TLR7 agonists and antibody-drug conjugates thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 429,096, filed on November 30, 2022, and U.S. Provisional Application No. 63 / 578,109, filed on August 22, 2023, both of which are incorporated herein by reference as if fully set forth herein.

[0003] Sequence Listing Incorporated by Reference

[0004] This application contains a sequence listing. This sequence listing has been submitted electronically as an XML file (titled "2387951.xml"). This sequence listing is 30,417 bytes in size and was created on November 27, 2023. This application is incorporated herein by reference in its entirety.

[0005] field

[0006] Provided herein are TLR7 agonists and antibody-drug conjugates (ADCs) thereof.

[0007] background

[0008] Toll-like receptors (TLRs) are a subgroup of pattern recognition receptors (PRRs) that play a key role in the innate immune response. TLRs are divided into two categories based on subcellular localization, of which endosomal TLRs have pharmaceutical benefits. 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 are reported to have antiviral and antibacterial activities, as well as activity as vaccine adjuvants and for the treatment of allergic diseases and asthma. It is important here to study TLR7 agonists as cancer immunotherapeutics. A TLR7 agonist (Imiquimod) has been approved by the US FDA for the treatment of actinic keratosis, superficial basal cell carcinoma, and external genital warts.

[0009] ADC combines the specificity of antibodies with the ability to target specific cell types or tissues with payloads. Research in this field has attracted significant attention, and some of the drugs that have been marketed include (brituximab vedotin) and KADCYLA TMADCs with TLR7 agonist payloads have been reported. For example, see U.S. Patents Nos. 10,472,420, 10,780,180, 10,548,985, 10,722,591, and 10,675,358; and PCT Publication No. WO 2020 / 181050. However, no such ADCs have been approved for use in humans.

[0010] Therefore, there is a continuing need for TLR agonists and their ADCs to treat various diseases, including cancer and chronic hepatitis B. Summary of the Invention

[0011] In one aspect, the present disclosure provides a TLR7 agonist of Formula I for use in the compositions and methods provided herein:

[0012]

[0013] or a pharmaceutically acceptable salt thereof, wherein:

[0014] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0015] R 2 is H, halogen or alkoxy;

[0016] 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;

[0017] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0018] R 23 is H, alkyl or aryl;

[0019] X is CH or N;

[0020] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0021] Z is -OH, alkoxy or -NR 7 R 8 ;

[0022] R 5 and R 6 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and

[0023] R 7 and R 8 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a heterocyclic ring.

[0024] In another aspect, the present disclosure provides a TLR7 agonist-linker of Formula II for use in preparing the ADCs provided herein:

[0025]

[0026] or a pharmaceutically acceptable salt thereof, wherein:

[0027] R 1 、R 2 and X is as defined in Formula I;

[0028] R 9 R as defined in claim 1 3 The group removes the terminal hydrogen (ie, away from the R 9 A divalent group formed by (a hydrogen of the attached phenyl group); and

[0029] L is any group or moiety that is connected, linked or bonded to the antigen binding domain ABD.

[0030] In certain embodiments, the ADCs provided herein can be used in therapeutic methods, imaging methods, or diagnostic methods.

[0031] Also provided herein 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 in treating diseases. In some embodiments, the antigen is HBV surface antigen (HBV sAg) and the disease is chronic hepatitis B.

[0032] In one aspect, the present disclosure provides an antibody-drug conjugate (ADC) comprising (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.

[0033] In some embodiments, the ADC further comprises a bivalent linker linking the ABD to the TLR7 agonist.

[0034] In some embodiments, the ADC is according to Formula IV:

[0035]

[0036] or a pharmaceutically acceptable salt thereof, wherein:

[0037] L 1 is a divalent linker;

[0038] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0039] R 2 is H, halogen or alkoxy;

[0040] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0041] R 9 For R 3 The divalent group formed by removing hydrogen, R 3 For R 9 The group at position 1 is connected to the phenyl group;

[0042] 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;

[0043] R 23 is H, alkyl or aryl;

[0044] X is CH or N;

[0045] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0046] Z is -OH, alkoxy or -NR 7 R 8 ;

[0047] R 5 and R 6 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a heterocyclic ring;

[0048] R 7 and R 8 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and

[0049] k is an integer from 1 to 30.

[0050] In some embodiments, the ADC comprises an ABD linked to a compound of Formula III, or a pharmaceutically acceptable salt thereof:

[0051]

[0052] in:

[0053] R 1 、R 2 and X is as described elsewhere for Formula I;

[0054] L is any group or moiety attached to ABD;

[0055] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0056] R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0057] R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0058] R 15 is hydrogen or alkyl;

[0059] R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and

[0060] x is 0, 1, 2, 3, 4, 5, or 6.

[0061] In some embodiments, ABD-L 1is 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.

[0062] In some embodiments, the ADC is according to Formula V:

[0063]

[0064] or a pharmaceutically acceptable salt thereof, wherein:

[0065] R 1 、R 2 and X is as described elsewhere for Formula I;

[0066] R 10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-;

[0067] The ABD is an antibody containing a Q295 residue, an N297Q mutation, and / or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG,LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15); and

[0068] k is an integer from 1 to 30.

[0069] In some embodiments, the ADC is according to Formula VI:

[0070]

[0071] or a pharmaceutically acceptable salt thereof, wherein:

[0072] L 1 is a divalent linker;

[0073] R 1 、R 2 、R 16 、R 11 、R 12 、R 13 、R 14 、R 15 , X and x are as defined in Formula III; and

[0074] k is an integer from 1 to 30.

[0075] 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 .

[0076] In some embodiments, k is 1, 2, 3, 4, or 5. In some embodiments, k is 2.

[0077] In some embodiments, the ABD comprises a heavy chain, and the C-terminus of the heavy chain is connected to the L 1 In some embodiments, the ABD comprises two heavy chains, and the C-termini of each of the two heavy chains are conjugated to L 1 In some embodiments, L 1 Linked to the cysteine ​​residue of ABD.

[0078] In some embodiments, the ABD is an antibody or fragment thereof directed 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 a scFv with binding specificity against HBV sAg. In some embodiments, the ABD comprises a V or Fv of an antibody directed against HBV sAg. H Chain and V L chain.

[0079] 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 an FcγR.

[0080] In another aspect, the present disclosure provides pharmaceutical compositions comprising an ADC disclosed herein and one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0081] In yet another aspect, the present disclosure provides a method of treatment comprising administering to a subject in need thereof an effective amount of an ADC or pharmaceutical composition disclosed herein.

[0082] In some embodiments, the subject has chronic hepatitis B. In some embodiments, the subject has elevated circulating HBV DNA or HBV sAg in serum prior to administration of the ADC or pharmaceutical composition.

[0083] In some embodiments, the method further comprises measuring circulating HBV DNA or HBVsAg in the subject's serum before administration. In some embodiments, the method further comprises measuring circulating HBV DNA or HBVsAg in the subject's serum after administration to assess the therapeutic effect of the ADC or pharmaceutical composition.

[0084] 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 at intervals of 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated at intervals of 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated at intervals of 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the step of administering the ADC or pharmaceutical composition is repeated at intervals of 1 month, 2 months, or 3 months.

[0085] In some embodiments, the ADC or pharmaceutical composition is administered orally, intravenously, intraperitoneally, by inhalation, intranasally, intramuscularly, or subcutaneously.

[0086] One aspect of the present disclosure provides an ADC or pharmaceutical composition for use in treatment. In some embodiments, the ADC or pharmaceutical composition is used to treat chronic hepatitis B in a subject in need thereof.

[0087] Another aspect of the present disclosure provides an ADC or pharmaceutical composition for use in preparing a medicament. In some embodiments, the medicament is used to treat chronic hepatitis B in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The in vitro plasma stability of the anti-HER2 Ab-LP1 ADC was demonstrated (Example 68).

[0089] Figure 2 The in vitro plasma stability of the anti-HER2 Ab-LP6A ADC was demonstrated (Example 68).

[0090] Figure 3 The in vitro plasma stability of the anti-HER2 Ab-LP11A ADC was demonstrated (Example 68).

[0091] Figure 4 The in vitro plasma stability of the anti-HER2 Ab-LP7A ADC was demonstrated (Example 68).

[0092] Figure 5 Conjugation schemes are shown for conjugating antibodies to linker-payloads provided herein (Examples 63, 64, and 69).

[0093] Figure 6 is a preparative SEC chromatogram of the conjugation mixture, showing complete separation of ADC monomers from aggregates (“HMW”) and unconjugated linker-payload (“free drug”).

[0094] Figure 7 is the analytical SEC chromatogram of the SEC-purified antibody-TLR7 conjugate, showing a monomer purity of 99.7%.

[0095] Figure 8 The LC-ESI-MS spectra of a deglycosylated and reduced ADC sample are shown. The calculated average DAR value is 1.84. The deconvoluted mass spectrum shows the light chain species (LC, LC1) and heavy chain species (HC, HC1, HC2, etc.). The average DAR can be calculated from the LC and HC drug loadings.

[0096] Figure 9 HIC chromatograms of the antibody and its LP11A conjugate are shown, showing a mixture of three species: DAR2 species (51%), DAR4 species (28%), and unconjugated antibody (21%). The average DAR for this ADC was 2.1.

[0097] Figure 10 Depicted are the results after a single treatment with an anti-HER2Ab-LP6A ADC (Table 3) in an N87 xenograft tumor model. Dosing was performed on day 0. Tumor regression was observed after treatment with 5 mg / kg (grey circles) of anti-HER2Ab-LP6A ADC compared to animals treated with saline (open circles), while tumor stagnation occurred after treatment with 1 mg / kg (grey squares) of anti-HER2Ab-LP6A ADC. No regression of N87 gastric tumors was observed in N87 xenograft mice treated with 5 mg / kg isotype control Ab-LP6A ADC (Table 3) (black circles) or 0.5 mg / kg (grey triangles) of anti-HER2Ab-LP6A ADC or 0.1 mg / kg (grey diamonds) of anti-HER2Ab-LP6A ADC compared to animals treated with saline (open circles). Data represent the mean tumor volume (mean + / - SEM) over time (after dosing).

[0098] Figure 11Depicted are the results of treatment of human N87 xenograft tumors with a single dose of anti-HER2Ab-LP6A ADC, anti-HER2Ab-LP11A ADC, or anti-HER2Ab-LP7A ADC (Table 3). Dosing was performed on day 0. Tumor regression was observed after treatment with 5 mg / kg anti-HER2Ab-LP6A ADC (grey circles), 5 mg / kg anti-HER2Ab-LP11A ADC (grey squares), or 5 mg / kg anti-HER2Ab-LP7A ADC (grey triangles) compared to animals treated with saline (open circles). No regression of N87 gastric tumors was observed in N87 xenograft mice treated with 5 mg / kg isotype control Ab-LP6A ADC (black circles) (Table 3), isotype control Ab-LP11A ADC (black squares) (Table 3), or isotype control Ab-LP7A ADC (black triangles) (Table 3) compared to animals treated with saline (open circles). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0099] Figure 12 Delineating trastuzumab-resistant HER2 中 Results after treatment of human JIMT-1 xenograft tumors. Dosing began on day 0 and continued every 7 days thereafter for a total of four doses of the anti-HER2 Ab-LP6A ADC (Table 3) or in combination with pertuzumab. Compared to animals treated with 5 mg / kg unconjugated mAb2 alone (open circles), tumor regression was observed after treatment with 5 mg / kg anti-HER2 Ab-LP6A ADC in combination with 5 mg / kg pertuzumab (grey squares), while treatment with 5 mg / kg anti-HER2 Ab-LP6A ADC alone (grey circles) resulted in tumor stasis for 45 days. No JIMT-1 breast tumor regression was observed following treatment with 5 mg / kg isotype control Ab-LP6A ADC (black circles) (Table 3), 5 mg / kg isotype control Ab-LP6A ADC in combination with 5 mg / kg Pertuzumab (black squares), or 5 mg / kg unconjugated mAb2 in combination with 5 mg / kg Pertuzumab (open squares), compared to animals treated with 5 mg / kg unconjugated mAb2 alone (open circles). Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0100] Figure 13Depict the results after treatment of MC38 tumors genetically engineered to express human CD20. Starting from day 0, tumor-bearing mice received a total of 3 anti-CD20Ab-LP6A ADC (Table 3) treatments, with a 7-day interval between each administration. Compared to animals treated with saline (open circles), after treatment with 5 mg / kg anti-CD20Ab-LP6A ADC (grey squares), 4 of 5 mice were observed to have tumor regression. Compared to animals treated with 5 mg / kg saline (open circles), no MC38hCD20 tumor regression was observed in MC38hCD20 syngeneic mice treated with 5 mg / kg anti-mIgG2aCtrl Ab-LP1ADC (black circles) (Table 3) or 5 mg / kg unconjugated anti-CD20Ab (open squares). Data represent the average tumor volume (mean + / - SEM) over time (after administration).

[0101] Figure 14 Depicted are HBV sAg levels measured in a mouse model of chronic hepatitis B (CHB) disease following three subcutaneous (SC) treatments (two weeks apart) with an anti-sAg mAb (mAb3), an anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS (Example 51; Experiment 1).

[0102] Figure 15 Depicted are HBV sAg levels measured in a mouse model of chronic hepatitis B (CHB) disease following three subcutaneous (SC) treatments (two weeks apart) with an anti-sAg mAb (mAb3), an anti-sAg mAb-TLR7 agonist (mAb3+LP1 or mAb4+LP1), a TLR7 agonist (LP1), or PBS (Example 51; Experiment 2).

[0103] Figure 16 Depicting initial clearance of MC38.hTAA Pos Mice with tumor transplantation ( Figure 13 The results were obtained after the parental MC38 tumor cells were re-challenged. Pos On day 60 after tumor cell inoculation, tumor-free mice (black squares) were re-challenged with parental MC38 cells that do not overexpress human TAAs. Mice previously treated with the anti-CD20-LP6A conjugate were protected against tumor re-challenge compared to control naive mice (open circles). Data represent mean tumor volume (mean + / - SEM) over time (after re-challenge).

[0104] Figure 17 Depicted are the results of a MC38.hTAA inoculation. PosResults of treatment of mice bearing tumor cells. Wild-type mice (solid solid symbols) and humanized IFNAR mice lacking the ability to respond to murine type I IFN (dashed open symbols) were treated with anti-CD20-LP11A conjugate three times every 7 days. Tumor regression was observed after treatment with 5 mg / kg anti-CD20-LP11A conjugate (solid triangles) compared to saline-treated animals (solid circles) and isotype control antibody conjugate (solid squares). No MC38.hTAA was observed in humanized IFNAR mice treated with 5 mg / kg anti-CD20-LP11A conjugate (open triangles), isotype control antibody conjugate (open squares), or saline-treated animals (open circles). Pos Tumor regression. Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0105] Figure 18 Depicts the MC38.hTAA inoculation Pos Results after treatment of mice with tumor cells in TAA and human CD3 humanized mice with anti-CD20-LP6A conjugate 3 times every 7 days, with or without anti-CD20xanti-hCD3 bispecific antibody 5 times every 4 days. Compared to mice treated with 2.5 mg / kg bispecific antibody isotype control (open circles) or 2.5 mg / kg isotype control antibody plus 2.5 mg / kg bispecific antibody isotype control (open squares), tumor regression was observed after treatment with 2.5 mg / kg anti-CD20-LP6A conjugate plus 2.5 mg / kg anti-CD20 x anti-hCD3 bispecific antibody (black triangles), while treatment with 2.5 mg / kg anti-CD20 plus 2.5 mg / kg bispecific antibody isotype control (open triangles), 2.5 mg / kg anti-CD20 x anti-hCD3 bispecific antibody alone (black circles), and 2.5 mg / kg isotype control antibody-(NC-1) plus 2.5 mg / kg anti-CD20 x anti-hCD3 bispecific antibody (black squares) delayed tumor growth. Data represent mean tumor volume (mean + / - SEM) over time (post-dose).

[0106] Figure 19 Depicted is the ring-opening of the imide bond in an antibody-drug conjugate formed by conjugating a cysteine ​​thiol to a maleimide linker-payload. Under physiological conditions, the ring-opening of the imide bond generates two regioisomers: one in which the thiol is linked to the alpha carbon of the carboxylic acid group and the other in which the thiol is linked to the beta carbon of the carboxylic acid group.

[0107] Figure 20 This article refers to Q o-Schematic diagram of a possible metabolic pathway of the compound LP11A.

[0108] Figure 21 This article refers to Q o -Schematic diagram of another possible metabolic pathway of the compound of LP11A.

[0109] Figure 22 Shown are hepatitis B virus surface antigen (HBV sAg) levels measured after five subcutaneous treatments (one week apart) with an anti-sAg monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate-buffered saline (PBS) in a mouse model of chronic hepatitis B (CHB) disease. mAb3+LP6A effectively reduced HBV sAg levels compared to PBS control.

[0110] Figure 23 Shown are anti-HBsAG IgG titers measured on day 120 (D120) after the first treatment with five subcutaneous doses (one week apart) of an anti-surface antigen (sAg) monoclonal antibody-TLR7 agonist (mAb3+LP6A) or phosphate-buffered saline (PBS) in a mouse model of chronic hepatitis B (CHB) disease. Mab3+LP6A-treated mice had higher titers compared to PBS-treated control mice. DETAILED DESCRIPTION

[0111] I. Definition

[0112] To facilitate understanding of the disclosure described herein, certain terms are defined below.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If a term has multiple definitions herein, the definition in this section will prevail unless otherwise stated.

[0114] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0115] As used herein, a "subject" is an animal, such as a mammal, including a human, such as a patient.

[0116] As used herein, "biological activity" refers to the activity of a compound in vivo, or the physiological response produced by a compound, composition, or other mixture after administration in vivo. Thus, biological activity encompasses both the therapeutic efficacy and pharmacokinetic behavior of such compounds, compositions, and mixtures. Biological activity can be observed in in vitro systems designed to test such activity.

[0117] As used herein, "antigen-binding domain" or "ABD" refers to any peptide, polypeptide, nucleic acid molecule, scaffold molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding to a particular target antigen. As used herein, "antigen-binding domain" includes antibodies and antigen-binding fragments of antibodies. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment, unless specifically indicated to be from a non-human species.

[0118] The phrases "specifically bind" or "specifically binds to" and the like refer to the formation of a complex between an antibody or antigen-binding fragment thereof and an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 -8 M or lower (e.g., smaller K D Indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, Real-time, label-free biolayer interferometry is performed on the HTX biosensor to identify antibodies that specifically bind to the target antigen. In addition, multispecific antibodies that bind to a domain of the target antigen and one or more other antigens, or bispecific antibodies that bind to two different regions of the target antigen, are still considered to be "specifically bound" antibodies when used herein. In addition to neutralizing antibodies, antibodies that specifically bind to the target antigen but are non-neutralizing can also be used to generate antibody-drug conjugates within the scope of the present disclosure. Such antibodies can play a role, for example, delivering a payload to cells expressing the target antigen.

[0119] The term "high affinity" refers to an antibody that has at least 10 -8 M; preferably 10 -9 M; more preferably 10 -10 M, even more preferably 10 -11 M, even more preferably 10 -12 The binding affinity of M to the target antigen (K D denoted) were detected by real-time label-free biolayer interferometry (e.g. HTX biosensors) or by surface plasmon resonance (e.g. BIACORE TM ) or measured by solution affinity ELISA.

[0120] The phrase or term "slow off-rate", "K off ” or “k d " refers to the rate constant for the dissociation of the antibody from the target antigen being 1x10 -3 s -1 or lower, preferably 1x10 -4 s-1 or lower, by real-time, label-free biolayer interferometry (e.g. HTX biosensors) or surface plasmon resonance (e.g. BIACORE TM ) determination.

[0121] As used herein, "unrelated antigens" are proteins, peptides, or polypeptides that have less than 95% amino acid identity with each other.

[0122] The term "antibody" as used herein refers to any antigen-binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds to or interacts with a specific antigen. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy chains (H) and two light chains (L), interconnected by disulfide bonds) and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region contains three domains: C H 1. C H 2 and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L) and the light chain constant region. The light chain constant region contains a domain (C L 1). V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L Each of them is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H The three CDRs are called HCDR1, HCDR2 and HCDR3, V L The three CDRs of the chromatin are called LCDR1, LCDR2 and LCDR3.

[0123] As used herein, the term "antigen-binding fragment" of an antibody refers to any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0124] The term "human antibody" as used herein refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies may still contain amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutations in vivo), for example, in CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from germlines of other mammalian species (e.g., mice) are transplanted onto human framework sequences.

[0125] The term "humanized antibody" as used herein refers to a chimeric antibody comprising the minimum sequence derived from a non-human antibody. A humanized antibody is typically a human antibody (recipient antibody) in which one or more CDR residues are replaced by one or more CDR residues of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody with desired specificity, affinity, or biological effect. In some cases, the selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues of the donor antibody. Humanized antibodies can also include residues not found in either the recipient antibody or the donor antibody. Such modifications can be used to further improve antibody function. For more 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, the entire contents of each of which are incorporated by reference.

[0126] The term "recombinant human antibody," as used herein, refers to all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (as further described below), antibodies isolated from a recombinant combinatorial human antibody library (as further described below), antibodies isolated from an animal (e.g., a mouse) 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 method involving splicing of human immunoglobulin gene sequences to other DNA sequences.

[0127] When referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantially identical" means that the nucleotide sequence identity of the nucleotide bases is at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99%, when optimally aligned with another nucleic acid (or its complementary strand) for appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm (e.g., FASTA, BLAST, or GAP), as described in WO 2016 / 100807 or US 2016 / 0176953 A1, each of which is incorporated herein by reference in its entirety. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may encode a polypeptide having an amino acid sequence that is identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0128] Herein, in the context of amino acid sequences, the phrase "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned (e.g., aligned using the default gap weights by the programs GAP or BESTFIT), have at least 90% sequence identity, more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0129] The term "surface plasmon resonance" as used herein 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 BIAcore TM System (Biacore Life Sciences division, GE Healthcare, Piscataway, NJ).

[0130] The term "K D " refers to the equilibrium dissociation constant for a specific protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise indicated, K values ​​disclosed herein are D The values ​​refer to K values ​​determined by surface plasmon resonance analysis at 25 °C. D value.

[0131] Pharmaceutically acceptable salts as used herein include, but are not limited to, amine salts such as, but not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine, and tris(hydroxymethyl)aminomethane; alkali metal salts such as, but not limited to, lithium, potassium, and sodium; 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 also include, but are not limited to, mineral acid salts such as, but not limited to, hydrochlorides and sulfates; and organic acid salts such as, but not limited to, acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, methanesulfonates, and fumarates.

[0132] As used herein, the terms "treat," "treatment," or "therapy" refer to the reduction or improvement in severity of at least one symptom or indication of a disease (e.g., cancer or hepatitis B infection) as a result of administering a therapeutic agent (e.g., a disclosed antibody) to a subject in need thereof. These terms include inhibiting the progression of a disease or worsening of an infection. These terms also include a positive prognosis for a disease, e.g., following administration of a therapeutic agent (e.g., a disclosed antibody or antibody-drug conjugate), the subject may not be infected, the subject's viral titer may decrease or disappear, or the subject's tumor may shrink. The therapeutic agent may be administered to a subject at a therapeutic dose.

[0133] The terms "prevent," "prophylactic," or "preventing" refer to inhibiting the appearance of any symptoms or signs of a disease (e.g., cancer or hepatitis B infection) following administration of a disclosed antibody or antibody-drug conjugate. The term includes preventing the spread of infection in a subject exposed to the virus or at risk for hepatitis B infection.

[0134] The phrase "therapeutically effective amount" refers to an amount that produces the desired effect of administration. The exact amount will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques (e.g., see Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0135] As used herein, amelioration of the symptoms of a particular disease by administration of a particular compound or pharmaceutical composition refers to any relief, whether permanent or temporary, long-lasting or transient, attributable to or associated with the administration of the compound or pharmaceutical composition.

[0136] As used herein, IC 50It refers to the amount, concentration, or dose of a particular test compound that achieves a 50% inhibition of the maximal response in an assay measuring such response.

[0137] When moieties are specified by their conventional chemical formula written from left to right, they equally include the chemically identical moieties that would result from writing the structure from right to left, for example, -CH2O- is equivalent to -OCH2-.

[0138] Unless otherwise indicated, the term "alkyl" by itself or as part of other substituents refers to a straight chain (i.e., unbranched) or branched saturated hydrocarbon group. The term "alkylene" by itself or as part of other substituents refers to a divalent group derived from an alkyl group. Typically, an alkyl (or alkylene) group has 1 to 24 carbon atoms, including those groups having 10 or fewer carbon atoms. "Lower alkyl" or "lower alkylene" refers to an alkyl or alkylene group with a shorter chain, typically having 6 or fewer carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and homologues and isomers thereof, such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0139] Unless otherwise indicated, the term "alkenyl" alone or as part of other substituents refers to a straight chain (i.e., unbranched) or branched hydrocarbon group having one or more carbon-carbon double bonds. The term "alkenylene" alone or as part of other substituents refers to a divalent group derived from an alkenyl group. Typically, an alkenyl (or alkenylene) group has 1 to 24 carbon atoms, including those with 10 or fewer carbon atoms. "Lower alkenyl" or "lower alkenylene" refers to an alkenyl or alkenylene group with a shorter chain, typically with 6 or fewer carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., vinyl), 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomers.

[0140] Unless otherwise indicated, the term "alkynyl", used alone or as part of another substituent, refers to a straight chain (i.e., unbranched) or branched hydrocarbon radical having one or more carbon-carbon triple bonds, including divalent and polyvalent radicals, with the specified number of carbon atoms (i.e., C1-C 10 represents 1 to 10 carbon atoms). Examples of alkynyl groups include, but are not limited to, ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0141] 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.

[0142] Unless otherwise indicated, the term "heteroalkyl", alone or in combination with other terms, means a straight or branched chain hydrocarbon radical containing in the chain at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atoms may bear alkyl substituents to satisfy the 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 radical (i.e., not at the point of attachment to the rest of the molecule). Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene," by itself or as part of another substituent, refers to a divalent radical derived from heteroalkyl, such as, but not limited to, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-O-CH2-CH2-NH-CH2-, -CH2-CH2-S-CH2-CH2-, and -CH2-S-CH2-CH2-NH-CH2-. For alkylene and heteroalkylene linking groups, the direction in which the linking group is written does not indicate the direction of the linking group. For example, the formula -C(O)2R'- refers to both -C(O)2R'- and -R'C(O)2-.

[0143] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl," alone or in combination with other terms, refer to cyclic forms of "alkyl" and "heteroalkyl," respectively, including bicyclic, tricyclic, and bridged bicyclic groups. Additionally, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the rest of the molecule. The terms "cycloalkylene" and "heterocycloalkylene," alone or as part of another substituent, refer to a divalent radical derived from a cycloalkyl or heterocycloalkyl group. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, bicyclo(2.2.2)octyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, 1- or 2-azabicyclo(2.2.2)octyl, and the like.

[0144] Unless otherwise indicated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent, which can be a monocyclic or polycyclic ring (in some embodiments, 1 to 3 rings), which are fused together or covalently linked. The term "heteroaryl" refers to an aryl group containing 1 to 4 heteroatoms selected from N, O and S in the ring, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group can be connected to the rest of the molecule through a carbon atom or a heteroatom. The terms "arylene" and "heteroarylene" alone or as part of other substituents refer to a divalent group derived from an aryl or heteroaryl group. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 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 in which one or more heteroatoms are positively charged.

[0145] Each of the above terms encompasses both substituted and unsubstituted forms of the group being described. Non-limiting examples of substituents for each group are listed below.

[0146] In some embodiments, the substituent moiety of the alkyl, heteroalkyl, alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups is selected from deuterium, -OR', =O, =NR', =N-OR', -NR'R", -SR', halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)N R'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and -NO2, in an amount ranging from zero to the number of hydrogen atoms in the group. In some embodiments, the substituent portion of the cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl groups also includes substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, and substituted and unsubstituted alkynyl. In some embodiments, R', R", R"' and R"" are each independently hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. For example, when the compounds provided herein contain more than one R group, each R group is independently selected, and when more than one R', R", R"' and R"" group is present, each R', R", R"' and R"" group is also independently selected. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-membered, 5-membered, 6-membered or 7-membered ring. For example, -NR'R" includes but is not limited to 1-pyrrolidinyl and 4-morpholinyl. Based on the above discussion of the substituents, those skilled in the art will understand that the term "alkyl" includes groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0147] In some embodiments, the substituent moieties of the aryl and heteroaryl groups are selected from deuterium, halogen, substituted and unsubstituted alkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR" R'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, the number of which ranges from zero to the total number of hydrogens on the aromatic ring system; wherein 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. For example, when the compounds provided herein contain more than one R group, each R group is independently selected, and when more than one R', R", R"' and R"" group is present, each R', R", R"' and R"" group is also independently selected.

[0148] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a ring of the formula -Q'-C(O)-(CRR') q -Q"- ring, wherein Q' and Q" are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a substituent of the formula -A-(CH2) r -B-, where 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 single bond of the new ring thus formed may be optionally replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by 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'-. In some embodiments, the substituents R, R', R" and R'" are 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.

[0149] Unless otherwise indicated, the term "halogen," by itself or as part of another substituent, refers to a fluorine, chlorine, bromine, or iodine atom. Furthermore, terms such as "haloalkyl" include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0150] As used herein, the term "oxo" refers to an oxygen atom double-bonded to a carbon atom.

[0151] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0152] Certain ADCs provided herein have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, tautomers, geometric isomers, and individual isomers are encompassed within the scope of this disclosure. The ADCs provided herein do not include ADCs known in the art that are too unstable to synthesize and / or separate.

[0153] II. TLR7 agonists

[0154] 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:

[0155]

[0156] or a pharmaceutically acceptable salt thereof, wherein:

[0157] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0158] R 2 is H, halogen or alkoxy;

[0159] 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;

[0160] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0161] R 23 is H, alkyl or aryl;

[0162] X is CH or N;

[0163] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0164] Z is -OH, alkoxy or -NR 7 R 8 ;

[0165] R 5 and R 6 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and

[0166] R 7 and R 8 Each is independently H or alkyl, or together with the N to which they are attached, forms a heterocyclic ring. In some embodiments, the compound of formula (I) is not a compound of the formula:

[0167] or

[0168]

[0169] In some embodiments, in the TLR7 agonist according to Formula I,

[0170] R 5 and R 6 Selected from (i), (ii) and (iii):

[0171] (i)R 5 and R 6 Each is H;

[0172] (ii)R 5 is H, and R 6 is an alkyl group;

[0173] (iii)R 5 and R 6 together with the nitrogen to which they are attached, form a heterocyclic ring; and

[0174] R 7 and R 8 Together with the nitrogen to which they are attached they form a heterocyclic ring.

[0175] In some embodiments, a TLR7 agonist is selected with the proviso that R 4 Not substituted by hydroxyl groups.

[0176] In some embodiments, a TLR7 agonist is selected with the proviso that R3 The alkylene and heteroalkylene portions are not substituted with oxo.

[0177] In some embodiments, a TLR7 agonist is selected provided 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.

[0178] In some embodiments, R 1 is H. In some embodiments, R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 , and is a straight chain of 6 atoms in length. In some embodiments, R 1 In some embodiments, R 1 NHR 4 In some embodiments, R 1 For-OR 4 In some embodiments, R 1 -NH-OR 4 In some embodiments, R 1 -R 4 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 It is -NH-CH2CH2-OEt.

[0179] In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 For H.

[0180] In some embodiments, R 3 -CO2R23 、-CONHR 23 , -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y. In some embodiments, R 3 for-CONHR 23 In some embodiments, R 3 In some embodiments, R 3 In some embodiments, R 3 is -heteroalkylene-arylene-Y. In some embodiments, R 3 In some embodiments, R 3 In some embodiments, R 3 In some embodiments, R 3 is -CONH2, -COOH, -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. In some embodiments, R 3 is -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. In some embodiments, R 3is -C(Me)2OH, -CO2H-CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2 CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2- In some embodiments, R 3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2 OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2N HC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH-CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2 or -CH2-1-piperazinyl.

[0181] In some embodiments, R 4 In some embodiments, R 4 In some embodiments, R 4 In some embodiments, R 4 In some embodiments, R 4 It is ethoxyethyl.

[0182] In some embodiments, R 5 and R 6 are each independently H or alkyl, or together with the nitrogen atom to which they are attached form a piperazinyl ring. 5 and R 6 Each is H. In some embodiments, R 5 H, R 6 In some embodiments, R 5 and R 6 Together with the nitrogen atom to which they are attached they form a 1-piperazinyl group.

[0183] In some embodiments, Y is OH. In some embodiments, Y is a divalent glycine group of formula -NHCH2C(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.

[0184] 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.

[0185] In some embodiments, R 7 and R 8 are each independently H or n-propyl, or together with the nitrogen atom to which they are attached form 1-piperazinyl. 7 and R 8 Together with the nitrogen atom to which they are attached they form a 1-piperazinyl group.

[0186] 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 them:

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] In some embodiments, the TLR7 agonist can be a known TLR7 agonist, such as 852A, imiquimod, resiquimod, gadiquimod, loxoribine, brospirin, 3M-011, 3M-052, DSR-6434, DSR-29133, SC1, SZU-101, SM-360320 and SM-276001. Exemplary TLR7 agonists are described in, for example, Chi et al., Front. Pharmacol. 8:34, May 31, 2017, which is incorporated herein by reference in its entirety.

[0194] III. Synthesis of TLR7 agonists

[0195] 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 shown in Schemes 1-7 herein. Scheme 1. Synthesis of intermediate Aa starting from compound 1

[0196]

[0197] Scheme 2. Synthesis of intermediates starting from compound 5

[0198]

[0199]

[0200] Scheme 3. Synthesis of payloads P1, P2, P20, P23, P27, P29, P32, P33, P37, and P39

[0201]

[0202] Scheme 4. Synthesis of payloads P3, P26, P28, P36, and P38.

[0203]

[0204] Scheme 5. Synthesis of payloads P22, P25, P31, P35, P21, P24, P30, and P34.

[0205]

[0206]

[0207] Scheme 6. Synthesis of payloads P4, P5, P6, P8, P9, P10, P11, P12, P17, P18, and P19.

[0208]

[0209]

[0210] Scheme 7. Synthesis of payloads P7, P13, P14, P15, and P16.

[0211]

[0212]

[0213] IV. Linker-TLR7-Agonist (Linker-Payload)

[0214] 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 or a pharmaceutically acceptable salt thereof:

[0215]

[0216] in:

[0217] R 1 、R 2 and X is as defined elsewhere for Formula I;

[0218] R 9 For R 3 The divalent group formed by removing hydrogen, R 3 For R 9 The group to which the phenyl group is attached; and

[0219] L is any group or moiety that connects, links 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) is not a compound of the formula:

[0220] or

[0221]

[0222] In some embodiments, R 9 -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 -alkylene-Y 1 -. In some embodiments, R 9 -heteroalkylene-Y 1 -. In some embodiments, R 9 -heteroalkylene-arylene-Y 1 -. In some embodiments, R 9 -(hydroxy)heteroalkylene-Y 1 In some embodiments, R 9 -(amino)heteroalkylene-Y 1 In some embodiments, R 9 -alkylene-PEG-Y 1 In another embodiment, R 9 -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 -. In another embodiment, R 9is -C(Me)2O-, -CO-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2 OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazine-4-yl-, -CH2OCH2NHC(O)CH2N H-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, -CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH- or -CH2-1-piperazin-4-yl.

[0223] In an embodiment, Y 1 In some embodiments, Y 1 is a divalent glycine group of formula -NHCH2C(O)-. In some embodiments, Y 1 -NR 5 -. In some embodiments, Y 1 for-COZ 1 , where Z 1 -O-, -NR 7 -, -O-alkylene- or 1-piperazin-4-yl. In some embodiments, Y 1 It is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl.

[0224] In some embodiments, Z 1 In some embodiments, Z 1 -NR 7 -. In some embodiments, R 7 is H. In some embodiments, R 7 It is an alkyl group.

[0225] In some embodiments, Z 1 In some embodiments, Y 1In some embodiments, Y 1 It is -CO-1-piperazin-4-yl.

[0226] In some embodiments, the linker L used herein can be found in, for example, Antibody-Drug Conjugates and Immunotoxins, Phillips, GL, Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates, Wang, J., Shen, W.-C., and Zaro, JL, Eds.; Springer International Publishing, 2015. In some embodiments, the L group of the ADC provided herein is sufficiently stable to take advantage of the circulating half-life of the antigen-binding domain while being able to release its payload upon antigen-mediated internalization of the ADC. The linker L can be cleavable or non-cleavable. Cleavable linkers used herein as L include linkers that are cleaved by intracellular metabolism after internalization, for example, by hydrolysis, reduction, or enzymatic cleavage. Non-cleavable linkers used herein as L include linkers that release the attached payload upon internalization by lysosomal degradation of the antigen binding domain. Suitable L linkers include, but are not limited to, acid-labile linkers, hydrolytically unstable linkers, enzymatically cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable L linkers also include, but are not limited to, being or comprising a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a maleimide-hexanoyl unit, a dipeptide unit, a valine-citrulline unit, and a p-aminobenzyl (PAB) unit.

[0227] Unless otherwise indicated, the term "PEG" or "polyethylene glycol", alone or in combination with other terms, refers to the group -(OCH2CH2O) n-, wherein n is an integer from about 1 to about 100, such as 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 [Insert ChemDraw Structure]. The PEG group can have any suitable molecular weight, such as from about 60 g / mol to about 6,000 g / mol, from about 60 g / mol to about 600 g / mol, from about 100 g / mol to about 500 g / mol, from about 300 g / mol to about 1,200 g / mol, from about 200 g / mol to about 800 g / mol, from about 200 g / mol to about 1,000 g / mol, from about 500 g / mol to about 1,000 g / mol, from about 500 g / mol to about 2,500 g / mol, or from about 800 g / mol to about 2,200 g / mol.

[0228] Any linker molecule or linker technology known in the art can be used as L to create or construct the ADC provided herein. In some embodiments, the L linker is a cleavable linker. In other embodiments, the L linker is a non-cleavable linker. In some embodiments, the L linker that can be used for the ADC provided herein includes a linker comprising the following groups or consisting of the following groups: for example, MC (6-maleimidocaproyl), MP (maleimidopropionyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide site in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide site in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl) and variants and combinations thereof. Other examples of L linkers that can be used for the ADC provided herein are disclosed in, for example, U.S. Patent No. 7,754,681 and Ducry, Bioconjugate Chem., 2010, 21: 5-13, and references cited therein.

[0229] In some embodiments, the L linker is stable under physiological conditions. In some embodiments, the L linker is cleavable, for example, capable of releasing at least a portion of the payload in the presence of an enzyme or at a specific pH range or value. 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 bonds, and hydrazones. In some embodiments, the L linker comprises a cathepsin-cleavable linker.

[0230] In some embodiments, the L linker comprises a non-cleavable moiety.

[0231] In some embodiments, the L linker comprises one or more amino acids. Suitable amino acids include natural amino acids, non-natural amino acids, standard amino acids, non-standard amino acids, proteinogenic amino acids, non-proteinogenic amino acids, 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, citrulline, derivatives thereof, or combinations thereof. In some embodiments, one or more side chains of an amino acid are attached to a side chain group described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the L linker comprises lysine, valine, and citrulline. In some embodiments, the L linker comprises lysine, valine, and alanine. In some embodiments, the L linker comprises valine and alanine.

[0232] In some embodiments, the L linker comprises a self-sacrificial group. The self-sacrificial group can be any such group known to those skilled in the art. In a specific embodiment, the self-sacrificial group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will appreciate that the self-sacrificial group can undergo a chemical reaction to release the remaining atoms of the L linker from the payload.

[0233] In other embodiments, the L group can be modified with one or more enhancing groups. In some embodiments, the enhancing group can be connected to the side chain of any amino acid in L. In one embodiment, the amino acid for connecting the enhancing group includes lysine, asparagine, aspartic acid, glutamine, glutamic acid and citrulline. The connection with the enhancing group can be a direct bond with the amino acid side chain, or the connection can be indirectly connected through a spacer and / or reactive group. In one embodiment, the spacer and reactive group include any one described herein. In some embodiments, the enhancing group can be any group that imparts a payload, a connector-payload or an ADC beneficial effect, including but not limited to biological, biochemical, synthetic, solubilizing, imaging, detection and reactive effects, etc. 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, heteroalkyl sulfonic acid, alkenyl sulfonic acid, heteroalkenyl sulfonic acid, heteroalkenyl taurine, heteroalkenyl phosphoric acid or phosphate, heteroalkenyl amine (e.g., quaternary amine) or heteroalkenyl sugar. In some embodiments, sugar includes but is 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, and also include conjugated forms thereof, such as glucuronides (i.e., by glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like. The cyclodextrin can be any cyclodextrin known to those 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 increase the solubility of the remaining portion 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 2 ) 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, wherein n is 1, 2, 3, 4 or 5, and m is 1, 2, 3, 4 or 5. In one embodiment, the alkyl or alkenyl sulfonic acid is -(CH2) 1-5 In some embodiments, the heteroalkyl or heteroalkylene sulfonic acid is -(CH2) n –NH-(CH2) 1-5 SO3H, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2) n –C(O)NH-(CH2) 1-5 SO3H, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, wherein m is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, wherein n is 1, 2, 3, 4, or 5. In some embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, wherein m is 1, 2, 3, 4 or 5.

[0234] In some embodiments, L comprises a maleimido group (for conjugation to a thiol of the antigen-binding domain, such as cysteine), an N-hydroxysuccinimide ester (for conjugation to an amine of the antigen-binding domain, such as lysine), or a cyclooctyne group (for conjugation to the antigen-binding domain using click chemistry). See, for example, WO 2020 / 132658; Chio et al. Methods Mol. Biol. 2020, 2078: 83-87.

[0235] In some embodiments, L contains a maleimido group. In these embodiments, the maleimido group in L reacts with a cysteine ​​residue on the antigen binding domain to form a carbon-sulfur bond.

[0236] In some embodiments, L contains an N-hydroxysuccinimide ester group. In these embodiments, the N-hydroxysuccinimide ester group reacts with a lysine residue on the antigen binding domain to form an amide bond.

[0237] In some embodiments, L comprises an alkyne, which can react with an azide by click chemistry, for example, to form a click chemistry product. In some embodiments, the alkyne group reacts with the azide on the modified antigen-binding domain. In some embodiments, L comprises a functional group or part capable of performing click chemistry reactions (see, for example, clickchemistry, 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 capable of performing 1,3-cycloaddition reactions with an azide. Alkynes useful in such embodiments include strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC), cycloalkynes (e.g., cyclooctyne), benzoannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of a copper catalyst. Alkynes useful in such embodiments also include, but are not limited to, dibenzazacyclooctyne, dibenzocyclooctyne, biarylazacyclooctynone, difluorinated cyclooctyne, substituted alkynes (e.g., fluorinated alkynes), azacycloalkynes, and bicyclo[6.1.0]nonyne. In other embodiments, alkynes can be used to conjugate antibodies that have been functionalized with an azide group. Such functionalized antibodies include antibodies functionalized with an azide-polyethylene glycol group. In some embodiments, such functionalized antibodies are derivatized 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 a transglutaminase.

[0238] In some embodiments, L is selected from 2-maleimido-1-ethyl, 2-maleimidoacetyl, and 3-maleimidopropionyl. In certain embodiments, L is selected from:

[0239]

[0240]

[0241]

[0242] In some embodiments, L is a group selected from the group consisting of 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropionyl,

[0243]

[0244] In some embodiments, the linker-TLR7 agonist is selected from those in Table 2 and pharmaceutically acceptable salts of any of these:

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] In some embodiments, when the payload (ie, TLR7 agonist) has an alcohol group (ie, -OH), the payload can be converted into a prodrug before being connected to the linker and forming the ADC. See, for example, WO2020 / 146541. In this embodiment, the payload of Formula I can be converted into a linker-TLR7 agonist of Formula III:

[0251]

[0252] or a pharmaceutically acceptable salt thereof, wherein:

[0253] L is a linker as defined elsewhere herein;

[0254] R 1 、R 2 and X is as defined elsewhere for Formula I;

[0255] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0256] R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0257] R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0258] R 15 is hydrogen or alkyl;

[0259] R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-;

[0260] and x is 0, 1, 2, 3, 4, 5, or 6.

[0261] In some embodiments, the linker-TLR7 agonist has Formula III, wherein R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5- or 6-membered heterocyclic group; R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5- or 6-membered heterocyclic group; R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 Bonded to form a 4-membered, 5-membered or 6-membered heterocyclic group; R 15 is hydrogen or alkyl; R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; x is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, R 16 R in Formula I 3 The part that connects the benzene ring and the oxygen atom of the alcohol.

[0262] In some embodiments, the TLR7 agonist used to prepare the linker-TLR7 agonist according to Formula III is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, or P39.

[0263] V. Synthesis of TLR7 Agonist-Linker

[0264] Scheme 8. Synthesis of linker-payloads LP1, LP2, LP3, and LP4.

[0265]

[0266]

[0267] Scheme 9. Synthesis of linker-payload LP5.

[0268]

[0269] Scheme 10. Synthesis of linker-payload LP6A.

[0270]

[0271]

[0272] Scheme 11. Synthesis of linker-payloads LP7A and LP10A.

[0273]

[0274] Scheme 12. Synthesis of linker-payload LP8A.

[0275]

[0276]

[0277] Scheme 13. Synthesis of linker-payloads LP9 and LP12.

[0278]

[0279]

[0280] Scheme 14. Synthesis of Linker-Payloads LP6A, LP6B, LP7A, LP7B, LP7C, LP7D, LP10A, LP10B, LP11A, LP11B, LP11C, LP11D, LP12, and LP14

[0281]

[0282]

[0283] Scheme 14A. Alternative Synthesis of Linker-Payload LP11A

[0284]

[0285]

[0286]

[0287] Scheme 15. Synthesis of Linker-Payload LP8B2

[0288] (SEQ ID NO: 16)

[0289]

[0290]

[0291] Scheme 16. Synthesis of Linker-Payload LP13

[0292]

[0293]

[0294] Scheme 16A: Connector-Payload Q c -LP7A,Q c -LP11A, Q o -LP7A and Q o Synthesis of LP11-A

[0295]

[0296] VI. ADCs for Use in Compositions and Methods

[0297] 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 (e.g., HBV sAg or a tumor-specific antigen)) and a TLR7 agonist. In some embodiments, the ADC further comprises a bivalent linker connecting the ABD and the TLR7 agonist. The ABD can be conjugated to the TLR7 agonist at any position along the ABD, with or without a linker, as long as the ABD is capable of binding to its target.

[0298] In some embodiments, the ADC is according to Formula IV:

[0299]

[0300] or a pharmaceutically acceptable salt thereof, wherein:

[0301] L 1 is a divalent linker;

[0302] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0303] R 2 is H, halogen or alkoxy;

[0304] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0305] R 9 For R 3 The divalent group formed by removing hydrogen, R 3 For R 9 The group at position 1 is connected to the phenyl group;

[0306] 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;

[0307] R 23 is H, alkyl or aryl;

[0308] X is CH or N;

[0309] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0310] Z is -OH, alkoxy or -NR 7 R 8 ;

[0311] R 5 and R 6 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a heterocyclic ring;

[0312] R 7 and R 8 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and

[0313] k is an integer from 1 to 30.

[0314] In some embodiments, the ABC comprises one TLR7 agonist molecule conjugated to an ABD with target antigen binding specificity. In some embodiments, the ADC comprises more than one TLR7 agonist molecule 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.

[0315] 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.

[0316] In some embodiments, R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 , and has a straight chain length of 6 atoms. In some embodiments, R 1 In some embodiments, R 1 NHR 4 In some embodiments, R 1 For-OR 4 In some embodiments, R 1 -NH-OR 4 In some embodiments, R 1 -R 4 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 It is -NH-CH2CH2-OEt.

[0317] In some embodiments, R 2In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 For H.

[0318] 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 For CONHR 23 In some embodiments, R 3 In some embodiments, R 3 In some embodiments, R 3 is -heteroalkylene-arylene-Y. In some embodiments, R 3 In some embodiments, R 3 In some embodiments, R 3 In some embodiments, R 3 is -CONH2, -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. In some embodiments, R 3 is -CH2-Y, -CH2-O-heteroalkylene-Y, or -CH2-O-alkylene-Y. In some embodiments, R 3is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2 CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2- In some embodiments, R 3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2 OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2N HC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH-CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2 or -CH2-1-piperazinyl.

[0319] In some embodiments, R 4 In some embodiments, R 4 In some embodiments, R 4 In some embodiments, R 4 In some embodiments, R 4 It is ethoxyethyl.

[0320] In some embodiments, R 5 and R 6 are each independently H or alkyl, or together with the nitrogen atom to which they are attached form a piperazinyl ring. 5 and R 6 Each is H. In some embodiments, R 5 H, R 6 In some embodiments, R 5 and R 6 Together with the nitrogen atom to which they are attached they form a 1-piperazinyl group.

[0321] In some embodiments, Y is OH. In some embodiments, Y is a divalent glycine group of formula -NHCH2C(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.

[0322] 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.

[0323] In some embodiments, R 7 and R 8 are each independently H or n-propyl, or together with the nitrogen atom to which they are attached form 1-piperazinyl. 7 and R 8 Together with the nitrogen atom to which they are attached they form a 1-piperazinyl group.

[0324] In some embodiments, ABD-L 1By selecting the R 3 The group at the position of is removed from hydrogen and connected to a compound selected from P1 to P43.

[0325] In some embodiments, R 9 -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 -alkylene-Y 1 -. In some embodiments, R 9 -heteroalkylene-Y 1 -. In some embodiments, R 9 -heteroalkylene-arylene-Y 1 -. In some embodiments, R 9 -(hydroxy)heteroalkylene-Y 1 In some embodiments, R 9 -(amino)heteroalkylene-Y 1 In some embodiments, R 9 -alkylene-PEG-Y 1 In some embodiments, R 9 -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 -. In some embodiments, R 9is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH 2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazine-4-yl-, -CH2OCH2NHC(O)CH2 NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, -CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH- or -CH2-1-piperazin-4-yl.

[0326] In some embodiments, Y 1 In some embodiments, Y 1 is a divalent glycine group of formula -NHCH2C(O)-. In some embodiments, Y 1 -NR 5 -. In some embodiments, Y 1 for-COZ 1 , where Z 1 -O-, -NR 7 -, -O-alkylene- or 1-piperazin-4-yl. In some embodiments, Y 1 It is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl.

[0327] In some embodiments, Z 1 In some embodiments, Z 1 -NR 7 -. In some embodiments, R 7 is H. In some embodiments, R 7 It is an alkyl group.

[0328] In some embodiments, Z 1 In some embodiments, Y 1In some embodiments, Y 1 It is -CO-1-piperazin-4-yl.

[0329] AL 1 divalent groups

[0330] In some embodiments, the ADC of the present disclosure comprises a TLR7 indirectly linked to an ABD via a linker. In some embodiments, the linker is a bivalent linker (L 1 ), which connects the ABD to the TLR7 agonist according to Formula IV. In some embodiments, when the ADC is conjugated to the TLR7 agonist indirectly through a linker, the bivalent linker (L 1 ) is generated by the reaction between the linker (L) and the ADC used for conjugation.

[0331] The linker (L 1 ) can be found in, for example, Antibody-Drug Conjugates and Immunotoxins, Phillips, GL, Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates, Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates, Wang, J., Shen, W.-C., and Zaro, JL, Eds.; Springer International Publishing, 2015. In certain embodiments, the L for ADC provided herein is 1 The group is stable enough to take advantage of the circulating half-life of the antigen binding domain while being able to release its payload upon antigen-mediated internalization of the ADC. 1 Can be cleavable or non-cleavable. 1 Cleavable linkers include linkers that are cleaved by intracellular metabolism after internalization, such as by hydrolysis, reduction, or enzymatic cleavage. 1 Non-cleavable linkers include linkers that release the attached payload upon internalization by lysosomal degradation of the antigen binding domain. Suitable L 1 Linkers include, but are not limited to, acid labile linkers, hydrolytically labile linkers, enzymatically cleavable linkers, reduction labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable L 1Linkers also include, but are not limited to, linkers that include or comprise peptides, carbohydrates, glucuronides, polyethylene glycol (PEG) units, hydrazones, maleimido-hexanoyl units, dipeptide units, valine-citrulline units, and p-aminobenzyl (PAB) units.

[0332] Any linker molecule or linker technology known in the art can be used as L 1 To create or construct the ADC provided herein. In certain embodiments, L 1 The linker is a cleavable linker. In other embodiments, L 1 The linker is a non-cleavable linker. In certain embodiments, L can be used in the ADCs provided herein. 1 Linkers include linkers comprising or consisting of, for example, MC (6-maleimidocaproyl), MP (maleimidopropionyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide site in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide site in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), and variants and combinations thereof. L can be used in the ADCs provided herein. 1 Other examples of linkers are disclosed in, for example, US Pat. No. 7,754,681 and Ducry, Bioconjugate Chem., 2010, 21: 5-13 and references cited therein.

[0333] In certain embodiments, L 1 The linker is stable under physiological conditions. 1 The linker is cleavable, for example, capable of releasing at least a portion of the payload in the presence of an enzyme or at a specific pH range or value. 1 The linker comprises an enzyme cleavable portion. In some embodiments, the enzyme cleavable L 1 Linkers include, but are not limited to, peptide bonds, ester bonds, and hydrazones. 1 The linker comprises a cathepsin cleavable linker.

[0334] In some embodiments, L 1 The linker comprises a non-cleavable portion.

[0335] In some embodiments, L 1 The linker comprises one or more amino acids. Suitable amino acids include natural amino acids, unnatural amino acids, standard amino acids, non-standard amino acids, proteinogenic amino acids, non-proteinogenic amino acids, 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, citrulline, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of an amino acid are linked to a side chain group as described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, L 1 The linker comprises lysine, valine, and citrulline. In some embodiments, L 1 The linker comprises lysine, valine, and alanine. In some embodiments, L 1 The linker contains valine and alanine.

[0336] In some embodiments, L 1 The linker comprises a self-immolative group. The self-immolative group may be any such group known to those skilled in the art. In a specific embodiment, the self-immolative group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). It will be appreciated by those skilled in the art that the self-immolative group is capable of chemically reacting to convert L 1 The remaining atoms of the linker are released from the payload.

[0337] In other embodiments, L 1 The group can be modified with one or more reinforcing groups. In certain embodiments, the reinforcing group can be attached to L 1In some embodiments, the amino acid for connecting the enhancing group includes lysine, asparagine, aspartic acid, glutamine, glutamic acid and citrulline. The connection with the enhancing group can be a direct bond with the amino acid side chain, or the connection can be indirectly connected through a spacer and / or reactive group. In some embodiments, the spacer and reactive group include any one described herein. In certain embodiments, the enhancing group can be any group that imparts a payload, a connector-payload or an ADC beneficial effect, and the beneficial effect includes but is not limited to biological, biochemical, synthetic, solubilizing, imaging, detection and reactive effects, etc. 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, assorted alkyl, alkenyl, assorted alkenyl sulfonic acid, assorted alkenyl taurine, assorted alkenyl phosphoric acid or phosphate, assorted alkenyl amine (e.g., quaternary amine) or assorted alkenyl sugar. In certain embodiments, sugar includes but is not limited to monosaccharides, disaccharides and polysaccharides. Exemplary monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose, and the like. In certain embodiments, sugars include sugar acids, such as glucuronic acid, and also include conjugated forms thereof, such as glucuronides (i.e., by glucuronidation). Exemplary disaccharides include maltose, sucrose, lactose, lactulose, trehalose, and the like. Exemplary polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose, and the like. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. In certain embodiments, the cyclodextrin is α-cyclodextrin. In certain embodiments, the cyclodextrin is β-cyclodextrin. In certain embodiments, the cyclodextrin is γ-cyclodextrin. In certain embodiments, the enhancing group can increase the solubility of the remaining portion of the ADC. In certain embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is substituted or unsubstituted. In certain embodiments, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is —(CH 2 ) 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, wherein n is 1, 2, 3, 4 or 5, and m is 1, 2, 3, 4 or 5. In some embodiments, the alkyl or alkene sulfonic acid is -(CH2) 1-5 In another embodiment, the heteroalkyl or heteroalkylene sulfonic acid is -(CH2) n –NH-(CH2) 1-5 SO3H, wherein n is 1, 2, 3, 4 or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl or heteroalkenyl sulfonic acid is -(CH2) n –C(O)NH-(CH2) 1-5 SO3H, wherein n is 1, 2, 3, 4 or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl or heteroalkenyl sulfonic acid is -(CH2CH2O) m –C(O)NH-(CH2) 1-5 SO3H, wherein m is 1, 2, 3, 4, or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl, or heteroalkenyl sulfonic acid is -(CH2) n –N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, wherein n is 1, 2, 3, 4 or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl or heteroalkenyl sulfonic acid is -(CH2) n –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, wherein n is 1, 2, 3, 4 or 5. In another embodiment, the alkyl, heteroalkyl, alkenyl or heteroalkenyl sulfonic acid is -(CH2CH2O) m –C(O)N((CH2) 1-5 C(O)NH(CH2) 1-5 SO3H)2, wherein m is 1, 2, 3, 4 or 5.

[0338] In another embodiment, L 1Contains a 3-sulfosuccinimidyl group (formed by conjugating a maleimide group to a thiol (e.g., cysteine) of the antigen-binding domain), an amide group (formed by conjugating an N-hydroxysuccinimide group to an amine (e.g., lysine) of the antigen-binding domain), or a triazolocyclooctyl group (formed by conjugating a cyclooctyne group to a modified antigen-binding domain containing an azide group using click chemistry). See, for example, WO 2020 / 132658; Chio et al. Methods Mol. Biol. 2020, 2078:83-87.

[0339] In certain embodiments, L 1 Contains a 3-sulfosuccinimide group. In these embodiments, L 1 The 3-sulfosuccinimide group in the antigen-binding domain is generated by the reaction of the cysteine ​​residue on the antigen-binding domain with the maleimide group of L to form a carbon-sulfur bond.

[0340] In certain embodiments, L 1 Derived from L containing a maleimido group. In these embodiments, the maleimido group in L reacts with a cysteine ​​residue on the antigen binding domain to form a carbon-sulfur bond.

[0341] In some embodiments, L 1 is derived from L containing an N-hydroxysuccinimide ester group. In these embodiments, the N-hydroxysuccinimide ester group reacts with a lysine residue on the antigen binding domain to form an amide bond.

[0342] In other embodiments, L 1Derived from L containing an alkyne, the alkyne can react with an azide by click chemistry, for example, to form a click chemistry product. In some embodiments, the alkyne group reacts with the azide on the modified antigen-binding domain. In certain embodiments, L comprises a functional group or part capable of performing click chemistry (see, for example, 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 capable of performing 1,3-cycloaddition reactions with an azide. Alkynes useful in such embodiments include strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC); cycloalkynes, such as cyclooctynes, benzocycloalkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of a copper catalyst. Alkynes useful in such embodiments also include, but are not limited to, dibenzazacyclooctynes, dibenzocyclooctynes, biarylazacyclooctynones, difluorinated cyclooctynes, substituted alkynes (e.g., fluorinated alkynes), azacycloalkynes, and bicyclo[6.1.0]nonynes. In other embodiments, alkynes can be used to conjugate antibodies that have been functionalized with an azide group. Such functionalized antibodies include antibodies functionalized with an azide-polyethylene glycol group. In certain 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 a transglutaminase.

[0343] In some embodiments, L 1 Contains an amide group. In these embodiments, L 1 The amide group in the peptide is generated by the reaction of the N-hydroxysuccinimide ester group of L with a lysine residue on the antigen-binding domain to form an amide bond.

[0344] In other embodiments, L 1The cyclic group is generated by reacting an alkyne with an azide by click chemistry, for example, to form a click chemistry product. In some embodiments, the alkyne group reacts with an azide on the modified antigen-binding domain. In some embodiments, the antigen-binding domain comprises an alkyne group that reacts with an azide on L. See, for example, click chemistry, Huisgen Proc. Chem. Soc. 1961, 357-396; Wang et al. J. Am. Chem. Soc. 2003, 125 (11), 3192-3193; and Agard et al. J. Am. Chem. Soc. 2004, 126 (46), 15046-15047. In some embodiments, the alkyne group is an alkyne that can undergo a 1,3-cycloaddition reaction with an azide. Alkynes useful in such embodiments include strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloaddition (SPAAC); cycloalkynes, such as cyclooctynes, benzocycloalkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of a copper catalyst. Alkynes useful in such embodiments also include, but are not limited to, dibenzazacyclooctynes, dibenzocyclooctynes, biarylazacyclooctynones, difluorinated cyclooctynes, substituted alkynes (e.g., fluorinated alkynes), azacycloalkynes, and bicyclo[6.1.0]nonynes. In other embodiments, alkynes can be used to conjugate antibodies that have been functionalized with an azide group. Such functionalized antibodies include antibodies functionalized with an azide-polyethylene glycol group. In certain 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 a transglutaminase.

[0345] In another embodiment, L 1 is a group derived from 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropionyl,

[0346]

[0347] In certain embodiments, L 1 is or contains a divalent group selected from:

[0348] In another embodiment, L 1 is or contains a group selected from the following

[0349]

[0350]

[0351]

[0352] In some embodiments, L 1 In some embodiments, L 1 Cleavable under physiological conditions. In some embodiments, L 1 is an acid labile linker, a hydrolytically labile linker, an enzymatically cleavable linker, a reduction labile linker, or a self-immolative linker. 1 is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a maleimido-hexanoyl unit, a dipeptide unit, a valine-citrulline unit, or a p-aminobenzyl (PAB) unit. In some embodiments, L 1 In some embodiments, L 1 Contains a self-immolative group. In some embodiments, L 1 Contains p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC). In some embodiments, L 1 Contains a maleimide group, an N-hydroxysuccinimide ester group, or a cyclooctyne group. In some embodiments, L 1 is a group derived from: 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropionyl,

[0353]

[0354]

[0355]

[0356] In some embodiments, the ADC comprises ABD-linked LP1-LP15.

[0357] B. Antigen Binding Domain (ABD)

[0358] In one embodiment, the antigen binding domain (ie, ABD in Formula IV) used in the ADCs provided herein includes any molecule that specifically interacts with a particular antigen.

[0359] In certain embodiments, the ABD is an antibody or an antigen-binding fragment of an antibody. In certain embodiments, the ABD is an antibody.

[0360] In some embodiments, ABD is an antibody comprising an Fc region modified to enhance binding affinity to FcγR. In some embodiments, ABD is an antibody having one or more mutations selected from F243L, R292P, Y300L, V305I, and P396L. In some embodiments, ABD is an antibody having one or more mutations selected from S239D and I332E. In some embodiments, ABD is an antibody having one or more mutations selected from S239D, I332E, and A330L. In some embodiments, ABD is an antibody having one or more mutations selected from S298A, E333A, and K334A. In some embodiments, ABD is an antibody having one or more mutations selected from L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, ABD is an antibody having one or more mutations selected from D270E, K326D, A330M and K334E. In some embodiments, 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, ABD is an antibody having one or more mutations selected from G236A, S239D and I332E. In some embodiments, ABD is an antibody having one or more mutations selected from M252Y, S254T and T256E. In some embodiments, ABD is an antibody having one or more mutations selected from M428L and N434S. In some embodiments, ABD is an antibody having one or more mutations selected from S267E and L328F. In some embodiments, the ABD is an antibody having one or more mutations selected from N325S and L328F.

[0361] In some embodiments, ABD is an antibody comprising glutamine residues. Antibodies comprising glutamine residues can be isolated from natural sources, or engineered to comprise one or more glutamine residues. The technology (glutamine-modified antibodies) in which glutamine residues are engineered into antibody polypeptide chains is well known to those skilled in the art. In other embodiments, ABD is an N297Q mutant antibody. In a further embodiment, Z is an antibody having one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15) sites. See, e.g., U.S. Patent No. 9,676,871 and U.S. Patent Application Publication No. 2003 / 0138785.

[0362] In certain embodiments, the antibody is aglycosylated. In some embodiments, the antibody is glycosylated.

[0363] In certain embodiments, the ABD 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), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass. In some embodiments, the ABD has a molecular weight of at least 500, 600, 700, 800, 900, 1000, 10,000, 50,000, or 100,000 Daltons.

[0364] In other embodiments, the antigen binding domains useful in the ADCs provided herein include antibodies, antigen binding fragments of antibodies, peptides that specifically interact with a specific antigen (e.g., peptibodies), receptor molecules that specifically interact with a specific antigen, proteins comprising a ligand binding portion of a receptor that specifically binds a specific antigen, antigen binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, triangular tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc. (see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22: 849-857 and references cited therein)), and aptamers or portions thereof. In some embodiments, the ABD comprises a scFv that has binding specificity for a target antigen.

[0365] Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used herein, an antigen binding domain includes a polypeptide that binds to a target antigen or portion thereof with 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. D , as measured in a surface plasmon resonance assay.

[0366] In certain embodiments, the framework regions (FRs) of the antibodies or antigen-binding fragments thereof used in the ADCs provided herein may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0367] 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. In general, 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, for example, 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 can also be used to identify CDR sequences within antibodies.

[0368] The antigen binding domains for ADC provided herein can comprise the antigen binding fragment of a complete antibody molecule or be composed of the antigen binding fragment of a complete antibody molecule. The antigen binding fragment of an antibody can be derived from a complete antibody molecule using, for example, any suitable standard technique (such as proteolytic digestion or recombinant genetic engineering technology), which involves operating and expressing the DNA encoding the antibody variable domains and optional constant domains. Such DNA is known and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or using molecular biology techniques, for example, one or more variable domains and / or constant domains are arranged into a suitable configuration, or codons are introduced, cysteine ​​residues are produced, modifications are made, additions or deletions of amino acids, etc.

[0369] Non-limiting examples of antigen-binding fragments for 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 that mimic the hypervariable region of an antibody (e.g., isolated complementary 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.

[0370] In certain embodiments, an antigen-binding fragment of an antibody will comprise at least one variable domain. A variable domain can be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in the same frame as one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and comprise V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a monomer V H or V L domain.

[0371] In certain embodiments, the antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the 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-CH 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ;(viii)V L -C H 1; (ix) V L -C H 2;(x)V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant domains, including any of the exemplary configurations described above, the variable and constant domains may be directly connected to each other, or may be connected by a complete or partial hinge region or connecting region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In a further embodiment, the antigen-binding fragment may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations described above, which are bound to each other and / or to one or more monomeric V H or V L The domains are non-covalently linked (eg, by disulfide bonds).

[0372] In another embodiment, the antigen binding domain used in the ADC provided herein may comprise or consist of a human antibody and / or a recombinant human antibody or an antigen binding fragment thereof.

[0373] In another embodiment, the antigen binding domain used in the ADC provided herein may comprise or consist of a recombinant human antibody or its antigen binding fragment. 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 are subjected to in vitro mutagenesis (or, when transgenic animals of human immunoglobulin sequences are used, in vivo somatic mutagenesis is performed), so that the V H and VL Although the amino acid sequence of the region is derived from human germline V H and V L sequences that are related to those of the human antibody germline but may not naturally occur in the human antibody germline repertoire in vivo.

[0374] In another embodiment, the antigen binding domains used in the ADC provided herein also include bispecific antigen binding molecules, such as bispecific antibodies. The method for preparing bispecific antibodies is known in the art and can be used to construct bispecific antigen binding molecules used herein. The exemplary bispecific forms that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or double antibody bispecific forms, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadroma (Quadroma), knobs-into-holes, shared light chains (for example, shared light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG and Mab 2Bispecific formats (for a review of the above formats, see, for example, Klein et al. 2012, mAbs 4: 6, 1-11 and references cited therein). See also, for example, US 2018 / 0134794, which discloses bispecific antigen binding molecules. In short, 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"). The bispecific antigen binding molecule simultaneously binds to two independent epitopes, thereby achieving effective ligand blocking while minimizing the activation of target signaling. In certain embodiments, the D1 and D2 domains of the bispecific antibody do not compete with each other. The non-competitiveness between D1 and D2 means that the monospecific antigen binding proteins from which D1 and D2 are each derived do not compete with each other for binding to the target. Exemplary antigen binding protein competition assays are known in the art. In certain embodiments, D1 and D2 bind to different (e.g., non-overlapping or partially overlapping) epitopes on the target. Bispecific antigen binding molecules can be constructed using the antigen binding domains of two independent monospecific antibodies. For example, monoclonal monospecific antibody sets can be prepared using standard methods known in the art. Each antibody thus produced can be tested in pairs to detect its cross-competitiveness with the target protein. If two different antibodies can bind to the target simultaneously (i.e., not competing with each other), the antigen-binding domains from the first antibody and the antigen-binding domains from the second non-competitive antibody can be engineered into a single bispecific antibody. Bispecific antigen binding molecules can be single multifunctional polypeptides, or can be multimeric complexes of two or more polypeptides, which are interconnected in a covalent or non-covalent manner. Any antigen-binding construct that can simultaneously bind to two independent, non-identical epitopes of the target molecule is considered as a bispecific antigen binding molecule. Bispecific antigen binding molecules or their variants can be constructed using standard molecular biology techniques (such as recombinant DNA and protein expression technology) known to those skilled in the art. In another embodiment, bispecific antibodies are also provided, wherein one arm of the bispecific antibody binds to an epitope on the first target protein, and the other arm of the bispecific antibody binds to a second epitope on the second target protein.Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab2 bispecific formats (for a review of the above formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. (Epub: Dec. 4, 2012)).

[0375] In another embodiment, the antigen binding domain used in the ADC provided herein also includes an antibody comprising any HCVR, LCVR and / or CDR amino acid sequence variant known in the art. In some embodiments, the variant includes a variant of any HCVR, LCVR and / or CDR amino acid sequence known in the art, and these variants have one or more conservative substitutions. For example, the antigen binding domain includes an antibody or antigen binding fragment thereof having a HCVR, LCVR and / or CDR amino acid sequence, and these sequences have, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any HCVR, LCVR and / or CDR amino acid sequence known in the art. In another embodiment, the antigen binding domain includes an antibody or antigen binding fragment thereof, and also includes a variant having substantial sequence identity with any HCVR, LCVR and / or CDR amino acid sequence known in the art. In certain embodiments, the non-identical residue positions differ due to conservative amino acid substitutions.

[0376] The sequence identity between two different amino acid sequences is usually measured using sequence analysis software. Sequence analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software includes programs such as GAP and BESTFIT, which can use default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different biological species), or the sequence homology or sequence identity between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. The FASTA program in GCG version 6.1 can also be used to compare polypeptide sequences using default or recommended parameters. FASTA (such as FASTA2 and FASTA3) provides alignment results and sequence identity percentages (Pearson (2000) supra) of the best overlapping region between the query sequence and the search sequence. When the sequence provided herein is compared with a database comprising a large number of sequences from different organisms, another algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, eg, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0377] The antigen-binding domains used in the ADCs provided herein encompass proteins whose amino acid sequences differ from those of the antibodies but still retain the ability to bind to the target protein. Such variant antigen-binding domains comprise one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit substantially the same biological activity as the antibody.

[0378] For example, if two antigen-binding domains are pharmaceutical equivalents or drug substitutes and do not differ significantly in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions (single or multiple doses), they would be considered bioequivalent. If some antigen-binding domains are absorbed equivalently to the extent but not the rate, they would still be considered bioequivalent because the difference in absorption rate is intentional and reflected in the labeling, is not critical to achieving effective in vivo drug concentrations (e.g., long-term use), and is not considered medically insignificant for the specific drug product under investigation.

[0379] In some embodiments, two antigen binding domains are bioequivalent if they do not have clinically meaningful differences in safety, purity, and potency.

[0380] In some embodiments, two antigen-binding domains are bioequivalent if a patient can switch one or more times between the reference product and the biologic without expected increased risk of adverse reactions, including clinically significant changes in immunogenicity, or decreased effectiveness, compared to continued treatment without such switching.

[0381] In some embodiments, if two antigen binding domains both function by a common mechanism or mechanisms of action for the conditions of use, then they are bioequivalent to the extent such mechanisms are known.

[0382] Bioequivalence can be demonstrated by both in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals, in which the concentration of the antigen-binding domain or its metabolites is measured in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro tests that have been correlated with and are reasonably predictive of in vivo human bioavailability data; (c) in vivo tests in humans or other mammals, in which the appropriate acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) rigorously controlled clinical trials to determine the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding domain.

[0383] Bioequivalent variants of the antigen binding domains of the ADCs provided herein can be constructed by, for example, performing various residue or sequence substitutions, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not necessary for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or erroneous intramolecular disulfide bonds after renaturation. In other cases, bioequivalent antigen binding domains can include variants that include amino acid changes that alter the glycosylation characteristics of the antigen binding domain (e.g., mutations that eliminate or remove glycosylation).

[0384] In certain embodiments, the antigen-binding domains for the ADC provided herein are combined with human target protein, but are not combined with the target protein of other species. In other embodiments, the antigen-binding domains for the ADC provided herein are combined with human target protein and target protein from one or more non-human species. For example, the antigen-binding domains for the ADC provided herein can be combined with human target protein, and can be combined or not combined with one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee target protein according to the circumstances. In some embodiments, the antigen-binding domains specifically bind to human target protein and cynomolgus monkey (for example, cynomolgus monkey (Macaca fascicularis)) target protein. In other embodiments, the antigen-binding domains used herein bind to human target protein, but do not bind or only weakly bind to cynomolgus monkey target protein.

[0385] 1.ABD sequence

[0386] In some embodiments, the ABD comprises the heavy and light chains of an antibody.

[0387] In some embodiments, the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0388] 2. Linking site

[0389] The ABD can be linked to the TLR7 agonist directly or indirectly through a linker via the linkage of specific amino acids within the ABD. Exemplary amino acid linkages that can be used in the context of the disclosed embodiments 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 2011 / 130598; US 2013 / 0101546; and US 2012 / 0585592). 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 2013 / 068874, and WO 2012 / 166559), and acidic amino acids (see, e.g., WO 2012 / 05982). The linker can also be conjugated to the ABD via attachment to a carbohydrate (see, for example, 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).

[0390] In some instances, 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 certain embodiments, the ABD is bound to the linker via a cysteine ​​residue. In certain embodiments, the linker maleimide portion is bound to the antibody cysteine ​​residue. In certain embodiments, the ABD is bound to the linker via a lysine residue. In certain embodiments, the linker N-hydroxysuccinimide portion is bound to the antibody lysine residue to form an amide bond.

[0391] In certain embodiments, the ABD is bound to a 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 comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues. In certain embodiments, antibodies or antigen-binding molecules are engineered by mutation (e.g., insertion or deletion) to facilitate the reaction by transglutaminase. In certain embodiments, antibodies or antigen-binding molecules are engineered to remove one or more glycosylation sites. In certain embodiments, antibodies or antigen-binding molecules are engineered to add one or more glutamine residues. In certain embodiments, glutamine residues are added to a TGase recognition tag, as described herein. The technology of transforming glutamine residues into antibody polypeptide chains (glutamine-modified antibodies or antigen-binding molecules) is within the skill of those skilled in the art. In certain embodiments, the antibody is non-glycosylated.

[0392] 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 heavy chain polypeptide containing a Gln295 or Q295 residue. In further embodiments, the ABD comprises one or more glutamine residues at positions other than position 295 of the heavy chain. Antibodies described herein carrying the N297Q mutation described herein are included in this section. In certain embodiments, a glutamine residue is added at the C-terminus of the heavy chain.

[0393] In certain embodiments, glutamine is a polypeptide that has been modified to have a glutamine-containing tag (e.g., a glutamine-containing peptide tag, a Q tag, or a TGase recognition tag). The term "TGase recognition tag" or "Q tag" refers to an amino acid sequence containing a glutamine residue that, when incorporated into (e.g., attached to) a polypeptide sequence under appropriate conditions, is recognized by transglutaminase ("TGase") and, through the reaction between the amino acid side chains in the amino acid sequence and the reactive groups, results in cross-linking by TGase. The recognition tag can be a non-naturally occurring peptide sequence in the polypeptide. In certain embodiments, the TGase recognition tag comprises at least one glutamine. In certain embodiments, the TGase recognition tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gin, Hexyl, Met, Pro, Thr, Lys, or Trp, or unconventional amino acids). In certain embodiments, the TGase recognition tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 2), LLQG (SEQ ID NO: 1), LSLSQG (SEQ ID NO: 17), GGGLLQGG (SEQ ID NO: 18), GLLQG (SEQ ID NO: 19), LLQ,GSPLAQSHGG (SEQ ID NO: 20), GLLQGGG (SEQ ID NO: 21), GLLQGG (SEQ ID NO: 22), GLLQ (SEQ ID NO: 23), LLQLLQGA (SEQ ID NO: 3), LLQGA (SEQ ID NO: 5), LLQYQGA (SEQ ID NO: 12), LLQGSG (SEQ ID NO: 6), LLQYQG (SEQ ID NO: 4), LLQLLQG (SEQ ID NO: 3), SLLQG (SEQ ID NO: 7), LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), and LLQGR (SEQ ID NO: 11). NO: 11). See, for example, WO2012059882, the entire contents of which are incorporated herein.

[0394] In some embodiments, 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, 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 is a pentapeptide sequence LLQGA (SEQ ID NO:5). In some embodiments, 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, 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 is a pentapeptide sequence LLQGA (SEQ ID NO:5).

[0395] ABD can also be modified at one or more glutamine residues by 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 the antibody can be coupled to a primary amine compound to provide a portion capable of reacting with a reactive group on a linker-payload. In certain embodiments, the primary amine compound provides a diene or a dienophile. In certain embodiments, the primary amine compound provides a diene or a dienophile, and the linker-payload provides a complementary dienophile or diene, respectively, for conjugation via a Diels-Alder reaction. In certain embodiments, the primary amine compound provides an azido group. In certain embodiments, the primary amine compound provides an azido group, and the linker-payload provides a complementary alkyne for conjugation via a click reaction.

[0396] In some embodiments, the ABD comprises a heavy chain, and the heavy chain is directly or indirectly connected to the ABD via a linker. In some embodiments, the ABD comprises a light chain, and the light chain is directly or indirectly connected to the ABD via a linker.

[0397] In some embodiments, ABD comprises a heavy chain, and the C-terminus of the heavy chain is directly or indirectly connected to ABD via a linker. In some embodiments, ABD comprises a light chain, and the C-terminus of the light chain is directly or indirectly connected to ABD via a linker.

[0398] In some embodiments, the ABD comprises two heavy chains, and each of the two heavy chains is directly or indirectly connected to the ABD via a linker. In some embodiments, the ABD comprises two light chains, and each of the two light chains is directly or indirectly connected to the ABD via a linker.

[0399] In some embodiments, the ABD comprises two heavy chains, and the C-terminus of each of the two heavy chains is directly or indirectly connected to the ABD via a linker. In some embodiments, the ABD comprises two light chains, and the C-terminus of each of the two light chains is directly or indirectly connected to the ABD via a linker.

[0400] 3. Epitope mapping and related technologies

[0401] The epitope bound by the antigen binding domain can be composed of a single continuous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the target protein. Alternatively, the relevant epitope can be composed 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.

[0402] Various techniques known to those of ordinary skill in the art can be used to determine the epitope that the antigen-binding domains used in the ADC provided herein interact with. Exemplary techniques that can be used to determine the epitope or binding domain of a specific 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. In addition, methods such as epitope excision, epitope extraction, and chemical modification can also be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids that interact with antigen-binding domains in a polypeptide is to detect hydrogen / deuterium exchange by mass spectrometry. In general, the hydrogen / deuterium exchange method includes labeling the target protein with deuterium, and then binding the antigen-binding domain to the deuterium-labeled protein. Next, the protein / antigen binding domain complex is transferred to water to allow hydrogen-deuterium exchange to occur for all residues except those protected by the antigen binding domain (which remain deuterium-labeled). After the antigen binding domain is dissociated, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to specific amino acids that interact with the antigen binding domain. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystal structure analysis can also be used to identify amino acids within a polypeptide that interact with the antigen binding domain.

[0403] 4. Synthesis of ABD

[0404] In one embodiment, the antibodies used in the ADCs provided herein are fully human antibodies. Methods for preparing monoclonal antibodies (including fully human monoclonal antibodies) are known in the art. Any such known methods can be used in the context of the present disclosure to prepare human antibodies that specifically bind to human protein targets.

[0405] For example, using VELOCIMMUNE TM The method of producing fully human monoclonal antibodies by the use of the technology or any other similar known method, first isolates a high-affinity chimeric antibody against a human protein target, which has a human variable region and a mouse constant region. The antibody is then characterized and screened for the desired properties, including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, the mouse constant region is replaced with the desired human constant region, such as wild-type or modified IgG1 or IgG4, to produce a fully human antibody. Although the constant region selected may vary depending on the specific use, the characteristics of high-affinity antigen binding and target specificity lie in the variable region. In some cases, fully human antibodies are isolated directly from antigen-positive B cells.

[0406] Monoclonal antibodies can be generated by any technique familiar to those of ordinary skill in the art. Such methods include, but are not limited to, transforming human peripheral blood cells (e.g., containing B lymphocytes) with Epstein-Barr virus (EBV), immunizing human B cells in vitro, fusing spleen cells from immune transgenic mice carrying inserted human immunoglobulin genes, separating from human immunoglobulin V region phage libraries, or other methods known in the art 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 stimulation. Methods for obtaining fully human antibodies from transgenic mice have been described, for example, by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; US 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 a mouse strain derived from an embryonic stem cell line 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 transposition sites on yeast artificial chromosomes, which undergo B cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissue. By immunizing transgenic mice, fully human monoclonal antibodies can be obtained, and these mice can then produce specific human antibodies against the target antigen. Lymphocytes from immunized transgenic mice can be used to produce hybridomas that secrete human antibodies according to the methods described herein. Polyclonal serum containing fully human antibodies can also be obtained from the blood of immunized animals.

[0407] Another method for producing human antibodies in this disclosure includes immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. This immortalized B cell line (or lymphoblastoid cell line) that produces monoclonal antibodies that specifically bind to the target antigen can be identified by immunoassay methods (e.g., ELISA) provided herein and then separated by standard cloning techniques. The stability of the lymphoblastoid cell line producing antibodies against the target antigen can be improved by fusing the transformed cell line with a mouse myeloma to produce 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 includes eliciting human spleen B cells with the target antigen and then fusing the elicited B cells with a heterologous fusion partner. See, for example, Boerner et al., 1991 J. Immunol. 147:86-95.

[0408] In certain embodiments, B cells that produce 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 (WO92 / 02551; US ​​Patent 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and as described herein. B cells of immunized animals can be isolated from spleen, lymph nodes, or peripheral blood samples by selecting cells that produce antibodies that specifically bind to the target antigen. B cells can also be isolated from humans (e.g., from peripheral blood samples).

[0409] Methods for detecting single B cells that produce antibodies with desired specificity are well known in the art, such as by plaque formation, fluorescence-activated cell sorting, and detection of specific antibodies after in vitro stimulation. Methods for screening B cells that produce specific antibodies include, for example, preparing a single cell suspension of B cells in soft agar containing the target antigen. The specific antibodies produced by the B cells bind to the antigen to form a complex, which may be visualized as an immunoprecipitate.

[0410] Methods for obtaining the antibodies of the present disclosure can also employ various phage display technologies known in the art. See, for example, Winter et al., 1994 Annu. Rev. Immunol. 12: 433-55; Burton et al., 1994 Adv. Immunol. 57: 191-280. Combinatorial libraries of human or murine immunoglobulin variable region genes can be created in phage vectors and screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to the target antigen or variants or fragments thereof. See, e.g., U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. For example, a library comprising a plurality of polynucleotide sequences encoding immunoglobulin variable region fragments can be inserted into the genome of a filamentous phage (e.g., M13 or a variant thereof) and in frame with a sequence encoding a phage coat protein. The fusion protein can be a fusion of a coat protein with a light chain variable region domain and / or a heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments can also be displayed on phage particles (see, e.g., U.S. Patent No. 5,698,426).

[0411] Antibody fragments fused to other proteins (e.g., minor coat proteins) can also be used to enrich phage antigens. Rearranged heavy chains (V chains) from mice immunized against the target antigen (e.g., HBVsAg, tumor-specific antigens) are then used to enrich phage antigens. H ) and light chain (V L ) random combinatorial libraries, displaying different antibody fragment libraries on the surface of phage. These libraries can be screened for complementary variable domains and purified, for example, using affinity columns. See Clackson et al., Nature, Vol. 352, pp. 624-628 (1991).

[0412] Heavy and light chain immunoglobulin cDNA expression libraries can also be prepared in lambda phage, for example using lambdalmmunoZapTM (H) and λImmunoZap TM (L) vector (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B cell population and used to create heavy and light chain immunoglobulin cDNA expression libraries in the λImmunoZap(H) and λImmunoZap(L) vectors. These vectors can be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can then be converted to non-lytic plasmids, which allow high-level expression of monoclonal antibody fragments from E. coli.

[0413] In some embodiments, nucleotide primers are used to amplify the variable region of the gene expressing the monoclonal antibody of interest in a hybridoma. These primers can be synthesized by one of ordinary skill in the art or purchased from commercial sources. (For example, see Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including primers for V Ha 、V Hb 、V Hc 、V Hd 、C H1 、V L and C L These primers can be used to amplify the heavy or light chain variable regions, which can then be inserted into vectors such as ImmunoZAP. TM H or ImmunoZAP TM L (Stratagene). These vectors can then be introduced into E. coli, yeast, or mammalian expression systems. These methods can be used to produce large quantities of V-containing H and V L A single-chain protein that is a fusion of the two domains (see Bird et al., Science 242: 423-426, 1988).

[0414] Once cells producing antibodies of the present disclosure have been obtained using any of the above-described immunization and other techniques, specific antibody genes can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures described herein. The antibodies thus produced can be sequenced, the CDRs identified, and the DNA encoding the CDRs can be manipulated as previously described to produce other antibodies of the present disclosure.

[0415] The binding agents disclosed herein preferably modulate the activity of the target antigen in the cell-based assays described herein and / or in the in vivo assays described herein, and / or bind to one or more of the domains described herein, and / or cross-block the binding of one of the antibodies described herein, and / or are cross-blocked from binding to the target antigen by one of the antibodies described herein. Thus, such binding agents can be identified using the assays described herein.

[0416] In certain embodiments, antibodies are generated by first identifying an antibody that binds to one or more domains provided herein and / or neutralizes and / or cross-blocks an antibody described herein and / or is cross-blocked from binding to a target antigen by one of the antibodies described herein in a cell-based and / or in vivo assay as described herein. The CDR regions of these antibodies are then used to insert into a suitable biocompatible framework to generate a binding agent directed against the target antigen. The non-CDR portion of the binding agent may be composed of amino acids or may be a non-protein molecule. The assay methods described herein can be used to characterize the binding agent. Preferably, the binding agent of the present disclosure is an antibody as defined herein.

[0417] Other antibodies according to the present disclosure can be obtained by conventional immunization and cell fusion procedures described herein and known in the art.

[0418] Molecular evolution of the complementary determining region (CDR) at the center of the antibody binding site has also been used to separate antibodies with enhanced affinity, for example, antibodies with enhanced affinity for c-erbB-2, as described in Schier et al., 1996, J. Mol. Biol. 263: 551. Therefore, such techniques can be used to prepare antibodies against target antigens. Antigen-binding proteins against target antigens can be used, for example, for in vitro or in vivo testing of the presence of target antigens. Antigen-binding proteins can also be used to purify target antigens by immunoaffinity chromatography.

[0419] Although human antibodies, partially human antibodies or humanized antibodies are suitable for a variety of applications, especially those involving the application of antibodies to human subjects, other types of antigen-binding proteins are suitable for certain applications. The non-human antibodies of the present disclosure can, for example, be derived from any animal producing antibodies, such as mice, rats, rabbits, goats, donkeys or non-human primates (such as monkeys (such as cynomolgus monkeys or rhesus monkeys) or apes (such as chimpanzees)). Antibodies from specific species can be produced in the following ways: for example, using a desired immunogen (such as HBV sAg, tumor-specific antigens) or using an artificial system for producing antibodies of the species (such as a system based on bacteria or phage display for producing antibodies of specific species), or converting antibodies from one species into antibodies from another species, such as by replacing the constant region of an antibody with a constant region from other species, or replacing one or more amino acid residues of an antibody so that it is more similar to the sequence of antibodies from other species. In some embodiments, the antibody is a chimeric antibody comprising an amino acid sequence derived from an antibody from two or more different species.

[0420] Antigen binding proteins can be prepared and screened for desired properties by any of a variety of conventional techniques. Certain techniques involve separating nucleic acids encoding polypeptide chains (or portions thereof) of target antigen binding proteins (e.g., anti-HBV sAg antibodies, tumor-specific antigens), and manipulating the nucleic acids by recombinant DNA techniques. The nucleic acids can be fused to another target nucleic acid, or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or replace one or more amino acid residues. In addition, antigen binding proteins can be purified from cells that naturally express them (e.g., antibodies can be purified from hybridomas that produce antibodies), or produced in a recombinant expression system using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0421] Any expression system known in the art can be used to prepare the recombinant polypeptides of the present disclosure. Expression systems have been described in detail above. Typically, host cells are transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Useful host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23: 175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70), as described by McMahan et al., 1991, EMBO J. 10: 2821. Cloning and expression vectors suitable for bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0422] It should be understood that the antibodies of the present disclosure may have at least one amino acid substitution, provided that the antibody retains binding specificity. Therefore, modifications to the antibody structure are also encompassed within the scope of the present disclosure. These modifications may include amino acid substitutions, which may be conservative or non-conservative and may not destroy the target binding ability of the antibody. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptide mimetics and other reverse or inverted amino acid moieties. Conservative amino acid substitutions may also involve replacing natural amino acid residues with conventional residues so that there is little or no effect on the polarity or charge of the amino acid residue at that position.

[0423] Non-conservative substitutions may involve replacing a member of one class of amino acids or amino acid mimetics with a member of another class having different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues can be introduced into regions of human antibodies that are homologous to non-human antibodies, or into non-homologous regions of the molecule.

[0424] In addition, those skilled in the art can generate test variants comprising a single amino acid substitution 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 found that a change in a particular amino acid residue results in a destroyed activity, an undesirable decrease in activity, or an inappropriate activity, variants with such changes can be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art can easily determine which amino acids should be avoided for further substitution, either alone or in combination with other mutations.

[0425] Those skilled in the art will be able to identify suitable variants of the polypeptides described herein using well-known techniques. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not believed to be important for activity. In certain embodiments, residues and portions of the molecule that are conserved among similar polypeptides can be identified. In certain embodiments, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without disrupting biological activity or adversely affecting the structure of the polypeptide.

[0426] In addition, one skilled in the art can consult structure-function studies to identify residues in similar polypeptides that are important for activity or structure. Based on such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues in similar proteins that are important for activity or structure. One skilled in the art can select chemically similar amino acids to replace these predicted important amino acid residues.

[0427] Those skilled in the art can also analyze the three-dimensional structure of similar polypeptides and the amino acid sequences associated with that structure. Given this information, one can predict how the amino acid residues of an antibody will be arranged relative to its three-dimensional structure. In certain embodiments, one can choose not to make radical changes to amino acid residues predicted to be located on the surface of the protein, as these residues may be involved in important interactions with other molecules.

[0428] In certain 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 certain embodiments, the variant contains a greater or lesser number of N-linked glycosylation sites than the native protein. N-linked glycosylation sites are characterized by the following sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substituting amino acid residues to create this sequence provides a potential new site for adding an N-linked carbohydrate chain. Alternatively, substitutions that 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 (typically naturally occurring sites) are eliminated and one or more new N-linked sites are created. Other preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or replaced with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when the antibody must be refolded into a biologically active conformation, for example, after the separation of insoluble inclusion bodies. Cysteine ​​variants typically have fewer cysteine ​​residues than the native protein and often have an even number of cysteine ​​residues to minimize interactions caused by unpaired cysteines.

[0429] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one skilled in the art when such substitutions are desired. According to certain embodiments, preferred amino acid substitutions are those that produce the following effects: (1) reduced susceptibility to proteolysis; (2) reduced susceptibility to oxidation; (3) altered binding affinity for forming protein complexes; (4) altered binding affinity; and / or (4) conferring or modifying other physicochemical or functional properties of such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain that forms intermolecular contacts). In certain embodiments, conservative amino acid substitutions generally do not significantly alter the structural characteristics of the parent sequence (e.g., the replaced amino acid should not tend to disrupt helices present in the parent sequence, or disrupt other types of secondary structures characteristic of the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH 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.

[0430] In certain embodiments, the antibodies of the present disclosure may be chemically bonded to a polymer, lipid, or other moiety.

[0431] The binding agent may comprise at least one CDR as 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, framework or scaffold, capable of displaying one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that bind to an antigen in a local surface area. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or may have one or more modifications relative to naturally occurring polypeptides or folds, such as additions, deletions, or substitutions of amino acids. These scaffolds may be derived from polypeptides of any species (or species), such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0432] Typically, biocompatible framework structures are based on protein scaffolds or backbones other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarcinomatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domains, and tendamistat domains can be used (see, e.g., Nygren and Uhlen, 1997, Curr. Opin. in Struct. Biol., 7, 463-469).

[0433] 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).

[0434] In addition, those skilled in the art will recognize that suitable binding agents include portions of these antibodies: LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and / or HCDR3. The non-CDR portion of an antibody can be a non-protein molecule, wherein the binding agent cross-blocks the binding of an antibody disclosed herein to a target antigen. The non-CDR portion of an antibody can be a non-protein molecule, wherein the antibody exhibits a similar binding pattern to the target antigen as at least one antibody disclosed herein in a competition binding assay. The non-CDR portion of an antibody can be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to a target antigen and / or neutralizes the target antigen. The non-CDR portion of an antibody can be composed of amino acids, wherein the antibody is a recombinant antibody, and the recombinant antibody exhibits a similar binding pattern to the target antigen as at least one antibody disclosed herein in a target epitope competition binding assay (as described below), and / or neutralizes the target antigen.

[0435] When the antibody comprises one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as described above, it can be obtained by expressing from a host cell containing DNA encoding these sequences. The DNA encoding each CDR sequence can be determined based on the amino acid sequence of the CDR, and appropriately synthesized together with any desired antibody variable region framework and constant region DNA sequence using oligonucleotide synthesis technology, site-directed mutagenesis and polymerase chain reaction (PCR) technology. Those skilled in the art can obtain the antibody by expressing from a host cell containing DNA encoding these sequences. DNA encoding variable region framework and constant region is widely available in gene sequence databases. In some embodiments, the heavy chain and light chain of the antibody are expressed by a single DNA construct. In some embodiments, the heavy chain and light chain of the antibody are expressed by two or more separate DNA constructs.

[0436] Once the DNA encoding the disclosed antibodies or fragments thereof is synthesized, it can be amplified and expressed using any number of known expression vectors according to any of a variety of well-known nucleic acid excision, ligation, transformation, and transfection procedures. Thus, in certain embodiments, expression of the antibody fragment may be preferably in a prokaryotic host, such as E. coli (see, for example, Pluckthun et al., 1989 Methods Enzymol. 178: 497-515). In certain other embodiments, expression of the antibody or fragment thereof may be preferably in a eukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells), or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma (e.g., mouse NSO line), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells.

[0437] One or more replicable expression vectors containing DNA encoding the variable and / or constant regions of the antibody can be prepared and used to transform a suitable cell line, such as a non-producing myeloma cell line, such as the mouse NSO line, or bacteria, such as E. coli, in which the antibody will be produced. In order to obtain 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, 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)) describes basic molecular biology procedures. DNA sequencing can be performed as described in Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International plc sequencing manual, and site-directed mutagenesis can be performed 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). In addition, numerous publications describe techniques suitable for preparing antibodies by manipulating DNA, creating expression vectors, and transforming and culturing appropriate cells (Mountain A and Adair, JR in Biotechnology and Genetic Engineering Reviews (ed. Tombs, MP, 10, Chapter 1, 1992, Intercept, Andover, UK); "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed.), Wiley Interscience, New York).

[0438] When it is desired to improve the affinity of an antibody of the present disclosure containing one or more of the above-mentioned CDRs, this can be achieved through a variety of affinity maturation protocols, including maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), using E. coli mutant strains (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7-88, 1996), and sexual PCR (Crameri, et al., Nature, 391, 288-291, 1998). All of these affinity maturation methods are discussed in Vaughan et al. (Nature Biotech., 16, 535-539, 1998).

[0439] Those skilled in the art will appreciate that certain proteins (e.g., antibodies) may undergo a variety of post-translational modifications. The type and extent of these modifications generally depend on the host cell line and culture conditions used to express the protein. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the deletion of carboxy-terminal basic residues (e.g., lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ Journal of Chromatography 705: 129-134, 1995).

[0440] 5. Bioequivalence

[0441] The antigen binding domains used in the ADCs provided herein encompass proteins whose amino acid sequences differ from those of the antibodies but still retain the ability to bind to the target protein. Such variant antigen binding domains comprise one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit substantially the same biological activity as the antibody.

[0442] For example, if two antigen-binding domains are pharmaceutical equivalents or drug substitutes and do not differ significantly in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions (single or multiple doses), they would be considered bioequivalent. If some antigen-binding domains are absorbed equivalently to the extent but not the rate, they would still be considered bioequivalent because the difference in absorption rate is intentional and reflected in the labeling, is not critical to achieving effective in vivo drug concentrations (e.g., long-term use), and is not considered medically insignificant for the specific drug product under investigation.

[0443] In one embodiment, two antigen binding domains are bioequivalent if they do not have clinically meaningful differences in safety, purity, and potency.

[0444] In one embodiment, two antigen-binding domains are bioequivalent if a patient can switch one or more times between the reference product and the biologic without expected increased risk of adverse reactions, including clinically significant changes in immunogenicity, or reduced effectiveness, compared to continued treatment without such switching.

[0445] In one embodiment, if two antigen binding domains both function by a common mechanism or mechanisms of action for the conditions of use, then they are bioequivalent to the extent such mechanisms are known.

[0446] Bioequivalence can be demonstrated by both in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals, in which the concentration of the antigen-binding domain or its metabolites is measured in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro tests that have been correlated with and are reasonably predictive of in vivo human bioavailability data; (c) in vivo tests in humans or other mammals, in which the appropriate acute pharmacological effect of the antigen-binding domain (or its target) is measured as a function of time; and (d) rigorously controlled clinical trials to determine the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding domain.

[0447] Bioequivalent variants of the antigen binding domains of the ADCs provided herein can be constructed by, for example, performing various residue or sequence substitutions, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not necessary for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or erroneous intramolecular disulfide bonds after renaturation. In other cases, bioequivalent antigen binding domains can include variants that include amino acid changes that alter the glycosylation characteristics of the antigen binding domain (e.g., mutations that eliminate or remove glycosylation).

[0448] 6. Species Selectivity and Species Cross-Reactivity

[0449] In certain embodiments, the antigen-binding domains for the ADC provided herein are combined with human target protein, but are not combined with the target protein of other species. In other embodiments, the antigen-binding domains for the ADC provided herein are combined with human target protein and target protein from one or more non-human species. For example, the antigen-binding domains for the ADC provided herein can be combined with human target protein, and can be combined or not combined with one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee target protein according to the circumstances. In one embodiment, the antigen-binding domains specifically bind to human target protein and cynomolgus monkey (for example, cynomolgus monkey (Macaca fascicularis)) target protein. In other embodiments, the antigen-binding domains used herein bind to human target protein, but are not combined or only weakly combined with cynomolgus monkey target protein.

[0450] 7. Exemplary Antibodies and Antigen Targets

[0451] The antigen binding domain (ABD) used in the ADC provided herein can have binding specificity for any antigen (target protein) deemed suitable by those skilled in the art. In certain embodiments, the antigen is a transmembrane molecule (eg, a receptor) or a surface protein.

[0452] ABD targeting hepatitis B virus (HBV) antigens

[0453] Chronic hepatitis B infection is typically associated with an increase in circulating HBV DNA and HBV surface antigen (HBV sAg) in the serum. A sustained decrease in circulating HBV sAg and HBV DNA is a sign of successful control of infection or "functional cure." Current treatment with nucleoside analogs can reduce HBV load in plasma, but is rarely accompanied by a decrease in HBV sAg. Therefore, nucleoside therapy needs to be taken lifelong to prevent viral rebound. Some embodiments of the present disclosure relate to ADCs targeting HBV antigens. The ADC can be used to treat hepatitis B. In some embodiments, the ADC comprises a specific ABD for HBV sAg, where HBV sAg can refer to non-infectious HBV sAg particles, infectious HBV virions, or cells expressing HBV sAg.

[0454] In some embodiments, the ABD comprises the heavy and light chains of an antibody specific for HBV sAg. In some embodiments, the ABD comprises the 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 under catalog number HBS-872.

[0455] ABD targeting tumor antigens

[0456] Some embodiments of the present disclosure relate to ADCs targeting tumor antigens. The ADCs can be used to treat cancer. In some embodiments, the ADCs comprise an ABD specific for a tumor antigen.

[0457] 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 for a certain type of tumor or an antigen that is shared, overexpressed, or modified on a particular type of tumor. In one embodiment, the antigen is expressed on a solid tumor. Exemplary antigens include, but are not limited to, 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 receptor, EphB receptor, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD 14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, or an antibody that binds to one or more tumor-associated antigens or cell surface receptors disclosed in U.S. Publication No. 2008 / 0171040 or U.S. Publication No. 2008 / 0305044; erythropoietin; osteoinductive 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, EGF RvIII, 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, L GR5, LMP2, MAGE protein, MART-1, mesothelin, ML-IAP, Muc1, Muc16, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, N Y-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE 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 scavenger receptors of class A, 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). 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. ;

[0458] Antibody scaffold

[0459] In certain embodiments, the antibody comprises a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In this 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 many antibody wild-type sequences. In other embodiments, the antibody can be modified to comprise a glutamine residue. In certain embodiments, the antibody comprises one or more N297Q mutations. Techniques for modifying antibody sequences to comprise glutamine residues are within the skill of those skilled in the art (see, for example, Ausubel et al. Current Protoc. Mol. Biol. (John Wiley & Sons)).

[0460] In embodiments where the antibody contains a Q295 residue, an N297Q mutation, or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15) sites, the payload of Formula I, wherein R 3is -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y; wherein Y is -NR 5 R 6 ; and R 5 and R 6 Each is H and can be directly conjugated to an antibody to form an ADC of Formula V:

[0461]

[0462] or a pharmaceutically acceptable salt thereof, wherein:

[0463] R 1 、R 2 and X is as defined elsewhere for Formula I;

[0464] R 10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-;

[0465] The ABD is an antibody containing a Q295 residue, an N297Q mutation, and / or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG, LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15) sites; and

[0466] k is an integer from 1 to 30.

[0467] In another embodiment, R 10 In another embodiment, R 10 In another embodiment, R 10 In another embodiment, R 10is -heteroalkylene-arylene-NH-. In another embodiment, R 10 In another embodiment, R 10 In another embodiment, R 10 It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl) or -CH2NH-.

[0468] In another embodiment, the ADC provided herein has the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, or ABD-P34, wherein ABD is linked to R 3 The payload is placed on the amino group of the TLR7 receptor agonist.

[0469] In another embodiment, the 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:

[0470]

[0471] or a pharmaceutically acceptable salt thereof, wherein:

[0472] R 1 、R 2 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 , L 1 , ABD and x are as defined elsewhere for Formula III; and

[0473] k is an integer from 1 to 30.

[0474] 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, of which ABD-L 1 ligated to a payload (ie, a TLR7 agonist).

[0475] VII. Synthesis of ADC

[0476] Also provided herein is a method 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.

[0477] In some embodiments, the method involves partially reducing the antigen-binding domain with tris (2-carboxyethyl) phosphine (TCEP), and then reacting the reduced cysteine ​​residues with a maleimide-functionalized linker-payload (i.e., a TLR7 agonist). In some embodiments, the antigen-binding domain is partially reduced by adding 1.5-3.0 times a molar excess of TCEP in PBS pH 7.4 and 2mM ethylenediaminetetraacetic acid (EDTA) and reacting for 2 hours at 37°C. The reduced antigen-binding domain can be buffer-exchanged into PBS containing 1% w / v polysorbate 20. The linker-payload can be added at a molar ratio of 5-10 linker-payload / antigen-binding domain and reacted for another 2 hours at 25°C in the presence of 12% v / v dimethyl sulfoxide (DMSO). The mixture can be purified, for example, by size exclusion chromatography (SEC) (AKTA pure, Superdex 200 Increase) to provide an ADC provided herein.

[0478] Alternatively, for the ADC of Formula V, in some embodiments, the reaction of the ABD with the payload (i.e., a TLR7 agonist) is mediated by transglutaminase. In another embodiment, the transglutaminase is a murine transglutaminase. In another embodiment, when the Gln of the ABD is Q295 of the N297 antibody, the ABD is reacted with PNGase (such as, but not limited to, PNGaseF) to deglycosylate N297 before reacting with the payload.

[0479] Chemical site-selective protein modification is becoming increasingly common for antibody-based bioconjugates. Among all the bioorthogonal reactions developed to date, the [4+2] cycloaddition of 1,2,4,5-tetrazine (s-tetrazine, Tz) with various dienophiles, known as the inverse electron demand Diels-Alder (IEDDA) reaction, is the reaction that meets most of the bioorthogonal criteria required for conjugation, such as rapidity, selectivity, biocompatibility, and catalyst-free. In this study, the tetrazine linker was designed to have two functions: (1) the tetrazine linker acts as a handle with an additional chemical moiety (e.g., amine) that can be linked to the antibody, and the tetrazine moiety can react with the linker-payload to generate ADC; (2) the tetrazine linker acts as a linker to the linker-payload, which can be connected to the antibody-handle (Titas Deb, et al., Chem. Rev. 2021, 121, 12, 6850–6914; Astrid-Caroline Knall and Christian Slugovc. Chem. Soc. Rev., 2013, 42, 5131).

[0480] In one embodiment, the ADC provided herein is selected from those in Table 3:

[0481]

[0482]

[0483]

[0484] VIII. Pharmaceutical Compositions

[0485] In one aspect, the present disclosure provides a pharmaceutical composition comprising an ADC as 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 an ADC and HBV sAg.

[0486] The TLR7 agonist or ADC can be formulated into a suitable pharmaceutical formulation. Typically, the above-mentioned TLR7 agonist or ADC is formulated into a pharmaceutical composition using techniques and procedures well known in the art (eg, see Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).

[0487] In the composition, an effective concentration of one or more TLR7 agonists or ADCs or pharmaceutically acceptable salts thereof is mixed with a suitable pharmaceutical carrier. In certain embodiments, the concentration of the TLR7 agonist or ADC in the composition is an amount that is effective to deliver, after administration, to treat, prevent, or improve one or more symptoms and / or progression of a disease or condition described herein.

[0488] Typically, these compositions are formulated for single-dose administration. To prepare the composition, the weight fraction of the TLR7 agonist or ADC is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration to alleviate or improve the condition being treated. Pharmaceutical carriers suitable for administration of the TLR7 agonists or ADCs provided herein include any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

[0489] In some embodiments, the TLR7 agonist or ADC is contained in a pharmaceutically acceptable carrier in an amount sufficient to treat the subject without producing adverse side effects. The therapeutically effective concentration can be determined empirically by testing the compound in in vitro and in vivo systems described herein and well known to those skilled in the art, and then inferring the dosage for humans therefrom. In some embodiments, the administration method of the ADC can achieve a therapeutically effective concentration of the payload. In some embodiments, companion diagnostics (see, for example, Olsen D and Jorgensen JT, Front. Oncol., 2014 May 16, 4: 105, doi: 10.3389 / fonC.2014.00105) are used to determine the therapeutic concentration and safety profile of the TLR7 agonist or ADC in a specific subject or subject population.

[0490] The concentration of the TLR7 agonist or ADC in the pharmaceutical composition depends on the absorption, tissue distribution, inactivation and excretion rate of the TLR7 agonist or ADC, the physicochemical properties of the TLR7 agonist or ADC, the dosing regimen, the dosage, and other factors known to those skilled in the art. For example, the amount delivered is sufficient to improve one or more symptoms of the disease or condition described herein.

[0491] The composition can be administered all at once or divided into multiple smaller doses administered at regular intervals. It will be understood that the precise dosage and duration of treatment will depend on the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​may also vary with the severity of the disease to be alleviated. It will also be understood that for any particular subject, the specific dosing regimen should be adjusted over time based on the individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0492] The compositions may contain other active compounds to achieve the desired combination of properties. The TLR7 agonists or ADCs provided herein, or pharmaceutically acceptable salts thereof as described herein, may also be advantageously administered in combination with other drugs known in the art to be valuable for treating one or more diseases or medical conditions described herein, to achieve therapeutic or preventive purposes. It should be understood that such combination therapies constitute another aspect of the compositions and treatment methods provided herein.

[0493] Pharmaceutical compositions can be in any form suitable for human or veterinary use, including liquids, oils, emulsions, gels, colloids, aerosols, or solids.

[0494] The pharmaceutical composition may be formulated for administration by any route of administration suitable for human or veterinary medicine, including enteral and parenteral routes of administration.

[0495] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.

[0496] IX. Administration

[0497] The compounds and pharmaceutical compositions provided herein can be administered in certain therapeutically or prophylactically effective amounts, at certain time intervals, in certain dosage forms, and in certain dosing regimens as described below.

[0498] The methods provided herein encompass treating patients regardless of the subject's age, although certain diseases or conditions are more common in certain age groups.

[0499] The TLR7 agonist or ADC provided by the present invention, or a pharmaceutically acceptable salt thereof, can be administered repeatedly as needed, for example, until the subject's condition stabilizes or subsides, or until the subject's condition progresses or unacceptable toxicity occurs.

[0500] The TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, can be administered once a day (QD) or divided into multiple daily doses, such as twice a day (BID), three times a day (TID) and four times a day (QID). In addition, administration can be continuous (i.e., daily administration for several consecutive days or daily administration), or intermittent, such as periodic (i.e., including drug withdrawal for several days, weeks or months). The term "daily" as used herein refers to the administration of a therapeutic compound (e.g., a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof) once or multiple times a day, for example, for a period of time. The term "continuous" refers to the administration of a therapeutic compound (e.g., a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof) every day for an uninterrupted period of at least 10 days to 52 weeks. The term "intermittent" or "intermittently" as used herein means 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 refers to administration 1 to 6 days per week, cyclic administration (e.g., daily administration for 2 to 8 consecutive weeks, followed by a maximum of 1 week of drug withdrawal) or administration every other day. The term "cycle" as used herein means daily or continuous administration of a therapeutic compound (e.g., a TLR7 agonist or ADC or a pharmaceutically acceptable salt thereof provided herein), but with a drug withdrawal period. In some such embodiments, administration is once a day for 2 to 6 days, followed by a drug withdrawal period of 5 to 7 days.

[0501] X. Treatment

[0502] In some embodiments, methods of treating a subject using a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, are provided. In some embodiments, methods of treating a subject using a pharmaceutical composition comprising a TLR7 agonist or ADC provided herein, or a pharmaceutically acceptable salt thereof, are provided. The pharmaceutical composition comprises any TLR7 agonist or ADC disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0503] The TLR7 agonists or ADCs provided herein are particularly useful for treating, preventing, and / or ameliorating any disease or condition associated with or mediated by the expression, signaling, or activity of the target protein of the antigen binding domain.

[0504] In certain embodiments, ADC provided herein is used to treat the primary tumor and / or metastatic tumor occurring in brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, hematopoietic cell and bone marrow, liver and urinary tract and special sense organs (such as eyes).In certain embodiments, TLR7 agonist provided herein or ADC are used to treat one or more of the following cancers: acute myeloid leukemia, adult T cell leukemia, astrocytoma, bladder cancer, breast cancer, PRLR positive (PRLR+) breast cancer, cervical cancer, bile duct cancer, chronic myeloid leukemia, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer (for example, head and neck squamous cell carcinoma (HNSCC)), Kaposi's sarcoma, renal cancer, leiomyosarcoma, liver cancer, lung cancer (for example, Small cell lung cancer, 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 (where "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anticancer 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.

[0505] In some embodiments, the subject has chronic hepatitis B. In some embodiments, the ADCs provided herein are used to treat chronic hepatitis B.

[0506] 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 serum before administering the ADC or pharmaceutical composition. In some embodiments, the treatment methods provided herein further include the step of measuring circulating HBV DNA or HBV sAg in the subject's serum before administering the ADC or pharmaceutical composition. In some embodiments, the treatment methods provided herein further include the step of measuring circulating HBV DNA or HBV sAg in the subject's serum after administration to assess the therapeutic effect of the ADC or pharmaceutical composition.

[0507] In the context of the treatment methods provided herein, a TLR7 agonist or ADC can be administered as monotherapy (ie, as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0508] VII. Combination Therapy with a Second Active Agent

[0509] Provided herein are compositions comprising any of the TLR7 agonists or ADCs provided herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject.

[0510] The TLR7 agonists or ADCs provided herein can be co-formulated and / or co-administered with one or more additional therapeutically active ingredients selected from a MET antagonist (e.g., an anti-MET antibody (e.g., onaluzumab, emibetuzumab, and H4H14639D) or a MET small molecule inhibitor), an EGFR antagonist (e.g., an anti-EGFR antibody (e.g., cetuximab or panitumumab) or an EGFR small molecule inhibitor (e.g., gefitinib or erlotinib)), an antagonist of another EGFR family member, such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., anti-ErbB2 (e.g., trastuzumab or T-DM1)), or an antagonist of a HER2 / ERB2, ErbB3, or ErbB4 family member (e.g., anti-ErbB2 (e.g., trastuzumab or T-DM1)). ), anti-ErbB3 or anti-ErbB4 antibodies or small molecule inhibitors of ErbB2, ErbB3 or 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, e.g., acetaminophen).

[00135] The present invention also includes but is not limited to: imatinib sulfonate 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, such as aflibercept, see, e.g., US 7,087,411 (also referred to herein as "VEGF inhibitory fusion protein"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib or pazopanib)), DLL4 antagonists (e.g., disclosed in US 2009 / 0142354), and VEGF inhibitors (e.g., VEGF-Trap, such as aflibercept, see, e.g., US 7,087,411 (also referred to herein as "VEGF inhibitory fusion protein"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib or pazopanib)), anti-DLL4 antibodies, such as REGN421), Ang2 antagonists (such as anti-Ang2 antibodies disclosed in US2011 / 0027286, such as H1H685P), FOLH1 antagonists (such as anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (such as anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (such as anti-TMPRSS2 antibodies), MSLN antagonists (such as anti-MSLN antibodies), CA9 antagonists (such as anti-CA9 antibodies), urinary plaque antagonists (such as anti-urinary plaque antagonists (e.g., anti-UPK3A) antibodies), MUC16 antagonists (e.g., anti-MUC16 antibodies), Tn antigen antagonists (e.g., anti-Tn antibodies), CLEC12A antagonists (e.g., anti-CLEC12A antibodies), TNFRSF17 antagonists (e.g., anti-TNFRSF17 antibodies), LGR5 antagonists (e.g., anti-LGR5 antibodies), monovalent CD20 antagonists (e.g., monovalent anti-CD20 antibodies, such as rituximab), CD20xCD3 bispecific antibodies, PD-1 blockers (e.g., anti-PD-1 antibodies, such as pembrolizumab or nivolumab), etc. Other agents that can be beneficially 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, IL-18, or their respective receptors.

[0511] For example, a PD-1 inhibitor (eg, an anti-PD-1 antibody) can be combined with a TLR7 agonist or ADC described herein.

[0512] In some embodiments, provided herein are pharmaceutical compositions comprising any TLR7 agonist or ADC provided herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide. TM ); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, methyldopa and uradopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphthazine, clofosamide, estramustine, ifosfamide, nitrogen mustard, nitrogen oxide hydrochloride, melphalan, cypermethrin, phenethyllactone, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, Fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, dactinomycin, oxamycin, azaserine, bleomycin, dactinomycin, calicheamicin, carbinomycin, carmomycin, chromomycin, dactinomycin D, daunorubicin, detoxib, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olivomycin, peplomycin, botoxomycin, puromycin, quinamycin, rhodoxorubicin, streptomycin melanocytin, streptozotocin, tuberculin, ubenimex, zinostatin, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as depoxetine, methotrexate, pterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine purine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calustratone, drostanolone propionate, epithioandrostenone Alkyl alcohol, mebilastane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folic acid; acetylglutamate; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestebacic acid; bisantrene; edatroxilate; difaramine; colchicine; diazinon; efosamil; elutinib acetate; etoglucil; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidamole; nitracrine; pentostatin; pheniramine; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK TM; Razoxane; Cizofuran; Spirogermanium; Tenoxetine; Triazinon; 2,2',2"-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitropol; Mitolactol; Pipobroman; Addacil; Cytosine; Cytarabine ("Ara-C"); Cyclophosphamide; Thiotepa; Taxanes, such as paclitaxel (Taxol TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere TM ; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; norantor; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above drugs. The definition also includes antihormonal drugs used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, troxifene, ketoxifen, LY117018, 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 foregoing.

[0513] The TLR7 agonists or ADCs provided herein can also be co-administered and / or co-formulated with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-γ, and / or NSAIDs.

[0514] Additional therapeutically active ingredients (e.g., any of the above-mentioned drugs or derivatives thereof) can be administered immediately before, simultaneously with, or shortly after administration of a TLR7 agonist or ADC as provided herein. In some embodiments, a pharmaceutical composition is provided wherein a TLR7 agonist or ADC as provided herein is co-formulated with one or more additional therapeutically active ingredients as described herein.

[0515] As used herein, the term "combination" includes the use of more than one therapy (e.g., one or more preventive and / or therapeutic agents). However, the use of the term "combination" does not limit the order in which the subject with a disease or illness is administered therapy (e.g., preventive and / or therapeutic agents). The first therapy (e.g., ADC provided herein) can be administered to the subject before the second therapy (e.g., preventive or therapeutic agent) is administered (e.g., before 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), simultaneously or afterwards (e.g., afterwards 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). Triple therapy is also contemplated herein.

[0516] The compounds provided herein or derivatives thereof and one or more second active agents can be administered to a subject simultaneously or sequentially via the same or different routes of administration. The suitability of a particular route of administration employed by a particular active agent depends on the active agent itself (e.g., whether it can be administered orally without decomposing before entering the bloodstream) and the disease or condition being treated.

[0517] XI. Additional Implementation Regulations and Terms

[0518] The present disclosure is further described by the following non-limiting embodiments.

[0519] Embodiment 1. Compound of formula I:

[0520]

[0521] or a pharmaceutically acceptable salt thereof, wherein:

[0522] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0523] R 2 is H, halogen or alkoxy;

[0524] 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;

[0525] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0526] R 23 is H, alkyl or aryl;

[0527] X is CH or N;

[0528] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0529] Z is -OH, alkoxy or -NR 7 R 8 ;

[0530] R 5 and R 6 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and

[0531] R 7 and R 8 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a heterocyclic ring;

[0532] Provided that the compound is not a compound of the formula:

[0533] or

[0534]

[0535] Embodiment 2. The compound of Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein:

[0536] R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0537] R 2 is halogen or alkoxy;

[0538] R 3 for-CONHR 23 , -alkylene-Y, -alkylene-arylene-Y, -heteroalkylene-Y, -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y;

[0539] R 23 is H, alkyl or aryl;

[0540] R4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0541] X is CH or N;

[0542] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0543] Z is -OH or -NR 7 R 8 ;

[0544] R 5 and R 6 Selected from (i), (ii) and (iii):

[0545] (i)R 5 and R 6 Each is H;

[0546] (ii)R 5 is H, and R 6 is an alkyl group;

[0547] (iii)R 5 and R 6 together with the nitrogen to which they are attached, form a heterocyclic ring; and

[0548] R 7 and R 8 Together with the nitrogen to which they are attached they form a heterocyclic ring.

[0549] Embodiment 3. The compound of Embodiment 1 or 2, provided that R 4 Not substituted by hydroxyl groups.

[0550] Embodiment 4. The compound of any one of Embodiments 1-3, provided that R 3 The alkylene and heteroalkylene portions of the alkylene and heteroalkylene groups are not substituted with oxo.

[0551] Embodiment 5. The compound of any one of Embodiments 1-4, provided 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.

[0552] Embodiment 6. The compound of any one of Embodiments 1-5, wherein R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4, and is a straight chain with a length of 6 atoms.

[0553] Embodiment 7. The compound of any one of Embodiments 1-6, wherein R 1 It is a halogen.

[0554] Embodiment 8. The compound of any one of Embodiments 1-6, wherein R 1 NHR 4 .

[0555] Embodiment 9. The compound of any one of Embodiments 1-6, wherein R 1 For-OR 4 .

[0556] Embodiment 10. The compound of any one of Embodiments 1-6, wherein R 1 -NH-OR 4 .

[0557] Embodiment 11. The compound of any one of Embodiments 1-6, wherein R 1 -R 4 .

[0558] Embodiment 12. The compound of any one of Embodiments 1-5, wherein R 1 It is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt.

[0559] Embodiment 13. The compound of any one of Embodiments 1-5, wherein R 1 It is -NH-n-pentyl.

[0560] Embodiment 14. The compound of any one of Embodiments 1-5, wherein R 1 It is -NH-O-n-butyl.

[0561] Embodiment 15. The compound of any one of Embodiments 1-5, wherein R 1 It is -O-n-pentyl.

[0562] Embodiment 16. The compound of any one of Embodiments 1-5, wherein R 1 It is -n-hexyl.

[0563] Embodiment 17. The compound of any one of Embodiments 1-5, wherein R 1 It is -NH-CH2CH2-OEt.

[0564] Embodiment 18. The compound of any one of Embodiments 1-17, wherein R 2 It is an alkoxy group.

[0565] Embodiment 19. The compound of any one of Embodiments 1-17, wherein R 2 It is a methoxy group.

[0566] Embodiment 20. The compound of any one of Embodiments 1-17, wherein R 2 For H.

[0567] Embodiment 21. The compound of any one of Embodiments 1-17, wherein R 2 It is a halogen.

[0568] Embodiment 22. The compound of any one of Embodiments 1-21, wherein R 3 for-CONHR 23 , -alkylene-Y, -heteroalkylene-Y, heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y.

[0569] Embodiment 23. The compound of any one of Embodiments 1-22, wherein R 3 For CONHR 23 .

[0570] Embodiment 24. The compound of any one of Embodiments 1-22, wherein R 3 is -alkylene-Y.

[0571] Embodiment 25. The compound of any one of Embodiments 1-22, wherein R 3 is -heteroalkylene-Y.

[0572] Embodiment 26. The compound of any one of Embodiments 1-22, wherein R 3 is -heteroalkylene-arylene-Y.

[0573] Embodiment 27. The compound of any one of Embodiments 1-22, wherein R 3 is -(hydroxy)heteroalkylene-Y.

[0574] Embodiment 28. The compound of any one of Embodiments 1-22, wherein R 3 is -(amino)heteroalkylene-Y.

[0575] Embodiment 29. The compound of any one of Embodiments 1-22, wherein R 3 is -alkylene-PEG-Y.

[0576] Embodiment 30. The compound of any one of Embodiments 1-22, wherein R 3 is -CONH2, -CH2-Y, -CH2-O-heteroalkylene-Y or -CH2-O-alkylene-Y.

[0577] Embodiment 31. The compound of any one of Embodiments 1-22, wherein R 3 is -CH2-Y, -CH2-O-heteroalkylene-Y or -CH2-O-alkylene-Y.

[0578] Embodiment 32. The compound of any one of Embodiments 1-22, wherein R 3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2 CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2- (4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2COOH, -CH2OCH2CH2OCH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -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.

[0579] Embodiment 33. The compound of any one of Embodiments 1-22, wherein R 3is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2O CH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NH C(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2 or -CH2-1-piperazinyl.

[0580] Embodiment 34. The compound of any one of Embodiments 1-33, wherein R 4 It is n-butyl, n-pentyl, n-hexyl or ethoxyethyl.

[0581] Embodiment 35. The compound of any one of Embodiments 1-33, wherein R 4 It is n-butyl.

[0582] Embodiment 36. The compound of any one of Embodiments 1-33, wherein R 4 It is n-pentyl.

[0583] Embodiment 37. The compound of any one of Embodiments 1-33, wherein R 4 It is a n-hexyl group.

[0584] Embodiment 38. The compound of any one of Embodiments 1-33, wherein R 4 It is ethoxyethyl.

[0585] Embodiment 39. The compound of any one of Embodiments 1-38, wherein R 5 and R 6 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a piperazinyl ring.

[0586] Embodiment 40. The compound of any one of Embodiments 1-38, wherein R5 and R 6 Each is H.

[0587] Embodiment 41. The compound of any one of Embodiments 1-38, wherein R 5 H and R 6 It is an alkyl group.

[0588] Embodiment 42. The compound of any one of Embodiments 1-38, wherein R 5 and R 6 Together with the nitrogen to which they are attached they form a 1-piperazinyl group.

[0589] Embodiment 43. A compound of any one of Embodiments 1-42, wherein Y is OH.

[0590] Embodiment 44. A compound of any one of Embodiments 1-42, wherein Y is Gly.

[0591] Embodiment 45. The compound of any one of Embodiments 1-42, wherein Y is -NR 5 R 6 .

[0592] Embodiment 46. A compound of any one of Embodiments 1-42, wherein Y is -COZ.

[0593] Embodiment 47. The compound of any one of Embodiments 1-42, wherein Y is -OH, Gly, -NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2, or -CO-1-piperazinyl.

[0594] Embodiment 48. A compound of any one of Embodiments 1-47, wherein Z is -OH.

[0595] Embodiment 49. A compound of any one of Embodiments 1-47, wherein Z is alkoxy.

[0596] Embodiment 50. The compound of any one of Embodiments 1-47, wherein Z is -NR 7 R 8 .

[0597] Embodiment 51. The compound of any one of Embodiments 1-47, wherein Z is -OH, ethoxy, -Nn-Pr2 or 1-piperazinyl.

[0598] Embodiment 52. A compound of any one of Embodiments 1-47, wherein Z is -OH or 1-piperazinyl.

[0599] Embodiment 53. The compound of any one of Embodiments 1-52, wherein R 7and R 8 Each is independently H or n-propyl, or together with the nitrogen to which they are attached forms 1-piperazinyl.

[0600] Embodiment 54. The compound of any one of Embodiments 1-52, wherein R 7 and R 8 Together with the nitrogen to which they are attached they form a 1-piperazinyl group.

[0601] Embodiment 55. A compound selected from the group consisting of:

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609] and pharmaceutically acceptable salts of any of these compounds.

[0610] Embodiment 56. Compound of Formula II:

[0611]

[0612] or a pharmaceutically acceptable salt thereof, wherein:

[0613] R 1 、R 2 and X is as defined in Formula I in Embodiment 1;

[0614] R 9 is R as defined in Embodiment 1 3 The group removes the terminal hydrogen (ie, away from the R 9 A divalent group formed by (a hydrogen of the attached phenyl group); and

[0615] L is any group or moiety that is connected, linked or bonded to the antigen binding domain ABD;

[0616] Provided that the compound is not a compound of the formula:

[0617] or

[0618]

[0619] Embodiment 57. The compound of embodiment 56, wherein R 9 -alkylene-Y 1 -, -heteroalkylene-Y 1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 or -alkylene-PEG-Y 1 .

[0620] Embodiment 58. The compound of embodiment 56 or 57, wherein R 9 -alkylene-Y 1 -.

[0621] Embodiment 59. The compound of embodiment 56 or 57, wherein R 9 -heteroalkylene-Y 1 -.

[0622] Embodiment 60. The compound of embodiment 56 or 57, wherein R 9 -heteroalkylene-arylene-Y 1 -.

[0623] Embodiment 61. The compound of embodiment 56 or 57, wherein R 9 -(hydroxy)heteroalkylene-Y 1 .

[0624] Embodiment 62. The compound of embodiment 56 or 57, wherein R 9 -(amino)heteroalkylene-Y 1 .

[0625] Embodiment 63. The compound of embodiment 56 or 57, wherein R 9 -alkylene-PEG-Y 1 .

[0626] Embodiment 64. The compound of embodiment 56 or 57, wherein R 9 -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 -.

[0627] Embodiment 65. The compound of embodiment 56 or 57, wherein R 9is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH 2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazine-4-yl-, -CH2OCH2NHC(O)CH2 NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, -CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH- or -CH2-1-piperazin-4-yl.

[0628] Embodiment 66. The compound of any one of Embodiments 55-65, wherein Y 1 It is -O-.

[0629] Embodiment 67. The compound of any one of Embodiments 55-65, wherein Y 1 For Gly.

[0630] Embodiment 68. The compound of any one of Embodiments 55-65, wherein Y 1 -NR 5 -.

[0631] Embodiment 69. The compound of any one of Embodiments 55-65, wherein Y 1 for-COZ 1 .

[0632] Embodiment 70. The compound of any one of Embodiments 55-65, wherein Y 1 It is -O-, glycine, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl.

[0633] Embodiment 71. The compound of embodiment 69, wherein Z 1 It is -O-.

[0634] Embodiment 72. The compound of embodiment 69, wherein Z1 -NR 7 -.

[0635] Embodiment 73. The compound of embodiment 69, wherein Z 1 It is -O- or 1-piperazin-4-yl.

[0636] Embodiment 74. The compound of embodiment 68, wherein R 5 For H.

[0637] Embodiment 75. The compound of embodiment 68, wherein R 5 It is an alkyl group.

[0638] Embodiment 76. The compound of any one of Embodiments 55-75, wherein L is not cleavable under physiological conditions.

[0639] Embodiment 77. The compound of any one of Embodiments 55-75, wherein L is cleavable under physiological conditions.

[0640] Embodiment 78. The compound of Embodiment 77, wherein L is an acid labile linker, a hydrolytically labile linker, an enzymatically cleavable linker, a reduction labile linker, or a self-immolative linker.

[0641] Embodiment 79. The compound of any one of Embodiments 55-78, wherein L is or comprises a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a maleimido-hexanoyl unit, a dipeptide unit, a valine-citrulline unit, or a p-aminobenzyl (PAB) unit.

[0642] Embodiment 80. A compound of any one of Embodiments 55-79, wherein L comprises one or more amino acids.

[0643] Embodiment 81. The compound of any one of Embodiments 55-80, wherein L comprises a self-immolative group.

[0644] Embodiment 82. The compound of any one of Embodiments 55-81, wherein L comprises p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC).

[0645] Embodiment 83. The compound of any one of Embodiments 55-82, wherein L comprises a maleimido group, an N-hydroxysuccinimidyl ester, or a cyclooctynyl group.

[0646] Embodiment 84. A compound of any one of embodiments 55-83, wherein L is a group selected from the group consisting of 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropionyl,

[0647]

[0648]

[0649]

[0650] Embodiment 85. A compound selected from the group consisting of:

[0651]

[0652]

[0653]

[0654]

[0655] and pharmaceutically acceptable salts of any of these compounds. Embodiment 86. Compounds of Formula III:

[0656]

[0657] or a pharmaceutically acceptable salt thereof, wherein:

[0658] R 1 、R 2 and X is as defined in Formula I in Embodiment 1;

[0659] L is any group or moiety that is connected, linked or bonded to the antigen binding domain ABD;

[0660] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0661] R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0662] R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0663] R 15 is hydrogen or alkyl;

[0664] R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and

[0665] x is 0, 1, 2, 3, 4, 5, or 6.

[0666] Embodiment 87. The compound of embodiment 86, wherein

[0667] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0668] R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0669] R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0670] R 15 is hydrogen or alkyl;

[0671] R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and

[0672] x is 0, 1, 2, 3, 4, 5, or 6.

[0673] Embodiment 88. The compound of Embodiment 86 or Embodiment 87, wherein the TLR7 agonist used to prepare the compound is P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, P39, P41, P42 or P43.

[0674] Embodiment 89. An antibody-drug-conjugate (ADC) comprising a compound according to any one of embodiments 1-88 or a compound of the formula:

[0675]

[0676] Embodiment 90. The ADC of Embodiment 89, which has Formula IV:

[0677]

[0678] or a pharmaceutically acceptable salt thereof, wherein:

[0679] R 1 、R 2 、R 9 and X is as defined by Formula I in Embodiment 1 and Formula II in Embodiment 56;

[0680] L 1 is a divalent linker;

[0681] ABD is the antigen binding domain; and

[0682] k is an integer from 1 to 30.

[0683] Embodiment 91. The ADC of embodiment 89 or 90, which is ABD-LP1, ABD-LP6A, ABD-LP7A, ABD-LP8A, ABD-LP10A or ABD-LP11A.

[0684] Embodiment 92. The ADC of Embodiment 89, which has Formula V:

[0685]

[0686] or a pharmaceutically acceptable salt thereof, wherein:

[0687] R 1 、R 2 and X is as defined in Formula I in Embodiment 1;

[0688] R 10is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-; and

[0689] The ABD is an antibody containing a Q295 residue, an N297Q mutation, and / or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG,LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15); and

[0690] k is an integer from 1 to 30.

[0691] Embodiment 93. The ADC of embodiment 92, wherein R 10 is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-.

[0692] Embodiment 94. The ADC of embodiment 92 or embodiment 93, wherein R 10 It is -alkylene-NH-.

[0693] Embodiment 95. The ADC of embodiment 92 or embodiment 93, wherein R 10 is -heteroalkylene-NH-.

[0694] Embodiment 96. The ADC of embodiment 92 or embodiment 93, wherein R 10 is -heteroalkylene-arylene-NH-.

[0695] Embodiment 97. The ADC of embodiment 92 or embodiment 93, wherein R 10 is -(hydroxy)heteroalkylene-NH-.

[0696] Embodiment 98. The ADC of embodiment 92 or embodiment 93, wherein R 10is -(amino)heteroalkylene-NH-.

[0697] Embodiment 99. The ADC of embodiment 92 or embodiment 93, wherein R 10 is -alkylene-PEG-NH-.

[0698] Embodiment 100. The ADC of embodiment 92 or embodiment 93, wherein R 10 is -CH2-NH-, -CH2-O-heteroalkylene-NH- or -CH2-O-alkylene-NH-.

[0699] Embodiment 101. The ADC of embodiment 92 or embodiment 93, wherein R 10 It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl), -CH2OCH(NH-)CH2OH or -CH2NH-.

[0700] Embodiment 102. The ADC of embodiment 92 or embodiment 93, wherein R 10 It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl) or -CH2NH-.

[0701] Embodiment 103. The ADC of any one of Embodiments 89-102, having the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, ABD-P34, or ABD-P41, wherein ABD is linked to R 3 The payload is placed on the amino group of the TLR7 receptor agonist.

[0702] Embodiment 104. The ADC of Embodiment 89, which has Formula VI:

[0703]

[0704] or a pharmaceutically acceptable salt thereof, wherein:

[0705] L1 is a divalent linker;

[0706] R 1 、R 2 、R 16 、R 11 、R 12 、R 13 、R 14 、R 15 , X and x are as defined in Formula III in Embodiment 86; and

[0707] k is an integer from 1 to 30.

[0708] Embodiment 105. The ADC of embodiment 104 having the formula ABD-L 1 -P1, ABD-L 1 -P2, ABD-L 1 -P6、ABD-L 1 -P8、ABD-L 1 -P17, ABD-L 1 -P18, ABD-L 1 -P19, ABD-L 1 -P20, ABD-L 1 -P23, ABD-L 1 -P27, ABD-L 1 -P29, ABD-L 1 -P32、ABD-L 1 -P33、ABD-L 1 -P37, ABD-L 1 -P39 or ABD-L 1 -P42, where ABD-L 1 Connect to R 3 The payload (i.e., TLR7 agonist) is placed on the alcohol group of

[0709] Embodiment 106. The ADC of any one of Embodiments 89-105, wherein the ABD has binding specificity for a transmembrane molecule (e.g., a receptor) expressed on a tumor.

[0710] Embodiment 107. A pharmaceutical composition comprising a compound according to any one of embodiments 1-88 or an ADC according to any one of embodiments 89-106, and a pharmaceutically acceptable carrier.

[0711] Embodiment 108. A method of treating or diagnosing a disease, comprising administering to a subject a compound of any one of embodiments 1-88, an ADC of any one of embodiments 89-106, or a pharmaceutical composition of embodiment 107.

[0712] Embodiment 109. The method of embodiment 108, wherein the method treats a disease.

[0713] Embodiment 110. The method of embodiment 108 or 109, wherein the disease is cancer.

[0714] The present disclosure is further described by the following non-limiting terms.

[0715] Item 1. An antibody-drug conjugate (ADC), comprising

[0716] a. having an antigen binding domain (ABD) that binds specifically to hepatitis B virus surface antigen (HBV sAg); and

[0717] b. Toll-like receptor 7 (TLR7) agonists.

[0718] Item 2. The ADC of Item 1, further comprising a bivalent linker linking the ABD to the TLR7 agonist.

[0719] Item 3. The ADC of Item 2, wherein the ADC is according to Formula IV:

[0720]

[0721] or a pharmaceutically acceptable salt thereof, wherein:

[0722] L 1 is a divalent linker;

[0723] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0724] R 2 is H, halogen or alkoxy;

[0725] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0726] R 9 For R 3 The divalent group formed by removing hydrogen, R 3 For R 9 The group at position 1 is connected to the phenyl group;

[0727] 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;

[0728] R 23 is H, alkyl or aryl;

[0729] X is CH or N;

[0730] Y is -OH, -Gly, or -NR 5 R 6 or -COZ;

[0731] Z is -OH, alkoxy or -NR 7 R 8 ;

[0732] R 5 and R 6 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a heterocyclic ring;

[0733] R 7 and R 8 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and

[0734] k is an integer from 1 to 30.

[0735] Clause 4. ADC of Clause 3, provided that ADC does not include

[0736] 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine or

[0737] (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3-methoxyphenyl)methanol.

[0738] Clause 5. The ADC of any one of clauses 1-3, wherein R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 , and has a straight chain length of 6 atoms.

[0739] Clause 6. ADC of Clause 5, wherein R 1 It's a halogen.

[0740] Clause 7. ADC of Clause 5, where R 1 Yes-NHR 4 .

[0741] Clause 8. The ADC of Clause 5, wherein R 1 Yes-OR 4 .

[0742] Clause 9. The ADC of Clause 5, wherein R 1 Yes-NH-OR 4 .

[0743] Item 10. ADC of Item 5, where R 1 Yes-R 4 .

[0744] Clause 11. ADC of Clause 5, where R 1 It is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt.

[0745] Clause 12. ADC of Clause 5, where R 1 It is -NH-n-pentyl.

[0746] Clause 13. ADC of Clause 5, where R 1 It is -NH-O-n-butyl.

[0747] Clause 14. ADC of Clause 5, where R 1 It is -O-n-pentyl.

[0748] Clause 15. ADC of Clause 5, wherein R 1 It is n-hexyl.

[0749] Clause 16. ADC of Clause 5, wherein R 1 It is -NH-CH2CH2-OEt.

[0750] Clause 17. The ADC of any one of clauses 3-16, wherein R 2 It is an alkoxy group.

[0751] Clause 18. The ADC of any one of clauses 3-16, wherein R 2 It's a methoxy group.

[0752] Clause 19. The ADC of any one of clauses 3-16, wherein R 2 It’s H.

[0753] Clause 20. The ADC of any one of clauses 3-16, wherein R 2 It's a halogen.

[0754] Clause 21. The ADC of any one of clauses 3-20, wherein R 3 Yes-CONHR 23, -alkylene-Y, -heteroalkylene-Y, or -heteroalkylene-arylene-Y, -(hydroxy)heteroalkylene-Y, -(amino)heteroalkylene-Y, or -alkylene-PEG-Y.

[0755] Clause 22. The ADC of Clause 21, wherein R 3 It is CONHR 23 .

[0756] Clause 23. The ADC of Clause 21, wherein R 3 It is -alkylene-Y.

[0757] Clause 24. The ADC of Clause 21, wherein R 3 It is -heteroalkylene-Y.

[0758] Clause 25. The ADC of Clause 21, wherein R 3 It is -heteroalkylene-arylene-Y.

[0759] Clause 26. The ADC of Clause 21, wherein R 3 It is -(hydroxy)heteroalkylene-Y.

[0760] Clause 27. The ADC of Clause 21, wherein R 3 It is -(amino)heteroalkylene-Y.

[0761] Clause 28. The ADC of Clause 21, wherein R 3 It is -alkylene-PEG-Y.

[0762] Clause 29. The ADC of any one of clauses 3-20, wherein R 3 is -CONH2, -CH2-Y, -CH2-O-heteroalkylene-Y or -CH2-O-alkylene-Y.

[0763] Clause 30. The ADC of any one of clauses 3-20, wherein R 3 is -CH2-Y, -CH2-O-heteroalkylene-Y or -CH2-O-alkylene-Y.

[0764] Clause 31. The ADC of any one of clauses 3-20, wherein R 3Is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2O CH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1- piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, -CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2 or -CH2-1-piperazinyl.

[0765] Clause 32. The ADC of any one of clauses 3-20, wherein R 3 Is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2 OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2N HC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH-CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2 or -CH2-1-piperazinyl.

[0766] Clause 33. The ADC of any one of clauses 3-32, wherein R 4It is n-butyl, n-pentyl, n-hexyl or ethoxyethyl.

[0767] Article 34. ADC of Article 33, wherein R 4 It is n-butyl.

[0768] Article 35. ADC of Article 33, wherein R 4 Is n-pentyl.

[0769] Article 36. ADC of Article 33, wherein R 4 It is the foundation of righteousness.

[0770] Article 37. ADC of Article 33, wherein R 4 It is ethoxyethyl.

[0771] Clause 38. The ADC of any one of clauses 3-37, wherein R 5 and R 6 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a piperazinyl ring.

[0772] Article 39. ADC of Article 38, wherein R 5 and R 6 Each is H.

[0773] Clause 40. ADC of Clause 38, wherein R 5 is H and R 6 It is an alkyl group.

[0774] Article 41. ADC of Article 38, where R 5 and R 6 Together with the nitrogen to which they are attached they form a 1-piperazinyl group.

[0775] Clause 42. The ADC of any one of clauses 3-41, wherein Y is OH.

[0776] Clause 43. The ADC of any one of clauses 3-41, wherein Y is glycine.

[0777] Clause 44. The ADC of any one of clauses 3-41, wherein Y is -NR 5 R 6 .

[0778] Clause 45. The ADC of any one of clauses 3-41, wherein Y is -COZ.

[0779] Clause 46. The ADC of any one of clauses 3-41, wherein Y is -OH, glycine, -NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2, or -CO-1-piperazinyl.

[0780] Clause 47. The ADC of any one of clauses 3-41, wherein Y is -OH, -NH2, 1-piperazinyl, -COOH, or -CO-1-piperazinyl.

[0781] Clause 48. The ADC of Clause 45, wherein Z is -OH.

[0782] Clause 49. The ADC of Clause 45, wherein Z is alkoxy.

[0783] Clause 50. The ADC of clause 45, wherein Z is -NR 7 R 8 .

[0784] Clause 51. The ADC of Clause 45, wherein Z is -OH, ethoxy, -Nn-Pr2 or 1-piperazinyl.

[0785] Clause 52. The ADC of Clause 45, wherein Z is -OH or 1-piperazinyl.

[0786] Clause 53. The ADC of any one of clauses 3-52, wherein R 7 and R 8 Each is independently H or n-propyl, or together with the nitrogen to which they are attached forms 1-piperazinyl.

[0787] Clause 54. The ADC of any one of clauses 3-53, wherein R 7 and R 8 Together with the nitrogen to which they are attached they form a 1-piperazinyl group.

[0788] Article 55. The ADC of Article 3, wherein ABD-L 1 By R 3 The groups at the corresponding positions are freed of hydrogen and connected to compounds selected from P1-P43 and pharmaceutically acceptable salts thereof, wherein the structures of P1-P43 are as follows:

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795] Clause 56. The ADC of any one of clauses 3-54, wherein R 9 -alkylene-Y 1 -, -heteroalkylene-Y1 -, -heteroalkylene-arylene-Y 1 -, -(hydroxy)heteroalkylene-Y 1 , -(amino)heteroalkylene-Y 1 or -alkylene-PEG-Y 1 -.

[0796] Article 57. ADC of Article 56, wherein R 9 is -alkylene-Y 1 -.

[0797] Clause 58. ADC of Clause 56, wherein R 9 is -heteroalkylene-Y 1 -.

[0798] Article 59. ADC of Article 56, wherein R 9 is-heteroalkylene-arylene-Y 1 -.

[0799] Clause 60. ADC of Clause 56, wherein R 9 is -(hydroxy)heteroalkylene-Y 1 .

[0800] Article 61. ADC of Article 56, where R 9 is -(amino)heteroalkylene-Y 1 .

[0801] Article 62. ADC of Article 56, wherein R 9 Y-alkylene-PEG-Y 1 -.

[0802] Article 63. ADC of Article 56, where R 9 Yes -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 -.

[0803] Article 64. ADC of Article 56, where R 9is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH 2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazine-4-yl-, -CH2OCH2NHC(O)CH2 NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, -CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH- or -CH2-1-piperazin-4-yl.

[0804] Clause 65. The ADC of any one of clauses 56-64, wherein Y 1 Yes -O-.

[0805] Clause 66. The ADC of any one of clauses 56-64, wherein Y 1 It's Gly.

[0806] Clause 67. The ADC of any one of clauses 56-64, wherein Y 1 Yes-NR 5 -.

[0807] Clause 68. The ADC of any one of clauses 56-64, wherein Y 1 Yes-COZ 1 , where Z 1 Yes -O-, -NR 7 -, -O-alkylene- or 1-piperazin-4-yl.

[0808] Clause 69. The ADC of any one of clauses 56-64, wherein Y 1 It is -O-, -NH-, 1-piperazin-4-yl, -COO- or -CO-1-piperazin-4-yl.

[0809] Article 70. ADC of Article 68, where Z 1 Yes -O-.

[0810] Article 71. ADC of Article 68, where Z 1 Yes-NR 7 -.

[0811] Clause 72. The ADC of Clause 71, wherein R 7 It’s H.

[0812] Article 73. ADC of Article 71, wherein R 7 It is an alkyl group.

[0813] Article 74. ADC of Article 68, where Z 1 It is 1-piperazin-4-yl.

[0814] Article 75. ADC of Article 69, where Y 1 It is 1-piperazin-4-yl.

[0815] Article 76. ADC of Article 69, where Y 1 It is -CO-1-piperazin-4-yl.

[0816] Clause 77. The ADC of any one of clauses 3-76, wherein L 1 Indestructible under physiological conditions.

[0817] Clause 78. The ADC of any one of clauses 3-76, wherein L 1 It can be cleaved under physiological conditions.

[0818] Article 79. ADC of Article 78, where L 1 is an acid-labile linker, a hydrolytically labile linker, an enzymatically cleavable linker, a reduction-labile linker, or a self-immolative linker.

[0819] Clause 80. The ADC of any one of clauses 3-79, wherein L 1 is or includes a peptide, a carbohydrate, a glucuronide, a polyethylene glycol (PEG) unit, a hydrazone, a maleimido-hexanoyl unit, a dipeptide unit, a valine-citrulline unit, or a p-aminobenzyl (PAB) unit.

[0820] Clause 81. The ADC of any one of clauses 3-80, wherein L 1 Contains one or more amino acids.

[0821] Article 82. ADC of Article 79, where L 1 Includes self-immolative groups.

[0822] Article 83. ADC of Article 80, where L 1 These include p-aminobenzyl (PAB) or p-aminobenzyloxycarbonyl (PABC).

[0823] Clause 84. The ADC of any one of clauses 3-76, wherein L 1 Contains maleimido, N-hydroxysuccinimidyl ester or cyclooctyne groups.

[0824] Clause 85. The ADC of any one of clauses 3-76, wherein L 1 It is derived from 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropionyl,

[0825]

[0826]

[0827] group.

[0828] Clause 86. The ADC of Clause 3, comprising an ABD linked to a compound selected from LP1, LP6-LP12, and pharmaceutically acceptable salts of these compounds, wherein the structures of LP1 and LP6-LP12 are as follows:

[0829]

[0830]

[0831]

[0832]

[0833] Clause 87. The ADC of Clause 3, comprising an ABD linked to a compound of Formula III:

[0834] or a pharmaceutically acceptable salt thereof, wherein:

[0835] R 1 、R 2 and X is as defined in Formula I in Clause 3;

[0836] L is any group or moiety attached to ABD;

[0837] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0838] R 14is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0839] R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0840] R 15 is hydrogen or an alkyl group;

[0841] R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene-, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and

[0842] x is 0, 1, 2, 3, 4, 5, or 6.

[0843] Article 88. The ADC of Article 87, wherein

[0844] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0845] R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0846] R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 4-, 5-, or 6-membered heterocyclic group;

[0847] R 15 is hydrogen or an alkyl group;

[0848] R 16is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-; and

[0849] x is 0, 1, 2, 3, 4, 5, or 6.

[0850] Article 89. The ADC of Article 3, wherein ABD-L 1 linked to a compound selected from P1, P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, and P39.

[0851] Clause 90. The ADC of clause 89, wherein the ABD is linked to a compound selected from LP1, LP6, LP7, LP8, LP10, and LP11.

[0852] Clause 90. The ADC of clause 3, wherein the ADC is according to formula V:

[0853]

[0854] or a pharmaceutically acceptable salt thereof, wherein:

[0855] R 1 、R 2 and X is as defined in Formula IV in Clause 3;

[0856] R 10 is -alkylene-NH-, -alkylene-arylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-;

[0857] The ABD is an antibody containing a Q295 residue, an N297Q mutation, and / or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG,LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15); and

[0858] k is an integer from 1 to 30.

[0859] Clause 92. The ADC of Clause 91, wherein R 10 is -alkylene-NH-, -heteroalkylene-NH-, -heteroalkylene-arylene-NH-, -(hydroxy)heteroalkylene-NH-, -(amino)heteroalkylene-NH-, or -alkylene-PEG-NH-.

[0860] Clause 93. The ADC of Clause 92, wherein R 10 It is -alkylene-NH-.

[0861] Clause 94. ADC of Clause 92, wherein R 10 It is -heteroalkylene-NH-.

[0862] Clause 95. The ADC of Clause 92, wherein R 10 It is -heteroalkylene-arylene-NH-.

[0863] Clause 96. ADC of Clause 92, wherein R 10 It is -(hydroxy)heteroalkylene-NH-.

[0864] Article 97. ADC of Article 92, wherein R 10 It is -(amino)heteroalkylene-NH-.

[0865] Article 98. ADC of Article 92, wherein R 10 It is -alkylene-PEG-NH-.

[0866] Article 99. ADC of Article 91 or 92, wherein R 10 It is -CH2-NH-, -CH2-O-heteroalkylene-NH- or -CH2-O-alkylene-NH-.

[0867] Clause 100. ADC of Clause 91, wherein R 10 It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl)-, -CH2OCH(NH-)CH2OH or -CH2NH-.

[0868] Article 101. ADC of Article 91, wherein R 10It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl)- or -CH2NH-.

[0869] Article 102. ADC of Article 3, including through R 3 The amino group of is connected to the ABD of a compound selected from the group consisting of P4, P5, P7, P9, P11, P12, P19, P21, P24, P30 and P34.

[0870] Clause 103. The ADC of Clause 2, wherein the ADC is according to Formula VI:

[0871]

[0872] or a pharmaceutically acceptable salt thereof, wherein:

[0873] L 1 is a divalent linker;

[0874] R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ;

[0875] R 2 is H, halogen or alkoxy;

[0876] R 4 is an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group;

[0877] R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0878] R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0879] R13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group;

[0880] R 15 is hydrogen or an alkyl group;

[0881] R 16 is alkylene, alkylene-arylene, heteroalkylene, heteroalkylene-arylene-, -(hydroxy)heteroalkylene-, -(amino)heteroalkylene-, or -alkylene-PEG-;

[0882] X is CH or N;

[0883] x is 0, 1, 2, 3, 4, 5, or 6; and

[0884] k is an integer from 1 to 30.

[0885] Clause 104. The ADC of clause 103, comprising 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 .

[0886] Clause 105. The ADC of clause 103 or 104, wherein k is 1, 2, 3, 4, or 5.

[0887] Clause 106. The ADC of clause 103, wherein k is 2.

[0888] Clause 107. The ADC of clause 103, wherein the ABD comprises a heavy chain and the C-terminus of the heavy chain is connected to L 1 conjugation.

[0889] Clause 108. The ADC of clause 103, wherein the ABD comprises two heavy chains and the C-termini of each of the two heavy chains are connected to L 1 conjugation.

[0890] Clause 109. The ADC of any of clauses 3-108, wherein L 1 Linked to the cysteine ​​residue of the ABD.

[0891] Clause 110. The ADC of any one of clauses 1-109, wherein the ABD is an antibody or fragment thereof directed against HBV sAg.

[0892] Clause 111. The ADC of any one of clauses 1-110, wherein ABD is a human or humanized antibody.

[0893] Clause 112. The ADC of any one of clauses 1-111, wherein the ABD is IgG1 or IgG2a.

[0894] Clause 113. The ADC of any one of clauses 1-110, wherein the ABD comprises a scFv with binding specificity for HBV sAg.

[0895] Clause 114. The ADC of any one of clauses 1-113, wherein the ABD comprises a V of an antibody directed against HBV sAg. H Chain and V L chain.

[0896] Clause 115. The ADC of any one of clauses 1-114, wherein the ABD comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody against HBV sAg.

[0897] Clause 116. The ADC of any one of clauses 1-102, wherein the ABD comprises an Fc region.

[0898] Clause 117. The ADC of Clause 116, wherein the Fc region comprises a modification for enhanced binding to FcγR.

[0899] Clause 118. The ADC of any one of clauses 1 to 114, wherein the ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence shown in SEQ ID NO: 25, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence shown in SEQ ID NO: 29.

[0900] Clause 119. The ADC of Clause 118, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:26, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:27, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:28, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:30, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32.

[0901] Clause 120. The ADC of clause 118 or 119, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 25.

[0902] Clause 121. The ADC of claim 120, wherein the HCVR is a component of a heavy chain comprising the amino acid sequence of SEQ ID NO: 33.

[0903] Clause 122. The ADC of clause 118 or 119, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 29.

[0904] Clause 123. The ADC of claim 120, wherein the LCVR is a component of a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0905] Clause 124. The ADC of any one of clauses 118-123, wherein the ABD is a component of an antibody or antigen-binding fragment thereof.

[0906] Clause 125. The ADC of Clause 1, wherein the 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.

[0907] Clause 126. A pharmaceutical composition comprising the ADC of any one of clauses 1-117 and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0908] Clause 127. A method of treatment comprising administering to a subject in need thereof an effective amount of the ADC of any one of Clauses 1-117 or the pharmaceutical composition of Clause 118.

[0909] Clause 128. The method of Clause 127, wherein the subject has hepatitis B.

[0910] Clause 129. The method of Clause 127, wherein the hepatitis B is chronic hepatitis B.

[0911] Clause 130. The method of Clause 127 or 128, wherein circulating HBV DNA or HBV sAg is elevated in the subject's serum prior to administration of the ADC or pharmaceutical composition.

[0912] Clause 131. The method of any one of clauses 127-130, further comprising measuring circulating HBV DNA or HBV sAg in the serum of the subject prior to administration.

[0913] Clause 132. The method of any one of clauses 127-131, further comprising measuring circulating HBV DNA or HBV sAg in the serum of the subject after administration to assess the therapeutic effect of the ADC or pharmaceutical composition.

[0914] Clause 133. The method of any one of clauses 127-132, wherein the step of administering the ADC or pharmaceutical composition is repeated.

[0915] Clause 134. The method of Clause 133, wherein the step of administering the ADC or pharmaceutical composition is repeated two, three or more times.

[0916] Clause 135. The method of Clause 133 or 134, wherein the step of administering the ADC or pharmaceutical composition is repeated at least at 1 week intervals, 2 week intervals, 3 week intervals, or 4 week intervals.

[0917] Clause 136. The method of Clause 133 or 134, wherein the step of administering the ADC or pharmaceutical composition is repeated at 1 week intervals, 2 week intervals, 3 week intervals, or 4 week intervals.

[0918] Clause 137. The method of Clause 133 or 134, wherein the step of administering the ADC or pharmaceutical composition is repeated at 1 month intervals, 2 month intervals, or 3 month intervals.

[0919] Clause 138. The method of any one of clauses 127-137, wherein the ADC or pharmaceutical composition is administered orally, intravenously, intraperitoneally, by inhalation, intranasally, intramuscularly, or subcutaneously.

[0920] Clause 139. The ADC of any one of clauses 1 to 117, or the pharmaceutical composition of clause 126, for use in therapy.

[0921] Clause 140. The ADC of any one of Clauses 1-117, or the pharmaceutical composition of Clause 126, for use in treating chronic hepatitis B in a subject in need thereof.

[0922] Clause 141. Use of the ADC of any one of clauses 1 to 117 or the pharmaceutical composition of clause 126 for the manufacture of a medicament.

[0923] Clause 142. Use of the ADC of any one of Clauses 1-117 or the pharmaceutical composition of Clause 126 for the manufacture of a medicament for treating chronic hepatitis B in a subject in need thereof.

[0924] XII. Examples

[0925] The following examples are intended to illustrate certain embodiments provided herein and are not intended to limit the scope of the present disclosure.

[0926] Example 1

[0927] Synthesis of intermediate Aa (see Scheme 1)

[0928] Methyl 4-{[2-chloro-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (3)

[0929]

[0930] To a mixture of compound 1 (0.50 g, 2.7 mmol) in acetonitrile (10 mL) were added bromide 2 (0.76 g, 2.9 mmol) and potassium carbonate (0.74 g, 5.3 mmol), and the suspension was stirred at room temperature for 6 hours. The reaction mixture was monitored by LCMS (ESI m / z: 366.1 (M+H) + ). Potassium carbonate (0.37 g, 2.7 mmol) and 1-pentylamine (0.70 g, 7.9 mmol) were added to the resulting mixture, and the reaction mixture was stirred at 85 ° C for 5 hours, 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 (x3). 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 / TFA aqueous solution (0.01%)) to give compound 3 (0.23 g, 21% yield) as a light yellow solid. ESI m / z: 417.2 (M+H) + .

[0931] Methyl 4-{[2-azido-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (4)

[0932]

[0933] 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 hours and monitored by LCMS. After cooling to room temperature, the mixture was diluted with ethyl acetate (40 mL) and saturated aqueous sodium bicarbonate solution (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 / TFA aqueous solution (0.01%)) to give compound 4 (60 mg, 30% yield) as a light yellow solid. ESI m / z: 424.2 (M + H) + .

[0934] Methyl 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Aa) (Method 1)

[0935]

[0936] Under nitrogen protection, zinc powder (0.56 g, 8.5 mmol) was added to a solution of compound 4 (0.12 g, 0.28 mmol) in anhydrous acetic acid (10 mL) at 0 ° C. The reaction mixture was then stirred at 85 ° C for 4 hours. 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 / TFA aqueous solution (0.05%)) to give intermediate Aa (65 mg, 58% yield) as a light yellow solid. ESI m / z: 398.3 (M+H) + .

[0937] Example 2

[0938] General Procedure I for the Synthesis of Compound 7

[0939]

[0940] To a mixture of compounds 5a,b (1.0 equiv) in acetonitrile (0.2 M) were added compounds 6a-c (1.1-1.2 equiv) and potassium carbonate (2.0 equiv). The suspension was stirred at room temperature for 16 hours, 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 afford compounds 7a-d (yields 34-94%) as light yellow solids.

[0941] Methyl 4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzoate (7a)

[0942]

[0943] Starting from compounds 5a (0.50 g, 3.0 mmol) and 6a (0.85 g, 3.3 mmol), following General Procedure I, compound 7a (0.85 g, 83% yield) was obtained as a light yellow solid after purification by silica gel flash chromatography (50-100% ethyl acetate / petroleum ether, 20 min). ESI m / z: 347.1 (M+H) + ,369.1(M+Na) + . 1H NMR (400MHz, CDCl3) δ7.57(d,J=1.3Hz,1H),7.52(dd,J=7.9,1.4Hz,1H),7.31(d,J=3.1Hz,1H),6. 57(d,J=7.9Hz,1H),6.39(d,J=3.1Hz,1H),5.58(s,2H),4.86(s,2H),3.95(s,3H),3.90(s,3H)ppm.

[0944] Methyl 4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzoate (7b)

[0945]

[0946] Starting from compounds 5a (0.55 g, 3.3 mmol) and 6b (0.90 g, 3.9 mmol), following General Procedure I, compound 7b (1.1 g, 94% yield) was obtained as a light yellow solid after purification by silica gel flash chromatography (5-10% methanol / DCM, 20 min). ESI m / z: 317.1 (M+H) + ,339.1(M+Na) + . 1 H NMR (400MHz, CDCl3) δ8.00(d,J=8.5Hz,2H),7.34(d,J=3.2Hz,1H),7.07(d,J= 8.5Hz, 2H), 6.42 (d, J = 3.2Hz, 1H), 5.62 (s, 2H), 4.87 (s, 2H), 3.90 (s, 3H) ppm.

[0947] Tert-butyl N-{[4-({2-amino-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)phenyl]methyl}carbamate (7c)

[0948]

[0949] Starting from compounds 5a (0.20 g, 1.2 mmol) and 6c (0.43 g, 1.4 mmol), following General Procedure I, compound 7c (0.30 g, 65% yield) was obtained as a light yellow solid after purification by silica gel flash chromatography (20-25% ethyl acetate / petroleum ether, 20 min). ESI m / z: 388.2 (M+H) + . 1H NMR (400MHz, CDCl3) δ7.31(d,J=3.2Hz,1H),7.23(d,J=8.0Hz,2H),7.01(d,J=8.0Hz,2H),6.37(d,J=3.1Hz,1H),5.55(s,2H),4.82(br s, 3H), 4.29 (d, J = 5.5Hz, 2H), 1.45 (s, 9H) ppm.

[0950] 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')

[0951]

[0952] Starting from compounds 5b (0.50 g, 2.9 mmol) and 6a (0.84 g, 3.2 mmol), following General Procedure I, white solid compound 7d (0.35 g, 34% yield) and its isomer 7d′ (0.15 g, 15% yield) were obtained after purification by silica gel flash chromatography (0-5% methanol / DCM).

[0953] 7d:ESI m / z:348.1(M+H) + ,717.3(2M+Na) + ; Retention time in LCMS: 1.64 min; 1 H NMR (400 MHz, DMSO d6 )δ8.48(s,1H),7.54(d,J=1.5Hz,1H),7.50(dd,J=8.0,1.5Hz,1H),6.72(d,J=8.0Hz,1H),6.70(s,2H),5.56(s,2H),3.93(s,3H),3.84(s,3H)ppm.

[0954] 7d':ESI m / z:348.1(M+H) + ; Retention time in LCMS: 1.67 min; 1 H NMR (400 MHz, DMSO d6 )δ8.17(s,1H),7.53(d,J=1.3Hz,1H),7.50(dd,J=7.8,1.4Hz,1H),6.94(s,2H),6.88(d,J=7.8Hz,1H),5.29(s,2H),3.93(s,3H),3.85(s,3H)ppm.

[0955] The structures of 7d and 7d' were confirmed by the NOE data of 9b and 9b'.

[0956] Example 3

[0957] General Procedure II for the Synthesis of Intermediates Aa, Ba, Bb, Bc, and Fa

[0958]

[0959] To a suspension of compound 7a-d (1.0 equiv) in acetonitrile (40-50 mM) was added potassium carbonate (4.0 equiv) and 1-pentylamine 8a (5.0 equiv) or O-butylhydroxylamine 8b (hydrochloride, 2.0 equiv) and the reaction mixture was stirred at 85° C. for 15 hours and monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by flash chromatography on silica gel (0-10% methanol / DCM) to afford intermediates Aa, Ba, Bb, Bc or Fa (yield 28-98%) as solids.

[0960] Methyl 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Aa) (Method 2)

[0961]

[0962] Starting from 7a (0.60 g, 1.7 mmol), 1-pentylamine 8a (0.75 g, 8.6 mmol) was used according to General Procedure II. After purification by silica gel flash chromatography (0-10% methanol / DCM), intermediate Aa (1.5 g, 83% yield) was obtained as a yellow solid. ESI m / z: 398.3 (M+H) + .

[0963] Methyl 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Ba)

[0964]

[0965] Starting from 7a (0.45 g, 1.3 mmol) and using O-butylhydroxylamine 8b (hydrochloride, 0.33 g, 2.6 mmol), following General Procedure II, intermediate Ba (0.51 g, 98% yield) was obtained as a gray solid after purification by flash chromatography on silica gel (0-10% methanol / DCM). ESI m / z: 400.3 (M+H) + ,821.5(2M+Na) + ; 1H NMR (400MHz, CDCl3) δ7.53-7.51(m,2H),6.87(d,J=8.0Hz,1H),6.79(d,J=2.9Hz,1H),6.07(d,J=2.9Hz,1H),5.46(s,2H),4.50(br s,1H),3.93(s,3H),3.90(s,3H),3.87(t,J=6.7Hz,2H),1.55-1.45(m,2H),1.37-1.18(m,2H),0.85(t,J=7.4Hz,3H)ppm.

[0966] Methyl 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}benzoate (Bb)

[0967]

[0968] Starting from 7b (0.40 g, 1.3 mmol) and using O-butylhydroxylamine 8b (hydrochloride, 0.32 g, 2.6 mmol), following General Procedure II, intermediate Bb (0.39 g, 83% yield) was obtained as an orange solid after purification by silica gel flash chromatography (0-10% methanol / DCM). ESI m / z: 370.3 (M+H) + ,761.4(2M+Na) + ; 1 H NMR (400MHz, CDCl3) δ7.95(d,J=8.3Hz,2H),7.18(d,J=8.3Hz,2H),6.74(d,J=2.9Hz,1H),6.07(d,J=2.9 Hz,1H),5.46(s,2H),3.94-3.77(m,5H),1.50-1.40(m,2H),1.30-1.20(m,2H),0.82(t,J=7.4Hz,3H)ppm.

[0969] tert-Butyl N-[(4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}phenyl)methyl]carbamate (Bc)

[0970]

[0971] Starting from 7c (0.14 g, 0.36 mmol), with O-butylhydroxylamine 8b (hydrochloride, 91 mg, 0.72 mmol), following General Procedure II, intermediate Bc (45 mg, 28% yield) was obtained as a white solid after purification by silica gel flash chromatography (0-10% methanol / DCM). ESI m / z: 441.1 (M+H)+ ,881.5(2M+Na) + .

[0972] Methyl 4-{[2-amino-6-(pentylamino)-7H-purin-7-yl]methyl}-3-methoxybenzoate (Fa)

[0973]

[0974] Starting from 7d (0.28 g, 0.81 mmol) and using 1-pentylamine 8a (0.21 g, 2.4 mmol), following General Procedure II, intermediate Fa (0.29 g, 90% yield) was obtained as a white solid after purification by silica gel flash chromatography (0-5% methanol / DCM). ESI m / z: 399.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ7.97(s,1H),7.53(d,J=1.2Hz,1H),7.49(dd,J=7.9,1.3Hz,1H),6.71(d,J=7.9Hz,1H),6.00(t,J=5.5Hz,1H),5.66(s,2H),5.58(s, 2H),3.93(s,3H),3.84(s,3H),3.35-3.22(m,2H),1.47-1.30(m,2H),1.25-1.07(m,2H),1.08-0.91(m,2H),0.75(t,J=7.3Hz,3H)ppm.

[0975] Example 4

[0976] General Procedure III for the Synthesis of Intermediates Ca, Cb, and Cc

[0977]

[0978] To a solution of compound 7a-c (1.0 equiv) in 1-butanol (0.10-0.15 M) was added DIPEA (4 equiv) and 2-ethoxyethane-1-amine 8c (2.0 equiv), and the reaction mixture was protected with argon and stirred in a sealed tube at 120° C. for 4-5 hours and 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 / TFA aqueous solution (0.01%)) to afford intermediate Ca-c (yield 51-61%) as a yellow solid.

[0979] Methyl 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzoate (Ca)

[0980]

[0981] Starting from 7a (0.17 g, 0.49 mmol), amine 8c (87 mg, 0.98 mmol) was used according to General Procedure III. After purification by reverse phase flash chromatography (0-100% acetonitrile / 0.01% aqueous TFA), intermediate Ca (0.12 g, 61% yield) was obtained as a yellow solid. ESI m / z: 400.3 (M+H) + .

[0982] Methyl 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzoate (Cb)

[0983]

[0984] Starting from 7b (0.16 g, 0.49 mmol), amine 8c (87 mg, 0.98 mmol) was used according to General Procedure III. After purification by reverse phase flash chromatography (0-100% acetonitrile / 0.01% aqueous TFA), intermediate Cb (0.12 g, 61% yield) was obtained as a yellow solid. ESI m / z: 370.3 (M+H) + .

[0985] Tert-butyl N-{[4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)phenyl]methyl}carbamate (Cc)

[0986]

[0987] Starting from 7c (0.18 g, 0.45 mmol), amine 8c (80 mg, 0.90 mmol) was used according to General Procedure III. After purification by reverse phase flash chromatography (0-100% acetonitrile / 0.01% aqueous TFA), intermediate Cc (90 mg, 51% yield) was obtained as a yellow solid. ESI m / z: 441.5 (M+H) + .

[0988] Example 5

[0989] Methyl 4-{[2-amino-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoate (Da)

[0990]

[0991] To a solution of compound 7a (0.50 g, 1.4 mmol) in 1-pentanol (5 mL) was added HCl / dioxane (4 M, 1 mL), the reaction mixture was sealed, stirred at 120° C. for 5 hours, and monitored by LCMS. After cooling, the mixture was concentrated in vacuo, and the residue was purified by silica gel flash chromatography (0-10% methanol / DCM) to give a mixture of compound Da and its hydrolysis product 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) + .

[0992] Example 6

[0993] General Procedure IV for the Synthesis of Intermediates 9a and 9b

[0994]

[0995] To a solution of compound 7a or 7d (1.0 equivalent) in dioxane and water (v / v=4, 0.10-0.12 M) was added potassium carbonate (5.0 equivalents), boronic acid 8e (3.0 equivalents) and tetrakis(triphenylphosphine)palladium (0.20 equivalents). The reaction mixture was protected by nitrogen and stirred at 80° C. for 15 hours, monitored by LCMS. The volatiles were removed in vacuo and the residue was directly separated by silica gel column chromatography (0-5% methanol / DCM) to give compound 9a or 9b (yield 53-90%) as a white solid.

[0996] Methyl 4-[(2-amino-4-(hex-1-en-1-yl)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]-3-methoxybenzoate (9a)

[0997]

[0998] Starting from 7a (0.40 g, 1.2 mmol), compound 9a (0.40 g, 90% yield) was obtained according to General Procedure IV as a yellow solid. ESI m / z: 395.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ7.70(d,J=3.0Hz,1H),7.57(s,1H),7.44(d,J=7.8Hz,1H),6.88-6.69(m,1H),6.35(m,2H),6.21(d,J=3.0Hz,1H), 5.83(s,2H),5.52(s,2H),3.98(s,3H),3.83(s,3H),2.05-1.95(m,2H),1.20-1.01(m,4H),0.77(t,J=7.0Hz,3H)ppm.

[0999] Methyl 4-{[2-amino-6-(hex-1-en-1-yl)-7H-purin-7-yl]methyl}-3-methoxybenzoate (9b)

[1000]

[1001] Starting from 7d (0.27 g, 0.78 mmol), compound 9b (0.18 g, 53% yield) was obtained according to general procedure IV as a yellow solid. ESI m / z: 396.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ8.39(s,1H),7.57(s,1H),7.47(d,J=7.8Hz,1H),6.92-6.77(m,1H),6.60(d,J=7.8Hz,1H),6.27(d,J=15.1Hz,1H), 6.14(s,2H),5.58(s,2H),3.97(s,3H),3.84(s,3H),2.08-1.95(m,2H),1.21-1.01(m,4H),0.77(t,J=7.0Hz,3H)ppm.

[1002] Methyl 4-{[2-amino-6-(hex-1-en-1-yl)-9H-purin-9-yl]methyl}-3-methoxybenzoate (9b')

[1003]

[1004] Starting from 7d' (0.10 g, 0.29 mmol), compound 9b' (60 mg, 53% yield) was obtained according to General Procedure IV as a yellow solid. ESI m / z: 396.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ8.09(s,1H),7.53-7.49(m,3H),6.80(d,J=7.4Hz,1H),6.66(d,J=16.3Hz,1H),6.40(s,2H),5.29 (s,2H),3.94(s,3H),3.85(s,3H),2.40-2.28(m,2H),1.37-1.31(m,4H),0.92(t,J=7.0Hz,3H)ppm.

[1005] Comparison of the NOESY data for 9b and 9b' revealed that the NOESY spectrum of compound 9b showed a signal between the benzyl CH2 (5.58 ppm, s, 2H) and CH=CH (6.27 ppm, d, J = 15.1 Hz, 1H), while the NOESY spectrum of compound 9b' showed no signal between the benzyl CH2 (5.29 ppm, s, 2H) and CH=CH (6.66 ppm, d, J = 16.3 Hz, 1H). This result indicated that compounds 7d and 9b were the desired intermediates.

[1006] Example 7

[1007] Methyl 4-({2-amino-4-hexyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzoate (Ea)

[1008]

[1009] To a solution of compound 9a (0.38 g, 0.96 mmol) in ethyl acetate (30 mL) was added palladium on carbon (containing 10% palladium, 40 mg) under nitrogen. The reaction mixture was stirred at room temperature under hydrogen for 15 hours and monitored by LCMS. The resulting mixture was filtered through celite, and the filtrate was concentrated in vacuo to afford compound Ea (0.38 g, 95% yield) as a white solid. ESI m / z: 397.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ7.66(d,J=2.9Hz,1H),7.56(s,1H),7.45(d,J=7.9Hz,1H),6.25(d,J=7.9Hz,1H),6.21(d,J=2.9Hz,1H),5.81(s,2H),5 .48(s,2H),3.96(s,3H),3.83(s,3H),2.49-2.41(m,2H),1.36-1.25(m,2H),1.21-0.93(m,6H),0.78(t,J=7.2Hz,3H)ppm.

[1010] Example 8

[1011] Methyl 4-[(2-amino-6-hexyl-7H-purin-7-yl)methyl]-3-methoxybenzoate (Fb)

[1012]

[1013] To a solution of compound 9b (0.20 g, 0.51 mmol) in ethanol (15 mL) was added palladium hydroxide (20 mg, 10 wt%) under nitrogen. The reaction mixture was stirred at 50° C. under a hydrogen balloon (1.1 atm) for 15 hours and monitored by LCMS. After cooling, the resulting mixture was filtered through celite and the filtrate was concentrated in vacuo to afford 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) + .

[1014] Example 9

[1015] General Procedure for the Reduction of Methyl Benzoate to Benzyl Alcohol V

[1016]

[1017] To the stirred suspension (0.04-0.20M) of lithium aluminum hydride (LAH, 2.0 equivalents) in anhydrous THF at 0 ℃ under nitrogen protection, dropwise add a solution (0.02-0.20M) of ester (1.0 equivalents) in anhydrous THF 5 minutes. The reaction mixture was stirred at 0 ℃ for 15 minutes, then stirred at room temperature for half an hour, until LCMS showed that the ester was completely reduced. The gained mixture was cooled to 0 ℃, and quenched carefully with saturated sodium bicarbonate aqueous solution (5% volume) and water (5% volume). The mixture was filtered, and the filtrate was extracted with ethyl acetate (x3). The organic solution merged was washed with salt water, and concentrated in vacuo. The crude product was purified through preparative HPLC (5-95% acetonitrile / TFA aqueous solution (0.05%)) to obtain payload (43% yield, TFA salt), which was a white solid.

[1018] Payload P1

[1019] (4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methanol (P1)

[1020]

[1021] Starting from intermediate Aa (65 mg, 0.16 mmol), following general procedure V, payload P1 (35 mg, 43% yield, TFA salt) was obtained as a white solid. ESI m / z: 389.3 (M+H)+ . 1 H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 3.2 Hz, 1H), 7.22 (br s, 2H), 7.02 (s, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 6.18 (d, J = 3.2 Hz, 1H), 5.50 (s, 2H), 5.21 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 5.6 Hz, 2H), 3.83 (s, 3H), 3.41-3.49 (m, 2H), 1.52-1.40 (m, 2H), 1.30-1.19 (m, 2H), 1.19-1.01 (m, 2H), 0.82 (t, J = 7.2 Hz, 3H) ppm. (COOH of TFA not shown) 19 F NMR (376 MHz, DMSO d6 )δ-73.4ppm.

[1022] Payload P20

[1023] (4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methanol (P20)

[1024]

[1025] Starting from intermediate Ba (0.15 g, 0.38 mmol), following general procedure V, payload P1 (0.13 g, 72% yield, TFA salt) was obtained as a white solid. ESI m / z: 372.5 (M+H) + . 1 H NMR (400MHz, CDCl3) δ12.06(s,1H),10.73(s,1H),7.90(s,2H),7.11(d,J=2. 9Hz,1H),6.98(s,1H),6.79(d,J=7.8Hz,1H),6.64(d,J=7.8Hz,1H),6.08(d, J=2.9Hz,1H),5.35(s,2H),5.17(s,1H),4.46(s,2H),3.95(t,J=6.6Hz,2H), 3.83(s,3H),1.59-1.48(m,2H),1.36-1.23(m,2H),0.85(t,J=7.4Hz,3H)ppm.

[1026] Payload P23

[1027] [4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxyphenyl]methanol (P23)

[1028]

[1029] Starting from intermediate Ca (30 mg, 75 μmol), following general procedure V, payload P23 (15 mg, 41% yield, TFA salt) was obtained as a white solid. ESI m / z: 372.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.30(s,1H),7.50-7.41(m,3H),7.39(d,J=3.0Hz,1H),7.02(s,1H),6.83(d,J=7.7Hz,1H),6.76(d,J=7.7Hz,1H),6.19(d,J=3.0Hz,1H),5 .48(s,2H),5.23(s,1H),4.47(s,2H),3.83(s,3H),3.66(q,J=7.0Hz,2H),3.48(t,J=5.9Hz,2H),3.42-3.38(m,2H),1.06(t,J=7.0Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.5ppm.

[1030] Payload P27

[1031] (4-{[2-amino-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methanol (P27)

[1032]

[1033] Starting from intermediate Da (60 mg, 0.15 mmol), following general procedure V, payload P27 (45 mg, 62% yield, TFA salt) was obtained as a white solid. ESI m / z: 371.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ13.17(s,1H),7.97(s,2H),7.68(d,J=2.9Hz,1H),7.00(s,1H),6.79(d,J=7.2Hz,1H),6.50(d,J=7.7Hz,1H),6.34(d,J=2.9Hz,1H),5. 43(s,2H),5.20(s,1H),4.46(s,2H),4.42(t,J=6.3Hz,2H),3.83(s,3H),1.66-1.54(m,2H),1.29-1.09(m,4H),0.81(t,J=7.1Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.5ppm.

[1034] Payload P29

[1035] [4-({2-amino-4-hexyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxyphenyl]methanol (P29)

[1036]

[1037] Starting from intermediate Ea (0.38 g, 0.96 mmol), following general procedure V, payload P29 (0.30 g, 65% yield, TFA salt) was obtained as a white solid. ESI m / z: 369.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ8.11-7.75(m,3H),7.07(s,1H),6.80(d,J=6.6Hz,1H),6.45(s,1H),6.30(d,J=6.6Hz,1H),5.52(s,2H),5.26(s, 1H),4.48(s,2H),3.87(s,3H),2.77-2.61(m,2H),1.49-1.35(m,2H),1.27-1.01(m,6H),0.83(t,J=6.5Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1038] Payload P32

[1039] (4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}phenyl)methanol (P32)

[1040]

[1041] Starting from intermediate Bb (82 mg, 0.22 mmol), following general procedure V, payload P32 (70 mg, 69% yield, TFA salt) was obtained as a white solid. ESI m / z: 342.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.12(s,1H),10.76(s,1H),7.93(s,2H),7.28(d,J=3.0Hz,1H),7.26(d,J= 8.0Hz,2H),7.16(d,J=8.0Hz,2H),6.08(d,J=3.0Hz,1H),5.38(s,2H),5.15(br s,1H),4.45(s,2H),4.03(t,J=6.6Hz,2H),1.66-1.53(m,2H),1.43-1.26(m,2H),0.89(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1042] Payload P33

[1043] [4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)phenyl]methanol (P33)

[1044]

[1045] Starting from intermediate Cb (28 mg, 75 μmol), following general procedure V, payload P33 (15 mg, 44% yield, TFA salt) was obtained as a white solid. ESI m / z: 342.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.29(s,1H),7.63(d,J=3.0Hz,1H),7.49(t,J=5.5Hz,1H),7.42(s,2H),7.26(d,J=8.0Hz,2H),7.05(d,J=8.0Hz,2H),6.24(d,J=3.0Hz,1H ),5.59(s,2H),5.21-5.14(m,1H),4.44(s,2H),3.63(q,J=6.8Hz,2H),3.43(t,J=7.0Hz,2H),3.39(t,J=7.0Hz,2H),1.07(t,J=7.0Hz,3H)ppm.19 F NMR (376 MHz, DMSO d6 )δ-73.5ppm.

[1046] Payload P37

[1047] (4-{[2-amino-6-(pentylamino)-7H-purin-7-yl]methyl}-3-methoxyphenyl)methanol (P37)

[1048]

[1049] Starting from intermediate Fa (0.28 g, 0.70 mmol), following general procedure V, payload P37 (0.13 g, 38% yield, TFA salt) was obtained as a white solid. ESI m / z: 371.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ13.27(s,1H),8.14(s,1H),7.70(t,J=5.4Hz,1H),7.56(s,2H),7.02(s,1H),6.97(d,J=7.6Hz,1H),6.86(d,J=7.6Hz,1H),5.58( s,2H),5.27(s,1H),4.48(s,2H),3.81(s,3H),3.60-3.40(m,2H),1.59-1.45(m,2H),1.34-1.10(m,4H),0.84(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.5ppm.

[1050] Payload P39

[1051] {4-[(2-amino-6-hexyl-7H-purin-7-yl)methyl]-3-methoxyphenyl}methanol (P39)

[1052]

[1053] Starting from intermediate Fb (0.21 g, 0.53 mmol), following general procedure V, payload P37 (0.13 g, 51% yield, TFA salt) was obtained as a white solid. ESI m / z: 370.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ8.40(s,1H),7.05(s,1H),6.80(d,J=7.8Hz,1H),6.43(d,J=7.8Hz,1H),6.32(br s,2H),5.45(s,2H),5.26(s,1H),4.48(s,2H),3.86(s,3H),2.59-2.48(m,2H),1.45-1.28(m,2H),1.24-1.04(m,6H),0.82(t,J=7.2Hz,3H)ppm.(COOH of TFA is not shown) 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1054] Example 10

[1055] Payload P2

[1056] 2-(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)propan-2-ol (P2)

[1057]

[1058] Under nitrogen protection, methylmagnesium bromide (3.0M / THF, 0.43mL, 1.3mmol) was added dropwise to a cooled (-5 to 5°C) solution of intermediate Aa (50mg, 0.13mmol) in anhydrous THF (5.0mL). The reaction mixture was stirred at 0°C for half an hour, then at room temperature for 2 hours and monitored by LCMS. The resulting mixture was quenched with methanol and the volatile substances 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 (5-100% acetonitrile / TFA aqueous solution (0.01%)) to give the payload P2 (10mg, 15% yield, TFA salt) as a white solid. ESI m / z: 398.3 (M+H) + . 1 H NMR (400 MHz, MeOD d4 )δ7.35(d,J=3.0Hz,1H),7.23(d,J=1.4Hz,1H),6.99(dd,J=8.0,1.5Hz,1H),6.73(d,J=7.9Hz,1H),6.19(d,J=3.0Hz ,1H),5.49(s,2H),3.92(s,3H),3.55-3.48(m,2H),1.50-1.44(m,8H),1.33-1.29(m,4H),0.87(t,J=6.3Hz,3H)ppm.19 F NMR (376 MHz, MeOD d4 )δ-73.5ppm.

[1059] Example 11

[1060] General Procedure VI for the Synthesis of Piperazine Analogs P3, P26, P28, P36, and P38

[1061]

[1062] At 0 ℃, to the suspension of alcohol payload P1, P27, P29, P37 or P39 (1.0 equivalent) in DCM (50mM), a solution of thionyl chloride (1.2 equivalent) in DCM (0.1M) was added dropwise. The reaction mixture was stirred at room temperature for 5 hours. Volatile substances were removed in vacuo to obtain rough corresponding chloride P3-1, P26-1, P28-1, P36-1 or P38-1, which was a yellow semi-solid and was dissolved in DMF (25mg / mL). Potassium carbonate (2.0 equivalent) and N-Boc piperazine (1.0 equivalent) were added to the solution, and the reaction mixture was stirred at room temperature for 15 hours, monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (0-95% methanol / TFA in water (0.01%)) to afford the Boc-payload P3-2, P26-2, P28-2, P36-2, or P38-2 as a white solid, which was dissolved in DCM (25 mg / mL). To this solution was added a solution of hydrochloric acid in dioxane (4 M, v / v DCM =2 / 3, pH <1). The reaction mixture was stirred at room temperature for 2 hours and monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse phase flash chromatography (0-95% acetonitrile / TFA in water (0.01%)) to afford the piperazine payload P3, P26, P28, P36, or P38 (31-72% yield) as a white solid.

[1063] Payload P3

[1064] 5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-N 4 -pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P3)

[1065]

[1066] Starting from P1, following General Procedure VI, payload P3 (0.23 g, 31% yield, TFA salt) was obtained as a white solid. ESI m / z: 438.4 (M+H) + . 1H NMR (400 MHz, DMSO d6 )δ12.81(s,1H),9.21(br s,1H),7.55(s,2H),7.41(d,J=3.0Hz,2H),7.15(s,1H),6.93(d,J=7.7Hz,1H),6.60(d,J=7.8Hz,1H),6 .21(d,J=3.0Hz,1H),5.56(s,2H),3.97(s,2H),3.85(s,3H),3.55-3.35(m,2H),3.27(brs,4H),2.99(br s,4H),1.55-1.34(m,2H),1.33-1.16(m,2H),1.15-1.04(m,2H),0.82(t,J=7.3Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-74.0ppm.

[1067] Payload P26

[1068] 5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-4-(pentyloxy)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (P26)

[1069]

[1070] Starting from P27, following General Procedure VI, payload P26 (15 mg, 31% yield, TFA salt) was obtained as a white solid. ESI m / z: 439.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ9.10(s,1H),8.21(s,2H),7.70(d,J=2.9Hz,1H),7.13(s,1H),6.90(d,J=7.6Hz,1H),6.52(d,J =7.7Hz,1H),6.36(d,J=2.9Hz,1H),5.46(s,2H),4.41(t,J=6.3Hz,2H)3.90-3.78(m,5H),3.24(br s,4H),2.95(brs,4H),1.66-1.47(m,2H),1.35-1.01(m,4H),0.80(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-74.0ppm.

[1071] Payload P28

[1072] 4-Hexyl-5-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-5H-pyrrolo[3,2-d]pyrimidin-2-amine (P28)

[1073]

[1074] Starting from P29, following General Procedure VI, payload P28 (46 mg, 53% yield, TFA salt) was obtained as a white solid. ESI m / z: 437.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ9.12(s,1H),8.01(br s,2H),8.00(d,J=3.0Hz,1H),7.21(s,1H),6.93(d,J=7.6Hz,1H),6.48(d,J=2. 9Hz,1H),6.37(d,J=7.7Hz,1H),5.56(s,2H),4.02(s,2H),3.89(s,3H),3.27(br s,4H),3.00(br s,4H),2.78-2.64(m,2H),1.50-1.38(m,2H),1.20-1.08(m,6H),0.83(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-74.2ppm.

[1075] Payload P36

[1076] 7-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-N 6 -Pentyl-7H-purine-2,6-diamine (P36)

[1077]

[1078] Starting from P37, following General Procedure VI, payload P36 (78 mg, 66% yield, TFA salt) was obtained as a white solid. ESI m / z: 439.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ9.02(br s,1H),8.15(s,1H),7.79(t,J=5.6Hz,1H),7.72(s,2H),7.10(s,1H),6.99(d,J=7.6Hz,1H) ,6.93(d,J=7.6Hz,1H),5.61(s,2H),3.82(s,3H),3.81(s,2H),3.53-3.45(m,2H),3.20(br s,4H),2.81(br s,4H),2.55(s,1H),1.58-1.42(m,2H),1.37-1.02(m,4H),0.84(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.9ppm.

[1079] Payload P38

[1080] 6-Hexyl-7-({2-methoxy-4-[(piperazin-1-yl)methyl]phenyl}methyl)-7H-purin-2-amine (P38)

[1081]

[1082] Starting from P39, following General Procedure VI, payload P38 (79 mg, 72% yield, TFA salt) was obtained as a white solid. ESI m / z: 438.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ8.72(s,2H),8.49(s,1H),7.10(s,1H),6.87(d,J=7.6Hz,1H),6.53(br s,1H),6.51(d,J=7.6Hz,1H),5.48(s,2H),3.87(s,3H),3.69(br s,2H),3.14(br s,4H),2.67(br s,4H),2.60-2.53(m,2H),1.40-1.28(m,2H),1.25-1.00(m,6H),0.83(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.8ppm.

[1083] Example 12

[1084] General Procedure for the Synthesis of Amides P22, P25, P31, and P35 VII

[1085]

[1086] To a solution (50-100 mM) of ester Ba, Ca, Bb or Cb (1.0 equivalent) in isopropanol or methanol is added sodium hydroxide or lithium hydroxide aqueous solution (1 M, 0.5 v alcohol solvent). The reaction mixture is stirred at room temperature for 1 hour and monitored by LCMS. The residual solution is neutralized to pH 6 with dilute aqueous hydrochloric acid (1 M) and the resulting mixture is concentrated. The residue is purified by reverse phase flash chromatography (0-95% acetonitrile / TFA aqueous solution (0.01%) to obtain acid P22-1, P25-1, P31-1 or P35-1 as a yellow solid, which is dissolved in DMF (50-80 mM). Ammonium chloride (2.0 equivalents), HATU (1.2 equivalents) and DIPEA (3.0 equivalents) are added to the solution and the reaction mixture is stirred at room temperature for 3 hours 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.

[1087] Payload P22

[1088] 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzamide (P22)

[1089]

[1090] Starting from Ba, following General Procedure VII, payload P22 (82 mg, 28% yield, TFA salt) was obtained as a white solid. ESI m / z: 385.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.09(s,1H),10.75(s,1H),7.95(s,1H),7.94(br s,2H),7.50(d,J=1.2Hz,1H),7.38(dd,J=7.6Hz and 1.2Hz,1H),7.36(s,1H),7.19(d,J=2.9Hz,1H),6.53(d,J=7.6Hz,1H),6.13(t,J=2.9Hz,1H),5 .41(s,2H),3.98-3.81(m,5H),1.49-1.36(m,2H),1.30-1.15(m,2H),0.80(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1091] Payload P25

[1092] 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)-3-methoxybenzamide (P25)

[1093]

[1094] Starting from Ca, following General Procedure VII, payload P25 (60 mg, 31% yield, TFA salt) was obtained as a white solid. ESI m / z: 385.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.35(s,1H),7.99(s,1H),7.52(d,J=1.3Hz,1H),7.47(d,J=3.0Hz,3H),7.43-7.36(m,3H),6.67(d,J=7.9Hz,1H),6.24(d,J= 3.0Hz,1H),5.57(s,2H),3.90(s,3H),3.63(q,J=5.7Hz,2H),3.44(t,J=5.9Hz,2H),3.33-3.28(m,2H),1.01(t,J=7.0Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.4ppm.

[1095] Payload P31

[1096] 4-{[2-amino-4-(butoxyamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}benzamide (P31)

[1097]

[1098] Starting from Bb, following General Procedure VII, payload P31 (5.0 mg, 4% yield, TFA salt) was obtained as a white solid. ESI m / z: 355.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ12.13(s,1H),10.76(s,1H),7.93(s,3H),7.81(d,J=8.3Hz,2H),7.35(s,1H),7.31(d,J=2.9Hz,1H),7.19(d,J=8.2Hz), 6.12(d,J=2.9Hz,1H),5.45(s,2H),3.97(t,J=6.5Hz,2H),1.55-1.44(m,2H),1.35-1.23(m,2H),0.85(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1099] Payload P35

[1100] 4-({2-amino-4-[(2-ethoxyethyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-yl}methyl)benzamide (P35)

[1101]

[1102] Starting from Cb, following General Procedure VII, payload P35 (50 mg, 51% yield, TFA salt) was obtained as a white solid. ESI m / z: 355.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.26(s,1H),7.93(s,1H),7.81(d,J=8.3Hz,2H),7.66(d,J=3.0Hz,1H),7.50-7.35(m,4H),7.08(d,J=8.1Hz,2H),6. 28(d,J=2.9Hz,1H),5.68(s,2H),3.61(d,J=5.9Hz,2H),3.41-3.37(m,2H),3.35-3.28(m,2H),1.02(t,J=7.0Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.5ppm.

[1103] 4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxybenzoic acid (P47)

[1104]

[1105] Starting from Aa, following General Procedure VII, but without amidation, crude acid P47 was collected. The crude acid was further purified by preparative HPLC to give pure P47 (5 mg, 60% yield) as a light yellow solid. ESI m / z: 384.2 (M+H) + .

[1106] Example 13

[1107] Payload P21

[1108] 5-{[4-(aminomethyl)-2-methoxyphenyl]methyl}-N 4 -Butoxy-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P21)

[1109]

[1110] Under nitrogen protection, at 0 ° C, to a stirred suspension of lithium aluminum tetrahydride (39 mg, 1.0 mmol) in anhydrous THF (6 mL) was added dropwise a solution of compound P22 (65 mg, 0.17 mmol) in anhydrous THF (4 mL) over 10 minutes. The reaction mixture was stirred at 65 ° C for 4 hours 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 (0-100% acetonitrile / TFA aqueous solution (0.01%)) to give P21 (31 mg, 38% yield, TFA salt) as a light yellow solid. ESI m / z: 371.4 (M + H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.53(s,1H),10.78(s,1H),8.14(br,4H),7.18(d,J=1.2Hz,1H),7.12(d,J=2.9Hz,1H) ,6.92(d,J=7.2Hz,1H),6.68(d,J=7.2Hz,1H),6.08(d,J=2.9Hz,1H),5.39(s,2H),4.00(br s,2H),3.93(t,J=6.5Hz,2H),3.85(s,3H),1.59-1.48(m,2H),1.38-1.22(m,2H),0.86(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.7ppm.

[1111] Example 14

[1112] Payload P24

[1113] 5-{[4-(aminomethyl)-2-methoxyphenyl]methyl}-N 4 -(2-Ethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P24)

[1114]

[1115] Under argon protection, a THF solution of borane (1M, 0.23 mL) was added dropwise to a solution of compound P25 (29 mg, 75 μmol) in anhydrous THF (4 mL) at 65 ° C. The reaction mixture was refluxed at 65 ° C for 4 hours under argon and monitored by LCMS. After cooling to 0 ° C, the resulting mixture was carefully quenched with saturated aqueous sodium bicarbonate solution (0.24 mL) and water (0.24 mL). The volatiles were removed in vacuo, and the residue was purified by reverse phase flash chromatography (0-100% acetonitrile / TFA aqueous solution (0.01%)) to give P24 (5 mg, 14% yield, TFA salt) as a light yellow solid. ESI m / z: 371.3 (M + H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.44(s,1H),8.19(s,2H),7.54(t,J=5.5Hz,1H),7.48(s,2H),7.39(d, J=3.0Hz,1H),7.20(s,1H),6.95(d,J=7.5Hz,1H),6.81(d,J=7.7Hz,1H),6. 21(d,J=3.0Hz,1H),5.52(s,2H),4.01(s,2H),3.84(s,3H),3.72-3.60(m,2 H), 3.50 (t, J = 5.9Hz, 2H), 3.42 (q, J = 7.0Hz, 2H), 1.07 (t, J = 7.0Hz, 3H) ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.5ppm.

[1116] Example 15

[1117] Payload P30

[1118] 5-{[4-(aminomethyl)phenyl]methyl}-N 4 -Butoxy-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P30)

[1119]

[1120] To a suspension of compound Bc (45 mg, 0.10 mmol) in DCM (1 mL) was added hydrochloric acid solution (4 M / dioxane, 0.5 mL) and the reaction mixture was stirred at room temperature for 1 hour until Boc 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 / TFA aqueous solution (0.01)) to afford P30 (45 mg, 97% yield, TFA salt) as a white solid. ESI m / z: 341.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.56(s,1H),10.82(s,1H),8.19(br s,4H),7.40(d,J=8.1Hz,2H),7.26(d,J=3.2Hz,1H),7.25(d,J=8.0Hz,2H),6.08(d,J=3.2Hz,1H) ,5.42(s,2H),4.10-3.90(m,4H),1.71-1.52(m,2H),1.42-1.28(m,2H),0.90(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.7ppm.

[1121] Example 16

[1122] Payload P34

[1123] 5-{[4-(aminomethyl)phenyl]methyl}-N 4 -(2-Ethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P34)

[1124]

[1125] Following a similar procedure to payload P30, but substituting Cc for Bc, payload P34 (0.10 g, 60% yield, TFA salt) was obtained as a white solid. ESI m / z: 341.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ12.60(s,1H),8.18(s,2H),7.64(d,J=3.0Hz,1H),7.61(t,J=5.6Hz,1H),7.51(s,2H), 7.39(d,J=8.2Hz,2H),7.14(d,J=8.2Hz,2H),6.24(d,J=3.0Hz,1H),5.63(s,2H),3.99(br s,2H),3.64(q,J=5.7Hz,2H),3.47-3.41(m,4H),1.09(t,J=7.0Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1126] Example 17

[1127] Intermediate P3-1 was synthesized following the procedure described for payload P3.

[1128] Example 18

[1129] 5-{[4-(bromomethyl)-2-methoxyphenyl]methyl}-N 4 -pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P4-1)

[1130]

[1131] To a solution of P1 (0.30 g, 0.81 mmol) in DCM was added phosphorus tribromide (0.38 mL), and the reaction mixture was stirred at room temperature for 3 hours, monitored by LCMS. The volatiles were removed in vacuo, and the residue was purified by reverse phase flash chromatography (0-100% acetonitrile / TFA aqueous solution (0.01%)) to afford P4-1 (0.32 g, 91% yield) as a yellow solid. ESI m / z: 432.1 (M+H) + .

[1132] Example 19

[1133] General Procedure for the Synthesis of Ethers by Reaction of Alcohols with Benzyl Halides VIII

[1134]

[1135] Under nitrogen protection, sodium hydride (60%, mineral, 2.5 equivalents) was added to a solution of alcohol P#-2 (2.0 equivalents) in anhydrous THF or DMF (40mM) at -15 to 0°C in one portion. The resulting suspension was stirred at -15 to 0°C for 10 minutes under nitrogen protection. Tetrabutylammonium iodide (TBAI) (0.05 equivalents) and benzyl halide (1.0 equivalents) were then added to the stirred mixture, and the reaction mixture was stirred at -15 to 0°C for 30 minutes, monitored by LCMS. The resulting mixture was quenched with methanol. The mixture (with Boc protection or propylene 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 / TFA aqueous solution (0.05%)) to give the desired ester.

[1136] Example 20

[1137] General Procedure IX for the synthesis of P4, P5, P10, P17, P18, P19, P41, and P42 by acidification with TFA

[1138]

[1139] The reaction mixture containing the quenching of P#-3 is diluted with DCM (for P4, P5, P10, P41 and P42) or THF (for P17, P18 and P19). TFA (25% volume) is added to the solution, and the reaction mixture is stirred at room temperature for half an hour, monitored by LCMS. The gained mixture is concentrated in vacuo, and the residue is purified by preparative HPLC (5-95% acetonitrile / TFA aqueous solution (0.05%)) to obtain payload P4, P5, P10, P17, P18, P19, P41 or P42 (20% yield), which is a white solid.

[1140] Example 21

[1141] Payload P4

[1142] 5-({4-[(2-aminoethoxy)methyl]-2-methoxyphenyl}methyl)-N 4 -pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P4)

[1143]

[1144] Following general procedures VIII and IX, starting from benzyl bromide P4-1, it was reacted with N-Boc-aminoethanol P4-2 to afford payload P4 (13 mg, 15% yield, di-TFA salt) as a white solid. ESI m / z: 413.4 (M+H)+ . 1 H NMR (400 MHz, DMSO d6 )δ12.43(br s,1H),7.87(br s,3H),7.50-7.35(m,4H),7.06(s,1H),6.88(d,J=7.6Hz,1H),6.60(d,J=7.6Hz,1H),6. 21(d,J=2.8Hz,1H),5.54(s,2H),4.50(s,2H),3.48(s,3H),3.70-3.40(m,4H),3.02(br s,2H),1.52-1.42(m,2H),1.27-1.18(m,2H),1.15-1.06(m,2H),0.81(t,J=7.2Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1145] Example 22

[1146] Payload P5

[1147] 5-({4-[(4-aminobutyloxy)methyl]-2-methoxyphenyl}methyl)-N 4 -pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P5)

[1148]

[1149] Following General Procedures VIII and IX, starting from benzyl bromide P4-1, the reaction with N-Boc-aminobutanol P5-2 was followed. After purification by preparative HPLC (5-95% acetonitrile / aqueous formic acid (0.1%)), the payload P5 (2 mg, 2% yield, formate salt) was obtained as a light yellow solid. ESI m / z: 441.5 (M+H) + . 1 H NMR (400 MHz, MeOD d4)δ7.26(d,J=3.0Hz,1H),6.97(s,1H),6.81(d,J=7.6Hz,1H),6.62(d,J=7.6Hz,1H),6.12(d,J=3.0Hz,1H),5.42(s,2H),4.51(br s,1H),4.41(s,2H),3.82(s,3H),3.51-3.38(m,4H),2.90-2.81(m,2H),1.70-1.55(m,4 H),1.42-1.33(m,2H),1.22-1.13(m,2H),1.10-0.99(m,2H),0.77(t,J=7.4Hz,3H)ppm.

[1150] Example 23

[1151] Payload P6

[1152] 2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]ethoxy}ethan-1-ol (P6)

[1153]

[1154] Following General Procedure VIII, starting from benzyl bromide P4-1, it was reacted with diethylene glycol P6-2 to afford payload P6 (12 mg, 23% yield, TFA salt) as an off-white solid. ESI m / z: 458.1 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.44(s,1H),7.44(s,2H),7.41(d,J=3.0Hz,1H),7.34(t,J=5.2Hz,1H),7.03(s,1H),6.83(d,J=7.6Hz,1H),6.56(d,J=7.6Hz,1H),6.20(d,J=3.0Hz,1H),5.53(s,2H),4.46(s,2H),3.84(s,3H),3.60-3.34(11H, covered or partially covered by water peak),1.52-1.34(m,2H),1.28-1.20(m,2H),1.19-1.00(m,2H),0.81(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.7ppm.

[1155] Example 24

[1156] Payload P8

[1157] 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)

[1158]

[1159] Following General Procedure VIII, starting from benzyl chloride P3-1, it was reacted with tetraethylene glycol P8-2 (CAS: 112-60-7) to afford payload P8 (36 mg, 6.4% yield, TFA salt) as an off-white solid. ESI m / z: 546.5 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.59(s,1H),7.49(s,2H),7.42(d,J=3.2Hz,1H),7.34(t,J=5.2Hz,1H),7.02(s,1H),6.83(d,J=7. 6Hz,1H),6.55(d,J=7.6Hz,1H),6.20(d,J=3.2Hz,1H),5.54(s,2H),4.46(s,2H),3.84(s,3H),3.80(br s,1H),3.58-3.36(m,18H),1.51-1.40(m,2H),1.26-1.18(m,2H),1.18-1.01(m,2H),0.81(t,J=7.4Hz,3H)ppm.

[1160] Payload P44

[1161] 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)

[1162]

[1163] Following General Procedure VIII, starting from benzyl chloride P3-1, hydroxy-PEG3-CH2CO2 t Bu (CAS: 518044-31-0) was reacted to obtain the payload P44 (4.6 mg, 13% yield) as a white solid (the tBu group was lost during the reaction). ESI m / z: 560.3 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ7.27(d,J=3.2Hz,1H),7.00(s,1H),6.80(d,J=7.6Hz,1H),6.46(d,J=7.6Hz,1H),6.28(s,1H),6.02(d,J=2.4Hz,1H),5.43(s,2H) ,4.44(s,2H),3.84(s,3H),3.54-3.50(m,17H),1.42-1.38(m,2H),1.23-1.18(m,2H),1.11-1.05(m,2H),0.80(t,J=7.6Hz,3H)ppm.

[1164] Payload P45

[1165] 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)

[1166]

[1167] Following General Procedure VIII, starting from benzyl chloride P3-1, hydroxy-PEG2-CH2CO2 t Bu (CAS: 149299-82-1) was reacted to obtain the payload P45 (3.9 mg, 15% yield) as a white solid (the tBu group was lost during the reaction). ESI m / z: 516.3 (M+H) + .

[1168] Payload P46

[1169] 2-{2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]ethoxy}acetic acid (P46)

[1170]

[1171] Following General Procedure VIII, starting from benzyl chloride P3-1, it was reacted with tert-butyl 2-(2-hydroxyethoxy)acetate (CAS: 287174-32-7) to afford payload P46 (3.2 mg, 12% yield) as a white solid (the tBu group was lost during the reaction). ESI m / z: 472.3 (M+H) + .

[1172] Payload P48

[1173] 2-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]ethan-1-ol (P48)

[1174]

[1175] Following General Procedure VIII, starting from benzyl chloride P3-1, it was reacted with ethylene glycol (CAS: 107-21-1) to afford payload P48 (5 mg, 10% yield, TFA salt) as a white solid. ESI m / z: 414.3 (M+H) + .

[1176] Example 25

[1177] Payload P9

[1178] 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)

[1179]

[1180] Following General Procedure VIII, starting from benzyl bromide P4-1, it was reacted with 11-azido-3,6,9-trioxaundecanol P9-2 (CAS: 86770-76-4) to afford compound P9-3 (3.0 g, 45% yield, TFA salt) as a yellow solid. ESI m / z: 571.5 (M+H) + .

[1181] 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)

[1182]

[1183] To a solution of compound P9-3 (1.7 g, 3.0 mmol) in methanol (50 mL) was added palladium on carbon (containing 10 wt% palladium, 0.20 g) under nitrogen. The reaction mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere 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 / TFA aqueous solution (0.05%)) to give P9 (0.40 g, 20% yield, di-TFA salt) as a light yellow solid. ESI m / z: 545.5 (M+H)+ ,273.4(M / 2+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.54(s,1H),7.82(br s, 3H), 7.46 (s, 2H), 7.40 (d, J = 3.2 Hz, 1H), 7.33 (t, J = 5.6 Hz, 1H), 7.01 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.58 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.2 Hz, 1H), 5.54 (s, 2H), 4.46 (s, 2H), 3.83 (s, 3H), 3.70-3.45 (16H, partially covered by water peak), 3.04-2.83 (m, 2H), 1.51-1.40 (m, 2H), 1.27-1.20 (m, 2H), 1.20-1.04 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H) ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.7ppm.

[1184] Example 26

[1185] Payloads P10 and P43

[1186] 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.

[1187] 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)

[1188]

[1189] Following General Procedure VIII, starting from benzyl bromide P4-1, reaction with P10-2 afforded payload P43 as a yellow solid. ESI m / z: 358.3 (M / 2+H)+.

[1190] 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)

[1191]

[1192] Following General Procedure IX, starting from P43, payload P10 (22 mg, 16% yield, di-TFA salt) was obtained as a yellow solid. ESI m / z: 308.0 (M / 2+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.67(s,1H),9.39(br s,2H),7.50(br s, 2H), 7.41 (d, J = 3.0 Hz, 1H), 7.34 (t, J = 5.4 Hz, 1H), 7.01 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H), 6.20 (d, J = 3.0 Hz, 1H), 5.54 (s, 2H), 4.46 (s, 2H), 3.83 (s, 3H), 3.75-3.25 (m, 26H, covered by water peak), 1.50-1.42 (m, 2H), 1.26-1.19 (m, 2H), 1.19-1.01 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H) ppm. 19 F NMR (376 MHz, DMSO d6 )δ-74.1ppm.

[1193] Example 27

[1194] Payload P17

[1195] (2R)-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]propane-1,2-diol (P17)

[1196]

[1197] Following General Procedures VIII and IX, starting from benzyl bromide P4-1, it was reacted with S-glycerol acetonide P17-2 to afford payload P17 (9 mg, 7% yield, TFA salt) as a light yellow solid. ESI m / z: 444.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6)δ12.36(br s,1H),7.41(d,J=3.0Hz,1H),7.40(s,2H),7.32(t,J=5.6Hz,1H),7.04(s,1H),6.83 (d,J=7.6Hz,1H),6.57(d,J=7.6Hz,1H),6.20(d,J=3.0Hz,1H),5.53(s,2H),4.72(br s,1H),4.54(br s, 1H), 4.46 (s, 2H), 3.83 (s, 3H), 3.71-3.25 (m, 7H, covered by water peak), 1.51-1.42 (m, 2H), 1.35-1.18 (m, 2H), 1.16-1.02 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H) ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1198] Example 28

[1199] Payload P18

[1200] (2S)-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]propane-1,2-diol (P18)

[1201]

[1202] Following general procedures VIII and IX, starting from benzyl bromide P4-1, it was reacted with R-glycerol acetonide P18-2 to afford payload P18 (9 mg, 7% yield, TFA salt) as a light yellow solid. 1 H NMR (400 MHz, DMSO d6 )δ12.31(s,1H),7.41(d,J=3.0Hz,1H),7.38(s,2H),7.31(t,J=5.6Hz,1H),7.04(s,1H),6.83(d,J=8.0Hz,1H),6.58(d,J=8.0Hz,1H),6.21(d,J=3.0Hz,1H),5.53(s,2H),4.46(s,2H),3.83(s,3H),3.71-3.25(m,9H, covered by water peak),1.51-1.42(m,2H),1.29-1.20(m,2H),1.20-1.03(m,2H),0.84(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6)δ-73.6ppm.

[1203] Example 29

[1204] Payload P19

[1205] 2-Amino-3-[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]propan-1-ol (P19)

[1206]

[1207] Following general procedures VIII and IX, starting from benzyl bromide P4-1, it was reacted with N-Boc-serinol P19-2 to afford payload P19 (13 mg, 6% yield, di-TFA salt) as a white solid. ESI m / z: 443.4 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.48(br s,1H),7.96(s,3H),7.45(s,2H),7.43-7.35(m,2H),7.05(s,1H),6.86(d,J=8.0Hz,1H),6.59(d,J=8.0Hz,1H),6.20(d,J=2.8Hz,1H),5.54(s,2H),5.32(br s,1H),4.50(s,2H),3.84(s,3H),3.62-3.23(7H,partially covered by water peak),1.51-1.44(m,2H),1.29-1.20(m,2H),1.20-1.05(m,2H),0.84(t,J=7.4Hz,3H)ppm. 19 F NMR (376 MHz, DMSO d6 )δ-73.6ppm.

[1208] Example 30

[1209] Payload P41

[1210] 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)

[1211]

[1212] Following General Procedure VIII, starting from benzyl bromide P4-1, it was reacted with N-Boc-PEG3-OH (P41-2, CAS: 139115-92-7) to afford compound P41-3 (51 mg, 71% yield) as a white solid. LC retention time: 2.05 min.; ESI m / z: 601.3 (M+H)+.

[1213] 5-{[4-({2-[2-(2-aminoethoxy)ethoxy]ethoxy}methyl)-2-methoxyphenyl]methyl}-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (P41)

[1214]

[1215] Following General Procedure IX, starting from P41-3, compound P41 (15 mg, 36% yield) was obtained as a white solid. LC retention time: 1.38 min.; ESI m / z: 501.1 (M+H)+. 1 H NMR (400 MHz, DMSO d6 )δ12.63(s,1H),9.07(s,2H),7.50(s,2H),7.44(t,J=5.5Hz,1H),7.40(d,J=3.0Hz,1H),7.26(d ,J=1.0Hz,1H),6.98(d,J=7.8Hz,1H),6.62(d,J=7.8Hz,1H),6.21(d,J=3.0Hz,1H),5.57(s,2H), 4.64(s,1H),4.14(s,2H),3.85(s,3H),3.67(t,J=5.1Hz,2H),3.56(s,4H),3.50-3.41(m,8H),3 .06(br,2H),1.53-1.43(m,2H),1.29-1.19(m,2H),1.17-1.08(m,2H),0.83(t,J=7.2Hz,3H)ppm.

[1216] Example 31

[1217] Payload P42

[1218] 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()

[1219]

[1220] Following General Procedure VIII, starting from benzyl bromide P4-1, reaction with N-Boc-PEG3-OH (P41-2) was carried out, but using 4 equivalents of sodium hydride (60% in mineral oil) instead of 2.5 equivalents, to give compound P41-3 (80 mg, 36% yield) and compound P42-3 (20 mg, 9% yield), respectively, as white solids after purification by preparative HPLC (5-95% acetonitrile / aqueous ammonium bicarbonate solution (10 mM)).

[1221] For 41-3: Retention time in LC: 2.05 min.; ESI m / z: 601.3 (M+H) + .

[1222] For 42-3: Retention time in LCMS: 1.84 min., ESI m / z: 601.3 (M+H) + .

[1223] 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)

[1224]

[1225] Following General Procedure IX, starting from P42-3, compound P42 (10 mg, 13% yield) was obtained as a white solid. Retention time in LC: 1.28 min.; ESI m / z: 501.1 (M+H) + . 1 H NMR (400 MHz, DMSO d6 )δ12.63(s,1H),9.07(s,2H),7.50(s,2H),7.44(t,J=5.5Hz,1H),7.40(d,J=3.0Hz,1H),7.26 (s,1H),6.98(d,J=7.8Hz,1H),6.62(d,J=7.8Hz,1H),6.21(d,J=3.0Hz,1H),5.57(s,2H),4.64 (s,1H),4.14(s,2H),3.85(s,3H),3.67(t,J=5.1Hz,2H),3.56(br,4H),3.50-3.42(m,6H),3.0 6(br,2H),1.53-1.42(m,2H),1.33-1.18(m,2H),1.16-1.06(m,2H),0.83(t,J=7.2Hz,3H)ppm.

[1226] Example 32

[1227] Payload P11

[1228] 2-Amino-N-{[(4-{[2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]methyl}-3-methoxyphenyl)methoxy]methyl}acetamide (P11)

[1229]

[1230] To a solution of P1 (0.30 g, 0.81 mmol) in anhydrous THF (5 mL) was added potassium tert-butoxide (0.18 g, 1.6 mmol) and compound P11-1 (CAS: 1599440-06-8) (0.45 g, 1.2 mmol) at 0°C under nitrogen, and the reaction mixture ...

Claims

1. A compound, wherein the compound has formula I: or a pharmaceutically acceptable salt thereof, in: R 1 H, halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ; R 2 is H, halogen 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 an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group; R 23 is H, alkyl or aryl; X is CH or N; Y is -OH, -Gly, or -NR 5 R 6 or -COZ; Z is -OH, alkoxy or -NR 7 R 8 ; R 5 and R 6 are each independently H or alkyl, or together with the nitrogen to which they are attached form a heterocycle; and R 7 and R 8 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a heterocyclic ring; Provided that the compound is not a compound of the formula: or 2. The compound of claim 1, wherein: R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 ; R 2 is halogen or alkoxy; R 3 for-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 an alkyl group optionally substituted with an alkoxy group or a heteroalkyl group; X is CH or N; Y is -OH, -Gly, or -NR 5 R 6 or -COZ; Z is -OH or -NR 7 R 8 ; R 5 and R 6 Selected from (i), (ii) and (iii): (i)R 5 and R 6 Each is H; (ii)R 5 is H, and R 6 is an alkyl group; (iii)R 5 and R 6 together with the nitrogen to which they are attached, form a heterocyclic ring; and R 7 and R 8 Together with the nitrogen to which they are attached they form a heterocyclic ring.

3. The compound of claim 1 or 2, provided that R 4 Not substituted by hydroxyl groups.

4. The compound of any one of claims 1 to 3, provided that R 3 The alkylene and heteroalkylene portions of the alkylene and heteroalkylene groups are not substituted with oxo.

5. The compound of any one of claims 1 to 4, provided that the compound is not 5-(2-methoxy-4-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine or (4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3-methoxyphenyl)methanol.

6. The compound of any one of claims 1 to 5, wherein R 1 Halogen, -NHR 4 、-OR 4 、-NH-OR 4 or -R 4 , and is a straight chain with a length of 6 atoms.

7. The compound of any one of claims 1 to 6, wherein R 1 It is a halogen.

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

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

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

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

12. The compound of any one of claims 1 to 5, wherein R 1 It is -NH-n-pentyl, -NH-O-n-butyl, -O-n-pentyl, -n-hexyl or -NH-CH2CH2-OEt.

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

14. The compound of any one of claims 1 to 5, wherein R 1 It is -NH-O-n-butyl.

15. The compound of any one of claims 1 to 5, wherein R 1 It is -O-n-pentyl.

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

17. The compound of any one of claims 1 to 5, wherein R 1 It is -NH-CH2CH2-OEt.

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

19. The compound of any one of claims 1 to 17, wherein R 2 It is a methoxy group.

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

21. The compound of any one of claims 1 to 16, wherein R 2 It is a halogen.

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

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

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

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

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

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

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

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

30. The compound of any one of claims 1 to 22, wherein R 3 is -CONH2, -CO2H, -CH2-Y, -CH2-O-heteroalkylene-Y or -CH2-O-alkylene-Y.

31. The compound of any one of claims 1 to 22, wherein R 3 is -CH2-Y, -CH2-O-heteroalkylene-Y or -CH2-O-alkylene-Y.

32. The compound of any one of claims 1 to 22, wherein R 3 -C(Me)2OH, -CO2H -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OC H2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2NHC(O)CH2NH2, -CH2OCH2-(4-NH2-1 -phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2COOH, -CH2OCH2CH2OCH2OCH2COOH, -CH2OCH2CH2OCH2OCH2OCH2COOH, -CH2OCH2COOEt, -CH2OCH2CON(n-Pr)2, -CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2, -CONH2 or -CH2-1-piperazinyl.

33. The compound of any one of claims 1 to 22, wherein R 3 is -C(Me)2OH, -CO2H, -CH2OCH2CH2NH2, -CH2OCH2CH2CH2CH2NH2, -CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2NH2, -CH2 OCH2CH2OCH2CH2OCH2CH2OCH2CH2OH, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazinyl, -CH2OCH2N HC(O)CH2NH2, -CH2OCH2-(4-NH2-1-phenyl), -CH2OCH2COOH, -CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH, -CH2OCH2CH2OCH2OCH2CH2OCH2COOH-CH2OCH2CO-1-piperazinyl, -(R)-CH2OCH(OH)CH2OH, -(S)-CH2OCH(OH)CH2OH, -CH2OCH(NH2)CH2OH, -CH2OH, -CH2NH2 or -CH2-1-piperazinyl.

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

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

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

37. The compound of any one of claims 1 to 33, wherein R 4 It is a n-hexyl group.

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

39. The compound of any one of claims 1 to 38, wherein R 5 and R 6 Each is independently H or alkyl, or together with the nitrogen to which they are attached, forms a piperazinyl ring.

40. The compound of any one of claims 1 to 38, wherein R 5 and R 6 Each is H.

41. The compound of any one of claims 1 to 38, wherein R 5 H and R 6 It is an alkyl group.

42. The compound of any one of claims 1 to 38, wherein R 5 and R 6 Together with the nitrogen to which they are attached they form a 1-piperazinyl group.

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

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

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

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

47. The compound of any one of claims 1-42, wherein Y is -OH, -NH2, 1-piperazinyl, -COOH, -COOEt, -CONPr2, or -CO-1-piperazinyl.

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

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

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

51. The compound of any one of claims 1-47, wherein Z is -OH, ethoxy, -Nn-Pr2 or 1-piperazinyl.

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

53. The compound of any one of claims 1 to 52, wherein R 7 and R 8 Each is independently H or n-propyl, or together with the nitrogen to which they are attached forms 1-piperazinyl.

54. The compound of any one of claims 1 to 52, wherein R 7 and R 8 Together with the nitrogen to which they are attached they form a 1-piperazinyl group.

55. A compound selected from the group consisting of: and pharmaceutically acceptable salts thereof.

56. A compound, wherein the compound has Formula II: or a pharmaceutically acceptable salt thereof, in: R 1 、R 2 and X is as defined in Formula I of claim 1; R 9 R as defined in claim 1 3 The group removes the terminal hydrogen (ie, away from the R 9 A divalent group formed by (a hydrogen of the attached phenyl group); and L is any group or moiety that is connected, linked or bonded to the antigen binding domain ABD; Provided that the compound is not a compound of the formula:

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

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

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

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

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

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

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

64. The compound of claim 56 or 57, wherein R 9 -CH2-Y 1 -, -CH2-O-heteroalkylene-Y 1 - or -CH2-O-alkylene-Y 1 -.

65. The compound of claim 56 or 57, wherein R 9 is -C(Me)2O-, C(O)-, -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH 2OCH2CH2OCH2CH2O-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2OCH2CH2-1-piperazine-4-yl-, -CH2OCH2NHC(O)CH2 NH-, -CH2OCH2-((4-NH-)-1-phenyl), -CH2OCH2COO-, -CH2OCH2CH2OCH2CO-, -CH2OCH2CH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CH2OCH2OCH2OCH2CO-, -CH2OCH2CO-1-piperazin-4-yl, -(R)-CH2OCH(OH)CH2O-, -(S)-CH2OCH(OH)CH2O-, -CH2OCH(NH2)CH2O-, -CH2O-, -CH2NH- or -CH2-1-piperazin-4-yl.

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

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

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

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

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

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

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

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

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

75. The compound of claim 68, wherein R 5 It is an alkyl group.

76. The compound of any one of claims 55-75, wherein L is not cleavable under physiological conditions.

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

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

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

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

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

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

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

84. The compound of any one of claims 55-83, wherein L is a group selected from the group consisting of 2-maleimido-1-ethyl, 2-maleimidoacetyl, 3-maleimidopropionyl, 85. A compound selected from the group consisting of: and pharmaceutically acceptable salts thereof.

86. A compound, wherein the compound has Formula III: or a pharmaceutically acceptable salt thereof, in: R 1 、R 2 and X as defined in claim 1; L is any group or moiety that is connected, linked or bonded to the antigen binding domain ABD; R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein when R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group; R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group; R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic group; 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, wherein R 11 and R 12 are independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, alkylene or heteroalkylene, wherein, When R 11 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group; R 14 is hydrogen, alkylene, heteroalkylene or an amino acid side chain, wherein when R 14 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 13 bonded to form a 4-, 5-, or 6-membered heterocyclic group; R 13 is hydrogen, alkyl, alkylene or heteroalkylene, wherein when R 13 When it is an alkylene or heteroalkylene group, the alkylene or heteroalkylene group is further 11 or R 14 bonded to form a 4-, 5-, or 6-membered heterocyclic group; 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.

88. The compound of claim 86 or claim 87, wherein the TLR7 agonist used to prepare the compound is P2, P6, P8, P17, P18, P19, P20, P23, P27, P29, P32, P33, P37, P39, P41, P42, or P43.

89. An antibody-drug-conjugate (ADC) comprising a compound according to any one of claims 1 to 88 or a compound of the formula:

90. The ADC of claim 89, wherein the ADC has Formula IV: or a pharmaceutically acceptable salt thereof, wherein: R 1 、R 2 、R 9 and X is defined as Formula I in claim 1 and Formula II in claim 56; L 1 is a divalent linker; ABD is the antigen binding domain; and k is an integer from 1 to 30.

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

92. The ADC of claim 89, wherein the ADC has Formula V: or a pharmaceutically acceptable salt thereof, wherein: R 1 、R 2 and X is as defined in Formula I of claim 1; 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 is an antibody containing a Q295 residue, an N297Q mutation, and / or one or more engineered LLQG (SEQ ID NO: 1), LLQGG (SEQ ID NO: 2), LLQLLQG (SEQ ID NO: 3), LLQYQG (SEQ ID NO: 4), LLQGA (SEQ ID NO: 5), LLQGSG (SEQ ID NO: 6), SLLQG (SEQ ID NO: 7), LQG,LLQLQ (SEQ ID NO: 9), LLQLLQ (SEQ ID NO: 10), LLQGR (SEQ ID NO: 11), LLQYQGA (SEQ ID NO: 12), LQGG (SEQ ID NO: 13), LGQG (SEQ ID NO: 14), or LLQLLQGA (SEQ ID NO: 15); and k is an integer from 1 to 30.

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

94. The ADC of claim 92 or claim 93, wherein R 10 It is -alkylene-NH-.

95. The ADC of claim 92 or claim 93, wherein R 10 is -heteroalkylene-NH-.

96. The ADC of claim 92 or claim 93, wherein R 10 is -heteroalkylene-arylene-NH-.

97. The ADC of claim 92 or claim 93, wherein R 10 is -(hydroxy)heteroalkylene-NH-.

98. The ADC of claim 92 or claim 93, wherein R 10 is -(amino)heteroalkylene-NH-.

99. The ADC of claim 92 or claim 93, wherein R 10 is -alkylene-PEG-NH-.

100. The ADC of claim 92 or claim 93, wherein R 10 is -CH2-NH-, -CH2-O-heteroalkylene-NH- or -CH2-O-alkylene-NH-.

101. The ADC of claim 92 or claim 93, wherein R 10 It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl), -CH2OCH(NH-)CH2OH or -CH2NH-.

102. The ADC of claim 92 or claim 93, wherein R 10 It is -CH2OCH2CH2NH-, -CH2OCH2CH2CH2CH2NH-, -CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2CH2OCH2CH2OCH2CH2NH-, -CH2OCH2NHC(O)CH2NH-, -CH2OCH2-(4-NH-1-phenyl) or -CH2NH-.

103. The ADC of any one of claims 89-102, having the formula ABD-P4, ABD-P5, ABD-P7, ABD-P9, ABD-P10, ABD-P11, ABD-P12, ABD-P19, ABD-P21, ABD-P24, ABD-P30, ABD-P34, or ABD-P41, wherein ABD is linked to R 3 The payload (i.e., TLR7 agonist) is placed on the amino group of 104. The ADC of claim 89, wherein the ADC has Formula VI: or a pharmaceutically acceptable salt thereof, wherein: L 1 is a divalent linker; R 1 、R 2 、R 16 、R 11 、R 12 、R 13 、R 14 、R 15 , X and x are as defined in Formula III of claim 86; and k is an integer from 1 to 30.

105. The ADC of claim 104 having the 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, where ABD-L 1 Connect to R 3 The payload (i.e., TLR7 agonist) is placed on 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. A pharmaceutical composition comprising a compound according to any one of claims 1 to 88 or an ADC according to any one of claims 89 to 106, and a pharmaceutically acceptable carrier.

108. A method of treating or diagnosing a disease comprising administering to a subject a compound of any one of claims 1-88 or an ADC of any one of claims 89-106 or a pharmaceutical composition of claim 107.

109. The method of claim 108, wherein the method treats a disease.

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

111. The method of any one of claims 108 to 110, wherein the disease is acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, breast cancer, PRLR positive (PRLR+) breast cancer, cervical cancer, bile duct cancer, 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)), 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. 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. Antibody-drug conjugate (ADC), comprising a. having an antigen binding domain (ABD) that binds specifically to hepatitis B virus surface antigen (HBV sAg); and b. Toll-like receptor 7 (TLR7) agonist linked to ABD with a bivalent linker.

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

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

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

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

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

120. The ADC of claim 114, wherein the ABD comprises a V of an antibody directed against HBV sAg. H Chain and V L 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 the ABD comprises an Fc region.

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

124. The ADC of any one of claims 114-123, wherein the ABD comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 25, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

29.

125. The ADC of claim 124, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:26, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:27, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:28, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:30, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:31, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

32.

126. The ADC of claim 124 or claim 125, wherein the HCVR comprises the amino acid sequence of SEQ ID NO:

25.

127. The ADC of claim 126, wherein the HCVR is a component of a heavy chain comprising the amino acid sequence of SEQ ID NO:

33.

128. The ADC of claim 124 or claim 125, wherein the LCVR comprises the amino acid sequence of SEQ ID NO:

29.

129. The ADC of claim 126, wherein the LCVR is a component of a light chain comprising the amino acid sequence of SEQ ID NO:

34.

130. The ADC of any one of claims 124-129, wherein the ABD is a component of an antibody or antigen-binding fragment thereof.

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

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

133. The method of claim 131, wherein the hepatitis B is chronic hepatitis B.

134. The method of claims 131-133, wherein circulating HBV DNA or HBV sAg is elevated in the subject's serum prior to administration of the ADC or pharmaceutical composition.

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

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

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