piperazin-4-yl.

66. Y 1 66. The compound of any one of claims 56-65, wherein is -O-.

67. Y 1 66. The compound of any one of claims 56-65, wherein is Gly.

68. Y 1 Ga-NR 5 66. The compound of any one of claims 56 to 65, wherein:

69. Y 1 Ga-COZ 1 66. The compound of any one of claims 56 to 65, wherein

70. Y 1 66. The compound of any one of claims 56-65, wherein is -O-, -NH-, 1-piperazin-4-yl, -COO-, or -CO-1-piperazin-4-yl.

71. Z 1 70. The compound of claim 69, wherein is -O-.

72. Z 1 Ga-NR 7 70. The compound of claim 69, wherein:

73. Z 1 70. The compound of claim 69, wherein is -O- or 1-piperazin-4-yl.

74. R 5 69. The compound of claim 68, wherein is H.

75. R 5 69. The compound of claim 68, wherein is alkyl.

76. 76. The compound of any one of claims 55-75, wherein L is non-cleavable under physiological conditions.

77. 76. The compound of any one of claims 55-75, wherein L is cleavable under physiological conditions.

78. 78. The compound of claim 77, wherein L is an acid labile linker, a hydrolytically labile linker, an enzyme-cleavable linker, a reduction-labile linker, or a self-immolative linker.

79. 79. The compound of any one of claims 55-78, wherein L is or comprises a peptide, carbohydrate, glucuronide, polyethylene glycol (PEG) unit, hydrazone, mal-caproyl unit, dipeptide unit, valine-citrulline unit, or para-aminobenzyl (PAB) unit.

80. 80. The compound of any one of claims 55-79, wherein L comprises one or more amino acids.

81. 81. The compound of any one of claims 55-80, wherein L comprises a self-immolative group.

82. 82. The compound of any one of claims 55-81, wherein L comprises p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC).

83. 83. The compound of any one of claims 55-82, wherein L comprises a maleimide, an N-hydroxysuccinimide ester, or a cyclooctynyl group.

84. L is 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropanoyl, 【Transformation 5】 【change】 【change】 84. The compound of any one of claims 55 to 83, which is the group selected from: 【Request Item 85】 【Table 2】 and pharmaceutically acceptable salts thereof.

86. The compound is of formula III: or a pharmaceutically acceptable salt thereof. 【Transformation 6】 (In the formula: R 1 , R 2 and X are as defined in claim 1; L is any group or moiety that links, connects or binds to the antigen binding domain ABD; R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 When is alkylene or heteroalkylene, the alkylene or heteroalkylene is 11 or R 14 further linked to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylene, alkylene-arylene, heteroalkylene or heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6).

87. R 11 and R 12 is independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylene, or heteroalkylene, where R 11 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 14 is hydrogen, alkylene, heteroalkylene, or an amino acid side chain, where R 14 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 13 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 13 is hydrogen, alkyl, alkylene, or heteroalkylene, where R 13 is alkylene or heteroalkylene, the alkylene or heteroalkylene is R 11 or R 14 further linked to form a 4-, 5-, or 6-membered heterocyclyl; R 15 is hydrogen or alkyl; R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and x is 0, 1, 2, 3, 4, 5, or 6; 87. The compound of claim 86.

88. 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. The compound of any one of claims 1 to 88 or a compound of the formula: 【Transformation 7】 An antibody drug conjugate (ADC) comprising:

90. 90. The ADC of claim 89, wherein the ADC is of formula IV: or a pharmaceutically acceptable salt thereof 【Transformation 8】 (In the formula: R 1 , R 2 , R 9 and X is as defined in claim 1 for formula I and in claim 56 for formula II; L 1 is a bivalent linker; ABD is the antigen binding domain; and and k is an integer from 1 to 30.

91. 91. The ADC of claim 89 or 90, which is ABD-LP1, ABD-LP6A, ABD-LP7A, ABD-LP8A, ABD-LP10A, or ABD-LP11A.

92. 90. The ADC of claim 89, wherein the ADC is of formula V: or a pharmaceutically acceptable salt thereof 【Chemistry 9】 (In the formula: R 1 , R 2 and X are as defined in claim 1 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-; The ABD may contain the Q295 residue, the N297Q mutation, and / or one or more modified 【Chemistry 10】 It is an antibody containing the and k is an integer from 1 to 30.

93. R 10 93. The ADC of claim 92, wherein is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-.

94. R 10 is -alkylene-NH-.

95. R 10 is -heteroalkylene-NH-.

96. R 10 is -heteroalkylene-arylene-NH-.

97. R 10 is -(hydroxy)heteroalkylene-NH-.

98. R 10 is -(amino)heteroalkylene-NH-.

99. R 10 is -alkylene-PEG-NH-.

100. R 10 But -CH 2 -NH-, -CH 2 -O-heteroalkylene-NH- or -CH 2 94. The ADC of claim 92 or claim 93, which is -O-alkylene-NH-.

101. 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 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- and is the ADC according to claim 92 or claim 93.

102. R 10 But -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 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 94. The ADC of claim 92 or claim 93, which is NH-.

103. 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 R 3 The ADC of any one of claims 89-102, wherein the payload (i.e., TLR7 agonist) is attached to the amino group of

104. 90. The ADC of claim 89, wherein the ADC is of formula VI: or a pharmaceutically acceptable salt thereof 【Chemistry 11】 (In the formula: L 1 is a bivalent linker; R 1 , R 2 , R 16 , R 11 , R 12 , R 13 , R 14 , R 15 , X, and x are as defined in claim 86 for formula III; and and k is an integer from 1 to 30.

105. Formula 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, wherein ABD-L 1 R 3 The ADC of claim 104, wherein the payload (i.e., TLR7 agonist) is attached to the alcohol group of

106. The ADC of any one of claims 89-105, wherein the ABD has binding specificity for a transmembrane molecule (e.g., a receptor) expressed on a tumor.

107. 107. A pharmaceutical composition comprising a compound of any one of claims 1 to 88 or an ADC of any one of claims 89 to 106 and a pharmaceutically acceptable carrier.

108. 107. A method of treating or diagnosing a disease, the method 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. 109. The method of claim 108, wherein the method treats a disease.

110. 110. The method of claim 108 or 109, wherein the disease is cancer.

111. The disease is acute myeloid leukemia, adult T-cell leukemia, astrocytoma, 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, glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lung cancer (e.g., small cell lung cancer, 111. The method of any one of claims 108-110, wherein the cancer being treated is selected from the group consisting of non-small cell lung cancer (NSCLC), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, malignant mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, castration-resistant prostate cancer, renal cell carcinoma, residual cancer, rhabdomyosarcoma, gastric cancer, synovial sarcoma, thyroid cancer, uterine cancer, and Wilms' tumor.

112. 112. The method of any one of claims 108 to 111, wherein the disease is breast cancer.

113. The method of any one of claims 108 to 111, wherein the disease is prostate cancer.

114. a. an antigen-binding domain (ABD) having binding specificity for hepatitis B virus surface antigen (HBV sAg); and b. A Toll-like receptor 7 (TLR7) agonist that links the ABD with a bivalent linker An antibody drug conjugate (ADC) comprising:

115. The ADC of claim 114, wherein said TLR7 agonist is any one of P2 to P39 and P41 to P48.

116. 115. The ADC of claim 114, wherein said TLR7 agonist having a bivalent linker is any one of LP1-5, LP6A-6B, LP7A-7E, LP8A-8B, LP9, LP10A-10B, LP11A-11D, and LP12-15.

117. 115. The ADC of claim 114, wherein the ABD is an antibody against HBV sAg or a fragment thereof.

118. 115. The ADC of claim 114, wherein the ABD is a human antibody or a humanized antibody.

119. 115. The ADC of claim 114, wherein the ABD comprises an scFv with binding specificity for HBV sAg.

120. The ABD is V of an antibody against HBV sAg. H Chain and V L 115. The ADC of claim 114, comprising a chain.

121. The ADC of claim 114, wherein the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody against HBV sAg.

122. The ADC of claim 114, wherein said ABD comprises an Fc region.

123. The ADC of claim 120, wherein the Fc region comprises a modification for enhanced binding to an FcγR.

124. The ADC of any one of claims 114 to 123, wherein the ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised 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) comprised 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 the 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. 126. The ADC of claim 124 or claim 125, wherein the 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. 130. The ADC of any one of claims 124-129, wherein the ABD is a component of an antibody or antigen-binding fragment thereof.

131. 131. A method of treatment comprising administering to a subject in need thereof an effective amount of the ADC of any one of claims 114-130.

132. 132. The method of claim 131, wherein the subject has chronic hepatitis B.

133. 132. The method of claim 131, wherein the 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 their serum prior to administration of the ADC or pharmaceutical composition.

135. 134. The method of any one of claims 131 to 133, further comprising measuring circulating HBV DNA or HBV sAg in the serum of said subject prior to administration.

136. The method of any one of claims 131 to 135, further comprising measuring circulating HBV DNA or HBV sAg in the serum of the subject after administration to assess the therapeutic efficacy of the ADC or pharmaceutical composition.

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