Targeted conjugates containing effector molecules and uses thereof

Abstract

Provided is a targeted conjugate comprising a targeting moiety and an effector molecule, where the effector molecule is conjugated to the targeting moiety via a conjugation moiety, and the effector molecule can be released from the targeted conjugate via cleavage. Also provided are therapeutic or diagnostic compositions and methods using the targeted conjugate.

Description

[Technical field]

[0001] The present application relates to targeted conjugates comprising effector molecules and uses thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of PCT application PCT / CN2021 / 077864, filed February 25, 2021, and PCT application PCT / CN2021 / 084952, filed April 01, 2021, the contents of both of which are incorporated by reference herein in their entireties.

[0003] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 199872000240SEQLIST.TXT, Recorded: February 24, 2021, Size: 163KB). [Background technology]

[0004] Antibodies have been successfully used as therapeutic and diagnostic agents for a variety of diseases. Native antibodies act by binding to a specific target. However, the antibody target may be expressed in both diseased (e.g., tumor) and normal tissues. When an antibody binds to a target expressed in a patient's normal tissues, the patient may experience undesirable toxic side effects, which may range in intensity from mild to severe or even life-threatening. Such side effects may lead to a reduction in the effective dosage of the antibody, which may reduce its therapeutic effectiveness. Antibody treatment may even be discontinued in some cases due to side effects that make the antibody therapeutic unavailable for use in human patients. Thus, there is an unmet need to engineer antibodies that preferentially bind to targets on diseased cells and tissues.

[0005] Antibody drug conjugates (ADCs) are targeted therapeutic agents designed to preferentially direct a drug ("payload") to diseased tissues that express the surface antigen recognized by the antibody. ADCs are often composed of an antibody linked to a therapeutically active agent (e.g., a cytotoxic drug) via a chemical linker that allows for the release of the therapeutically active agent in the diseased environment. Currently, five ADCs have been approved by the FDA for therapeutic use, and more than 100 are under clinical investigation. Among the most widely used ADCs in cancer treatment are trastuzumab emtansine (KADCYLA®) and brentuximab vedotin (ADCETRIS®) (see, e.g., Jackson et al., Pharm Res (2015) 32:3458-3469). The majority of ADCs currently approved or undergoing clinical evaluation are small molecule drug-antibody conjugates. Current ADCs incorporate standard chemotherapeutic agents, such as antimitotics and antimetabolites, including auristatin monomethyl auristatin E (MMAE), calicheamicin, and derivatives of maytansine 1 (DM1). See, e.g., Polakis, Pharma Rev, 2016, 68(1) 3-19.

[0006] Although ADCs have conceptual advantages and promising clinical results, the development of effective ADC therapeutics remains significantly challenging. The overall design of the ADC, the selection of the target tissue, the antibody, the chemical linker, the site of drug attachment, and the nature of the cargo all affect the efficacy and risks of the ADC (see, e.g., Chau et al., Lancet 2019;394:793-804). For example, early ADCs in clinical trials have suffered from the immunogenicity of the murine antibodies used, and recent advances in the development of ADCs have relied on humanized antibodies. Other unsolved challenges in ADCs include a) ineffectiveness of the drug cargo; b) premature release of the drug leading to loss of efficacy and toxicity; c) the level of target expression; d) suboptimal target selectivity; and e) side effects such as thrombocytopenia and neuropathy, as seen with KADCYLA® and ADCETRIS®. These limitations highlight the need for further improvement of ADCs in search of highly potent payloads, stable chemical linkers, efficient and highly specific cargo release, as well as increased antibody-target selectivity. The ADC platform as a highly specific cargo delivery system in vivo lends itself to applications beyond small molecule drug delivery. For example, siRNA can be fused to an antibody and developed into an efficient method for mRNA knockdown in vivo (see Baumer et al., Nature Protocols, 2016, 11:22-36). Such antibody-mediated siRNA delivery has shown promising results in colon cancer treatment (see Baumer et al., Clin Cancer Res 2015 15;21(6):1383-94). A broader scope of design, antibody type, and therapeutic agent spread in the ADC platform is still needed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jackson et al.,Pharm Res(2015)32:3458-3469 [Non-Patent Document 2] Polakis,Pharma Rev,2016,68(1)3-19 [Non-Patent Document 3] Chau et al.,Lancet 2019;394:793-804 [Non-Patent Document 4] Baumer et al.,Nature Protocols,2016,11:22-36 [Non-Patent Document 5] Baumer et al., Clin Cancer Res 2015 15;21(6):1383-94 Summary of the Invention [Means for solving the problem]

[0008] The present application provides targeted conjugates comprising a targeting moiety and one or more effector molecules, compositions, methods of preparation and methods of use thereof.

[0009] In some embodiments, a targeted conjugate is provided that includes a targeting moiety and an effector molecule, wherein the effector molecule is conjugated to the targeting moiety via a conjugation moiety, and the effector molecule can be released from the targeted conjugate via cleavage. In some embodiments, the targeted conjugate has the structure of Formula I: [ka] (wherein A1 is a first targeting moiety; A2 is a second targeting moiety; P1 is a first cleavage site; P2 is a second cleavage site; P3 is a third cleavage site; C is a conjugation moiety; L is a linker; D is an effector molecule; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20). In some embodiments, z=0. In some embodiments, z=1. In some embodiments, y=0. In some embodiments, y=1. In some embodiments, x=0. In some embodiments, x=1. In some embodiments, u=0. In some embodiments, u=1. In some embodiments, a is 1 or more. In some embodiments, b is 1 or more.

[0010] In some embodiments according to any one of the targeting conjugates described above, the targeting conjugate comprises a targeting peptide. In some embodiments, the targeting conjugate comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monospecific antibody, a multispecific antibody, a diabody, a nanobody, an scFv, a scFab, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a dsFv. In some embodiments, the targeting moiety comprises an antibody-peptide fusion protein. In some embodiments, the antibody-peptide fusion protein comprises an Fc region. In some embodiments, the peptide is fused to the C-terminus of the Fc region.

[0011] In some embodiments according to any one of the targeting conjugates described above, the targeting conjugate comprises a targeting moiety that specifically binds to a target molecule (e.g., a cell surface molecule) at a target site. In some embodiments, the target site is a site of a disease. In some embodiments, the disease is a tumor. In some embodiments, the target molecule is selected from the group consisting of PD1, PD-L1, Trop2, CTLA-4, LAG-3, TIM-3, 4-1BB, CD40, OX40, CD47, SIRPα, HER2, HER3, EGFR, VEGF, VEGR2, CD19, CD20, CD22, CD30, CD33, CD38, CD79, integrin αvβ3, αvβ6, MUC1, PMSA, uPAR, and angiopep-2.

[0012] In some embodiments of any one of the targeting conjugates described above, cleavage is triggered by conditions at the target site for the targeting moiety. In some embodiments, the conditions at the target site are selected from the group consisting of proteases, pH changes, redox changes, hypoxia, oxidative stress, hyperthermia, and extracellular ATP concentration. In some embodiments, the target site is a site of disease. In some embodiments, the disease is a tumor.

[0013] In some embodiments of any one of the above-mentioned targeted conjugates, cleavage is by a protease. In some embodiments, the protease is selected from the group consisting of urokinase plasminogen activator (uPA), legumain, plasmin, TMPRSS3, TMPRSS4, TMPRSS6, MMP1, MMP2, MMP-3, MMP-9, MMP-8, MMP-14, MT1-MMP, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, TACE, human neutrophil elastase, beta-secretase, fibroblast-associated protein, matriptase, PSMA, and PSA. In some embodiments, the protease is uPA. In some embodiments, the cleavage site comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 50-55.

[0014] In some embodiments according to any one of the targeted conjugates described above, the effector molecule is selected from the group consisting of a therapeutic agent, an oligonucleotide, and a detectable label. In some embodiments, the effector molecule is a therapeutic agent. In some embodiments, the effector molecule is selected from the group consisting of a protein-based drug, a small molecule drug, a cytotoxic agent, a toxin, an immunomodulatory agent, an anti-inflammatory agent, an anti-infective agent, and an epigenetic modulator. In some embodiments, the effector molecule is an oligonucleotide. In some embodiments, the oligonucleotide is about 2 to about 100 nucleotides in length. In some embodiments, the oligonucleotide is selected from the group consisting of double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, antisense RNA, small interfering RNA, microRNA, short hairpin RNA (shRNA), and CpG (cytosine-phosphodiester-guanine) oligonucleotide. In some embodiments, the oligonucleotide is a CpG oligonucleotide. In some embodiments, the oligonucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-67.

[0015] In some embodiments according to any one of the above-described targeted conjugates, the targeted conjugate comprises two or more effector molecules.

[0016] In some embodiments of any one of the targeted conjugates described above, the effector molecule is a cytotoxic agent selected from the group consisting of anthracyclines, auristatins, camptothecins, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterins, spliceostatins, pladienolides, and stereoisomers, isosteres, analogs, or derivatives thereof. In some embodiments, the effector molecule is SN38 or a derivative thereof. In some embodiments, the effector molecule is MMAE or a derivative thereof. In some embodiments, the effector molecule is selected from the compounds of formulas (1)-(7). In some embodiments, the targeted conjugate comprises two or more small molecule effector molecules.

[0017] In some embodiments according to any one of the targeting conjugates described above, the targeting conjugate comprises the structure of formula I, where A1 is a first targeting peptide or a first antibody or an antigen-binding fragment thereof that recognizes a first target molecule, and A2 is a second targeting peptide or a second antibody or an antigen-binding fragment thereof that recognizes a second target molecule. In some embodiments, the first target molecule and the second target molecule are the same. In some embodiments, the first target molecule and the second target molecule are different. In some embodiments, A1 is connected to the N-terminus of the conjugation site (C) and A2 is connected to the C-terminus of the conjugation site (C).

[0018] In some embodiments according to any one of the targeting conjugates described above, the conjugation site is a covalent conjugation site. In some embodiments, the conjugation site is an endogenous conjugation site. In some embodiments, the conjugation site is an engineered conjugation site introduced into the targeting site. In some embodiments, the conjugation site is present on a peptide fused to the targeting site. In some embodiments, the conjugation site is a transglutaminase conjugation site, e.g., a glutamine-containing tag. In some embodiments, the conjugation site comprises multiple glutamine-containing tags fused in tandem to each other. In some embodiments, the conjugation site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0019] In some embodiments of any one of the targeting conjugates described above, the conjugation site is a non-covalent conjugation site. In some embodiments, the conjugation site comprises an oligonucleotide binding polypeptide. In some embodiments, the conjugation site comprises a CpG binding polypeptide. In some embodiments, the conjugation site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-65.

[0020] In some embodiments according to any one of the above-mentioned targeting conjugates, wherein the targeting conjugate comprises the structure of formula I, L is represented by the formula: SH2-spacer, MAL-spacer, NH2-spacer, or Osu-spacer (Osu: oxysuccinimide). In some embodiments, L is represented by the formula: (Gly) n -(PEG) m -VC-PAB-(DMAE) k (Formula II) (wherein n≧1, m≧2, and k=0 or 1). In some embodiments, L is represented by the formula: (Gly) n -(PEG) m-Val-Ala-PAB-(DMAE) k (wherein n≧1, m≧2, and k=0 or 1). In some embodiments, L is represented by (Gly) n -(PEG) m -P-PAB-(DMAE) k (Formula III) (wherein n, m, and k are integers, n≧1, m≧2, k=0 or 1, and P is a cleavage site).

[0021] In some embodiments, a targeted conjugate is provided that comprises a targeting moiety conjugated to a therapeutic agent having a formula selected from the group consisting of the compounds of formulas (1)-(7).

[0022] Also described herein is a composition comprising any one of a plurality of the above-mentioned targeting conjugates. In some embodiments, the average ratio of effector molecule and targeting moiety in the composition is at least about 1:1. In some embodiments, at least two of the targeting conjugates in the composition comprise different numbers of effector molecules. In some embodiments, the effector molecule is a therapeutic agent or an oligonucleotide, the composition is a pharmaceutical composition. In some embodiments, the effector molecule is a detectable label, the composition is a diagnostic composition.

[0023] In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of any one of the above-described pharmaceutical compositions.

[0024] In some embodiments, a method of diagnosing a disease in an individual is provided, comprising administering to the individual an effective amount of a diagnostic composition described above, wherein detection of the detectable label indicates the presence of the disease.

[0025] Also provided herein is a method of making any one of the targeted conjugates described above, the method comprising conjugating an effector molecule to the targeting moiety. [Brief description of the drawings]

[0026] [Figure 1-1] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=0; z=0; u=0; v=1; a=1-20; b=1-20. [Figure 1-2]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=0; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 1-3] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=1; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 2-1]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=0; z=1; u=0; v=1; a=1-20; b=1-20. [Figure 2-2] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=0; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 2-3]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=1; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 3-1] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=0; z=0; u=0; v=1; a=1-20; b=1-20. [Figure 3-2]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=0; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 3-3] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=1; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 4-1]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=0; y=0; z=1; u=0; v=1; a=1-20; b=1-20. [Figure 4-2] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=0; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 4-3]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=1; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 5-1] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=0; z=0; u=0; v=1; a=1-20; b=1-20. [Figure 5-2]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=0; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 5-3] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=1; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 6-1]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=0; z=1; u=0; v=1; a=1-20; b=1-20. [Figure 6-2] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=0; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 6-3]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=1; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 7-1] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=0; z=0; u=0; v=1; a=1-20; b=1-20. [Figure 7-2]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=0; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 7-3] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=1; z=0; u=1; v=1; a=1-20; b=1-20. [Figure 8-1]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=0; y=0; z=1; u=0; v=1; a=1-20; b=1-20. [Figure 8-2] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=0; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 8-3]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=0; y=1; z=1; u=1; v=1; a=1-20; b=1-20. [Figure 9-1] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=0; z=0; u=0; v=0; a=1-20; b=1-20. [Figure 9-2]1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeted conjugates are shown, where x=1; y=0; z=0; u=1; v=0; a=1-20; b=1-20. [Figure 9-3] 1 shows schematic structures of exemplary targeting conjugates, including: A1: a first targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; A2: a second targeting moiety, e.g., an antibody or antigen-binding fragment or a targeting peptide (including multispecific antibodies and fusion proteins), with or without a protease cleavage site; D: an effector molecule, e.g., a small molecule or oligonucleotide; P1: protease cleavage site 1; P2: protease cleavage site 2; P3: protease cleavage site 3; C: a conjugation site, e.g., a covalent conjugation site, or a non-covalent conjugation site (e.g., a cationic peptide or a neutrally charged peptide); L: a linker; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20. Exemplary targeting conjugates are shown, where x=1; y=1; z=0; u=1; v=0; a=1-20; b=1-20. [Figure 10-1]Exemplary targeted conjugates are shown, where x=0; y=0; z=0; u=0; v=0; a=1-20; b=1-20. [Figure 10-2] Exemplary targeted conjugates are shown, where x=0; y=0; z=0; u=1; v=0; a=1-20; b=1-20. [Figure 10-3] Exemplary targeted conjugates are shown, where x=0; y=1; z=0; u=1; v=0; a=1-20; b=1-20. [Figure 11] 1 shows a gel shift assay for CpG ODN linkers non-covalently conjugated to antibodies. [Figure 12] 1 shows a gel shift assay for CpG ODN linkers covalently conjugated to antibodies. [Figure 13] 1 shows a gel shift assay for CpG ODN linkers covalently conjugated to antibodies. [Figure 14] 1 shows dose-response curves for PD-1 / PD-L1 immune blockade reporter assay. [Figure 15-1] 1 shows dose-response cytotoxicity curves of compounds using HCT15-hPDL1 cells. [Figure 15-2] 1 shows antibody dose-response cytotoxicity curves using HCT15-hPDL1 cells. [Figure 15-3] 1 shows the response cytotoxicity curves of ADCs using HCT15-hPDL1 cells. [Figure 16] 1 shows the in vivo anti-tumor activity of antibodies and conjugates in the MDA-MB-231 CDX model. [Figure 17] 1 shows the dose-response cytotoxicity curves of drugs using PC3-hPDL1-hTROP2 cells. [Figure 18] 1 shows dose-response cytotoxicity curves of ADCs using PC3-hPDL1-hTROP2 cells. [Figure 19] 1 shows drug dose-response cytotoxicity curves using HCT15-hPDL1-hTROP2 cells. [Figure 20] Shows in vivo antitumor activity in the MC38-hPDL1-hTROP2 mouse syngeneic tumor model. [Figure 21] Shows in vivo antitumor activity in the PBMCs-PC3-hPDL1-hTROP2 CDX model. [Figure 22] Shows in vivo antitumor activity in HCT15-hPDL1-hTROP2 CDX model. [Figure 23] Response of HEK-Blue hTLR9-hPDL1 cells to different CpG ODN-linkers is shown. [Figure 24] Response of HEK-Blue hTLR9-hPDL1 cells to AOC is shown. [Diagram 25] Shows in vivo antitumor activity in the MC38-hPDL1 mouse syngeneic tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present application provides compositions and methods of treatment or diagnosis using a targeting conjugate comprising a targeting moiety and an effector molecule, the effector molecule being conjugated to the targeting moiety via a conjugation moiety, and the effector molecule being capable of being released from the targeting conjugate via cleavage. The targeting moiety specifically recognizes and binds to a target, such as a cell surface molecule expressed on diseased tissue or diseased cells (e.g., tumor cells). Binding of the targeting moiety can trigger cleavage of one or more cleavage sites in the targeting conjugate, leading to the release of the effector molecule at the target site. In some embodiments, the targeting conjugate is multispecific. In some embodiments, the targeting conjugate comprises a first targeting moiety and a second targeting moiety, the first targeting moiety and the second targeting moiety being fused to each other via a conjugation moiety to which one or more effector molecules are conjugated. For example, the targeting moiety can be designed by fusing one or more scFvs or scFabs to a monoclonal antibody with proven clinical efficacy and safety (e.g., anti-PDL1 mAb or anti-Trop2 mAb) to provide multispecificity in target recognition to provide improved clinical benefit. The targeted conjugates described herein can have multiple types of cargo (e.g., oligonucleotides and small molecule drugs) conjugated to the conjugation moiety, which can be a targeting peptide, or an antibody or antigen-binding fragment thereof (including multispecific antibodies), or a combination thereof. Thus, the targeted conjugates described herein can have improved selective target recognition ability and efficacy compared to conventional ADCs. Incorporating multiple conjugation sites and different types of cargo into one targeted conjugate opens up the potential for more powerful therapeutic effects. The possibility of combining oligonucleotides and other therapeutic agents (e.g., small molecule drugs) expands the range of diseases that can be targeted by the targeted conjugates described herein.

[0028] Thus, one aspect of the present application provides a targeted conjugate comprising a targeting moiety and an effector molecule, wherein the effector molecule is conjugated to the targeting moiety via a conjugation moiety, and the effector molecule can be released from the targeted conjugate via cleavage. In some embodiments, the cleavage occurs outside the cell. In some embodiments, the targeted conjugate comprises a structure of formula (I), where x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20.

[0029] Also provided are compositions, kits and articles of manufacture for use in any one of the above methods.

[0030] I. Definition Terms are used herein as commonly used in the art unless otherwise defined below.

[0031] The term "targeting moiety," as used herein, refers to a polypeptide-based binding molecule that specifically binds to a target molecule, or a portion thereof that contributes to specific binding. Both antibody- and non-antibody-based binding molecules or portions thereof are contemplated herein.

[0032] The term "therapeutic agent," as used herein, refers to a molecule that has a therapeutic effect. A therapeutic agent can be of any suitable molecular entity, except for oligonucleotides.

[0033] The term "effector molecule" as used herein refers to a molecule that can be used for therapeutic and / or diagnostic purposes. Exemplary effector molecules include, but are not limited to, therapeutic agents, diagnostic labels, and oligonucleotides.

[0034] The term "conjugation" refers to the chemical connection of two chemical groups or moieties to each other through one or more covalent or non-covalent bonds. Conjugation can be direct between the two chemical groups or moieties, or can be indirect through a third chemical group or moiety (e.g., a linker) that bridges the two chemical groups or moieties.

[0035] The term "conjugation site" refers to a site that directly connects two chemical moieties.

[0036] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof. Antibodies and / or antigen-binding fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelid antibody variable domains.

[0037] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. The variable regions in both chains usually contain three highly variable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs comprising HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are sandwiched between adjacent extensions known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant regions of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, e.g., lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0038] The term "antigen-binding fragment" as used herein refers to antibody fragments including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that the parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0039] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the heavy and light chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0040] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0041] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions were identified by Kabat et al., J.Biol.Chem.252:6609-6616(1977);Kabat et al.,USDept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991);Chothia et al.,J.Mol.Biol.196:901-917(1987);Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997);MacCallum et al.,J.Mol.Biol.262:732-745(1996);Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008);Lefranc MPet al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The contents of the references cited in this paragraph in which CDR prediction algorithms and interfaces are known in the art, including, for example, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015), are incorporated by reference in their entirety for use in this application and for possible inclusion in one or more claims herein.In some embodiments, the CDR sequences provided herein are based on IMGT definition.For example, CDR sequences can be determined by VBASE2 tool (www.vbase2.org / vbase2.php; see also Retter I, Althaus HH, Munch RM, MMuller M W: VBASE2, an integrative V gene database. Nucleic Acids Res. 2005 Jan 1; 33 (Database issue): D671-4 (incorporated herein in its entirety by reference)).

[0042] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, attenuating the extent of a disease, stabilizing a disease (e.g., preventing or slowing the worsening of a disease), preventing or slowing the spread of a disease, preventing or slowing the onset or recurrence of a disease, slowing or slowing the progression of a disease, improving a disease state, providing remission (whether partial or total) of a disease, reducing the dose of one or more other pharmaceutical agents required to treat a disease, slowing the progression of a disease, improving quality of life, and / or prolonging survival. "Treatment" also encompasses a reduction in the pathological consequences of a disease. The methods of this application contemplate any one or more of these aspects of treatment.

[0043] The terms "individual," "subject," and "patient" are used interchangeably herein to describe a mammal, including a human. In some embodiments, the individual is a human. In some embodiments, the individual is suffering from a disease or condition (e.g., cancer). In some embodiments, the individual is in need of treatment.

[0044] As understood in the art, an "effective amount" refers to an amount of an agent (e.g., a targeted conjugate) sufficient to produce a desired therapeutic outcome (e.g., reducing the severity or duration of cancer, stabilizing the severity of cancer, or eliminating one or more symptoms of cancer) or a desired diagnostic outcome. For therapeutic use, beneficial or desired results include, for example, reducing one or more symptoms resulting from a disease (biochemical, histological, and / or behavioral), including its complications and intermediate pathological phenotypes exhibited during the progression of the disease, improving the quality of life of a person suffering from the disease, reducing the dose of other pharmaceutical agents required to treat the disease, enhancing the effect of another pharmaceutical agent, slowing the progression of the disease, and / or prolonging the survival of a patient. In some embodiments, an effective amount of an agent may extend survival (including overall survival and progression-free survival); result in an objective response (including a complete or partial response); alleviate to some extent one or more signs or symptoms of a disease or condition; and / or improve the quality of life of a subject.

[0045] "Percent amino acid sequence identity" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences and considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004, each of which is incorporated by reference herein in their entirety and for all purposes.

[0046] Amino acid substitutions can include, but are not limited to, the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the subject antibody and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0047] The terms "polypeptide" or "peptide" are used herein to encompass all types of naturally occurring and synthetic proteins, including all length protein fragments, fusion proteins, and modified proteins, including but not limited to glycoproteins, as well as any other type of modified protein, such as proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, and the like.

[0048] The term "fusion" refers to the genetic joining of two polypeptide fragments to provide a single continuous polypeptide (a "fusion polypeptide"). The two polypeptide fragments can be directly joined to each other or can be joined via another polypeptide placed between them. Conventional recombinant DNA techniques or chemical gene synthesis can be used to provide a nucleic acid that genetically encodes the fusion polypeptide.

[0049] The term "epitope" as used herein refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies or antigen-binding fragments may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen.

[0050] As used herein, the terms "specifically bind," "specifically recognize," and "specific" refer to a measurable and reproducible interaction, e.g., binding, between a target and a targeting moiety (e.g., a targeting peptide or an antibody or antigen-binding fragment thereof). In certain embodiments, specific binding determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules (e.g., cell surface receptors). For example, a targeting moiety that specifically recognizes a target (which may be an epitope) is a targeting moiety (e.g., an antibody) that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than its binding to other molecules. In some embodiments, the extent of binding of the targeting moiety to unrelated molecules is less than about 10% of the binding of the targeting moiety to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, a targeting moiety that specifically binds to a target is less than about 10% of the binding of the targeting moiety to the target. -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M, or ≤ 10 -12 The targeting moiety has a dissociation constant (KD) of M. In some embodiments, the targeting moiety specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of the targeting moiety can be experimentally determined by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORETM, and peptide scan.

[0051] As used herein, "link," "conjugate," "ligate," and "fuse" are used interchangeably to refer to the joining of two chemical moieties, either covalently or non-covalently. The joining can be direct or indirect, e.g., through a linker.

[0052] The term "pharmaceutical composition" refers to a preparation which is in a form such that the biological activity of the active ingredients contained therein is effective, and which does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation will be administered.

[0053] "Pharmaceutically acceptable carrier" refers to one or more ingredients in a pharmaceutical formulation, other than the active ingredient, that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, cryoprotectants, isotonicity agents, preservatives, and combinations thereof. A pharmaceutically acceptable carrier or excipient preferably meets the required standards of toxicity and manufacturing testing and / or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration or other state / state governments, or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more specifically humans.

[0054] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosages, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic products.

[0055] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., an agent for the treatment of a disease or condition (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit may be promoted, distributed, or sold as a unit for performing the methods described herein.

[0056] It will be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0057] Reference herein to "about" a value or parameter includes (and describes) the variation relative to that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0058] As used herein, reference to a value or parameter "is not" generally means and describes a value or parameter "other than." For example, a method is not used to treat a disease of type X means that the method is used to treat a disease of a type other than X.

[0059] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0060] As used in this specification and the appended claims, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise.

[0061] As used herein, phrases such as "and / or," "A and / or B" are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, phrases such as "and / or," "A, B and / or C" are intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] II. Targeting Conjugates One aspect of the present application provides a targeted conjugate comprising a targeting moiety and an effector molecule, the effector molecule being conjugated to the targeting moiety via a conjugation moiety, the effector molecule being capable of being released from the targeted conjugate via cleavage. The cleavage may occur at the diseased site, e.g., at the tumor microenvironment. In some embodiments, the cleavage occurs outside the cell, e.g., outside the tumor cell in the tumor microenvironment.

[0063] In some embodiments, the structure of Formula I: [ka] (wherein A1 is a first targeting moiety; A2 is a second targeting moiety; P1 is a first cleavage site; P2 is a second cleavage site; P3 is a third cleavage site; C is a conjugation site; L is a linker; D is an effector molecule; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1, v=0 or 1; a=1-20; and b=1-20).

[0064] Exemplary targeting conjugates are shown in Figures 1A-10C and described below in Section E, "Exemplary Targeting Conjugates."

[0065] The targeting moiety (A1) of the present application is conjugated to one or more effector molecules (D), which may be therapeutic agents, oligonucleotides, detectable labels, and combinations thereof. In some embodiments, the effector molecule is conjugated to the targeting moiety at the conjugation site (C) via a linker (L). In some embodiments, the effector molecule is directly conjugated to the targeting moiety at the conjugation site (C) without a linker (L). In some embodiments, a cleavage site (P1, e.g., a protease cleavage site) is located between the conjugation site (C) and the effector molecule (D). In some embodiments, there is no cleavage site (P1, e.g., a protease cleavage site) located between the conjugation site (C) and the effector molecule (D). In some embodiments, the targeting conjugate comprises a second targeting moiety (A2). In some embodiments, the second targeting moiety (A2) is fused to the conjugation moiety (C) via a cleavage site (P2, e.g., a protease cleavage site). In some embodiments, there is no cleavage site (P2, e.g., a protease cleavage site) located between the second targeting moiety (A2) and the conjugation moiety (C). In some embodiments, the effector molecule (D) is fused to the linker via a cleavage site (P3, e.g., a protease cleavage site). In some embodiments, there is no cleavage site (P3, e.g., a protease cleavage site) located between the linker (L) and the effector molecule (D).

[0066] In some embodiments, the structure of Formula I: [ka] wherein A1 is a first antibody or antigen-binding fragment thereof; A2 is a second antibody or antigen-binding fragment thereof; P1 is a first cleavage site (e.g., a protease cleavage site, e.g., a uPA cleavage site); P2 is a second cleavage site (e.g., a protease cleavage site, e.g., a uPA cleavage site); P3 is a third cleavage site (e.g., a protease cleavage site, e.g., a uPA cleavage site); C is a conjugation site (e.g., a transglutaminase conjugation site); L is a linker; D is an effector molecule (e.g., a therapeutic agent, an oligonucleotide, or a detectable label); x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; and b=1-20. In some embodiments, A1 is an antibody or antigen-binding fragment (e.g., a full-length antibody) that specifically binds to an immune checkpoint molecule, e.g., PD-L1. In some embodiments, A2 is an antibody or antigen-binding fragment (e.g., a full-length antibody) that specifically binds to a tumor antigen, e.g., TROP-2.

[0067] In some embodiments, a targeted conjugate is provided that comprises: (a) a first polypeptide chain comprising a first heavy chain of an anti-PD-L1 antibody, a first transglutaminase conjugation site and a first antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab, or nanobody); (b) a second polypeptide chain comprising a second heavy chain of an anti-PD-L1 antibody, a second transglutaminase conjugation site and a second antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab, or nanobody); (c) a third polypeptide chain comprising a first light chain of an anti-PD-L1 antibody; (d) a fourth polypeptide chain comprising a second light chain of an anti-PD-L1 antibody; (e) a first effector molecule conjugated to the first transglutaminase conjugation site via a first linker; and (f) a second effector molecule conjugated to the second transglutaminase conjugation site via a second linker. In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: a first heavy chain of an anti-PD-L1 antibody, a first protease cleavage site (e.g., a uPA cleavage site), a first transglutaminase conjugation site, a second protease cleavage site (e.g., a uPA cleavage site), and a first antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab, or nanobody); and the second polypeptide chain comprises, from N-terminus to C-terminus: a second heavy chain of an anti-PD-L1 antibody, a third protease cleavage site (e.g., a uPA cleavage site), a second transglutaminase conjugation site, a fourth protease cleavage site (e.g., a uPA cleavage site), and a second antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab, or nanobody). In some embodiments, the first polypeptide chain and the second polypeptide chain each comprise, from N-terminus to C-terminus: a heavy chain of an anti-PD-L1 antibody, a protease cleavage site (e.g., a uPA cleavage site), a transglutaminase conjugation site, and an antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab or nanobody).In some embodiments, the first polypeptide chain and the second polypeptide chain each comprise, from N-terminus to C-terminus: a heavy chain of an anti-PD-L1 antibody, a transglutaminase conjugation site, a protease cleavage site (e.g., a uPA cleavage site), and an antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab, or nanobody). In some embodiments, the first polypeptide chain and the second polypeptide chain each comprise, from N-terminus to C-terminus: a heavy chain of an anti-PD-L1 antibody, a transglutaminase conjugation site, and an antigen-binding fragment of an anti-TROP2 antibody (e.g., an scFv, scFab, or nanobody). In some embodiments, the first effector molecule and the second effector molecule are identical. In some embodiments, the first effector molecule and the second effector molecule are different. In some embodiments, the first effector molecule (including the first linker) and / or the second effector molecule (including the second linker) are therapeutic agents selected from SN38, MMAE and derivatives thereof, e.g., compounds of formula (1)-(7). In some embodiments, the first effector molecule and / or the second effector molecule are oligonucleotides, e.g., CpG oligonucleotides. In some embodiments, the CpG oligonucleotides comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-67. In some embodiments, the first effector molecule and / or the second effector molecule are detectable labels. In some embodiments, two or more effector molecules are conjugated to the first transglutaminase conjugation site and / or the second transglutaminase conjugation site. In some embodiments, the first transglutaminase conjugation site and / or the second transglutaminase conjugation site comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. In some embodiments, the first protease cleavage site and / or the second protease cleavage site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:50-55.In some embodiments, the first polypeptide and / or the second polypeptide comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the third polypeptide and / or the fourth polypeptide comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 117, 119, 121, 123, 125, 127, and 129.

[0068] In some embodiments, a targeted conjugate is provided that comprises, from N-terminus to C-terminus: (a) a first polypeptide chain comprising a first heavy chain of an anti-PD-L1 antibody and a first transglutaminase conjugation site; (b) a second polypeptide chain comprising, from N-terminus to C-terminus: a second heavy chain of an anti-PD-L1 antibody and a second transglutaminase conjugation site; (c) a third polypeptide chain comprising a first light chain of an anti-PD-L1 antibody; (d) a fourth polypeptide chain comprising a second light chain of an anti-PD-L1 antibody; (e) a first effector molecule conjugated to the first transglutaminase conjugation site via a first linker; and (f) a second effector molecule conjugated to the second transglutaminase conjugation site via a second linker. In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: a first heavy chain of an anti-PD-L1 antibody, a first protease cleavage site (e.g., a uPA cleavage site) and a first transglutaminase conjugation site, and the second polypeptide chain comprises, from N-terminus to C-terminus: a second heavy chain of an anti-PD-L1 antibody, a second protease cleavage site (e.g., a uPA cleavage site) and a second transglutaminase conjugation site. In some embodiments, the first polypeptide chain and the second polypeptide chain each comprise, from N-terminus to C-terminus: a first heavy chain of an anti-PD-L1 antibody and a first transglutaminase conjugation site. In some embodiments, the first effector molecule and the second effector molecule are the same. In some embodiments, the first effector molecule and the second effector molecule are different. In some embodiments, the first effector molecule (including a first linker) and / or the second effector molecule (including a second linker) is a therapeutic agent selected from SN38, MMAE, and derivatives thereof, e.g., a compound of formula (1)-(7). In some embodiments, the first effector molecule and / or the second effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide.In some embodiments, the CpG oligonucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-67. In some embodiments, the first effector molecule and / or the second effector molecule is a detectable label. In some embodiments, the two or more effector molecules are conjugated to a first transglutaminase conjugation site and / or a second transglutaminase conjugation site. In some embodiments, the first transglutaminase conjugation site and / or the second transglutaminase conjugation site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. In some embodiments, the first protease cleavage site and / or the second protease cleavage site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-55. In some embodiments, the anti-PD-L1 antibody is duravalumab, atezolizimab or a derivative thereof. In some embodiments, the first polypeptide and / or the second polypeptide comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 100, 102, 104, 106, 108, 110, and 112. In some embodiments, the third polypeptide and / or the fourth polypeptide comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 101, 103, 105, 107, 109, 111, and 113.

[0069] In some embodiments, the targeting conjugate may include any suitable number of effector molecules (D), conjugation sites (C), protease cleavage sites (P1, P2, and P3), and linkers (L). In some embodiments, the number of conjugation sites C may be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of effector molecules (D) may be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of first protease cleavage sites P1 can be any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of second protease cleavage sites P2 can be any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of third protease cleavage sites P3 can be any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of linkers L can be any one of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0070] The targeted conjugate may comprise any one of the targeting moieties, effector molecules, conjugation moieties, cleavage sites (e.g., protease cleavage sites), and linkers described in Sections A-D below.

[0071] In some embodiments, the targeted conjugate comprises a single effector molecule. In some embodiments, the targeted conjugate comprises multiple effector molecules. In some embodiments, the targeted conjugate comprises a single molecule of an effector molecule. In some embodiments, the targeted conjugate comprises two or more of the same effector molecule. In some embodiments, the targeted conjugate comprises two or more different effector molecules. In some embodiments, the targeted conjugate comprises a single copy of each effector molecule. In some embodiments, the targeted conjugate comprises two or more copies of each effector molecule.

[0072] In some embodiments, the targeting conjugate has high drug loading. The term "drug loading" refers to the ratio between the number of effector molecules to the targeting moiety (e.g., antibody or its antigen-binding fragment) in the targeting conjugate. For example, an antibody conjugated to a total of eight effector molecules has eight drug loadings. Each molecule of the targeting conjugate has an integer value of drug loading. However, in a composition, different molecules of the targeting conjugate can have different values ​​of drug loading. Thus, the composition can have an average drug loading of an integer value or a non-integer value.

[0073] In some embodiments, the targeted conjugate has a ratio of effector molecule to targeting moiety (e.g., first targeting moiety and / or second targeting moiety) of at least about any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the targeted conjugate has a ratio of effector molecule to targeting moiety (e.g., first targeting moiety and / or second targeting moiety) of no more than about any one of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the targeted conjugate has a ratio of effector molecule to targeting moiety (e.g., first targeting moiety and / or second targeting moiety) of any one of about 1:1 to 2:1, 2:1 to 4:1, 4:1 to 8:1, 1:1 to 10:1, 1:1 to 16:1, 4:1 to 20:1, 10:1 to 20:1, 1:1 to 20:1, or 2:1 to 20:1.

[0074] In some embodiments, the drug loading of the targeted conjugate is at least about any one of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1 or more. In some embodiments, the drug loading of the targeted conjugate is less than or equal to about any one of 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. In some embodiments, the drug loading of the targeted conjugate is any one of about 2:1 to 4:1, 2:1 to 8:1, 2:1 to 10:1, 2:1 to 16:1, 4:1 to 20:1, 10:1 to 20:1, 20:1 to 40:1, 40:1 to 100:1, 2:1 to 20:1, 2:1 to 40:1, or 10:1 to 40:1.

[0075] In some embodiments, the targeted conjugate has a ratio of oligonucleotide to targeting site (e.g., first targeting site and / or second targeting site) of at least about any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the targeted conjugate has a ratio of oligonucleotide to targeting site (e.g., first targeting site and / or second targeting site) of no more than about any one of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the targeted conjugate has a ratio of oligonucleotide to targeting moiety (e.g., first targeting moiety and / or second targeting moiety) of any one of about 1:1 to 2:1, 2:1 to 4:1, 4:1 to 8:1, 1:1 to 10:1, 1:1 to 16:1, 4:1 to 20:1, 10:1 to 20:1, 1:1 to 20:1, or 2:1 to 20:1.

[0076] Also provided is a method of preparing a targeted conjugate, comprising conjugating an effector molecule to a targeting moiety.

[0077] A. Targeting site The targeting conjugate of the present application comprises one or more targeting moieties. In some embodiments, the targeting conjugate comprises a first targeting moiety that specifically binds to a first target molecule, a second targeting moiety that specifically binds to a second target molecule, and a conjugation moiety, where the first targeting moiety is fused to the second targeting moiety via the conjugation moiety. In some embodiments, the first targeting moiety and the second targeting moiety are identical. In some embodiments, the first targeting moiety and the second targeting moiety are different. In some embodiments, the first targeting moiety and the second targeting moiety specifically bind to the same target molecule. In some embodiments, the first targeting moiety and the second targeting moiety specifically bind to different target molecules. In some embodiments, a cleavage site (e.g., a protease cleavage site) is disposed between the first targeting moiety and the conjugation moiety. In some embodiments, a cleavage site (e.g., a protease cleavage site) is disposed between the second targeting moiety and the conjugation moiety.

[0078] Antibodies or antigen-binding fragments thereof In some embodiments, the targeting moiety comprises an antibody, or an antigen-binding fragment thereof. In some embodiments, the targeting moiety is a fusion protein comprising an antibody or an antigen-binding fragment thereof. In some embodiments, the targeting moiety is a monoclonal antibody. In some embodiments, the targeting moiety is a full-length antibody. In some embodiments, the targeting moiety is an antigen-binding fragment. Exemplary antigen-binding fragments include, but are not limited to, single chain Fv (scFv), Fab, Fab', F(ab')2, Fv, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, single domain antibodies (e.g., VHH), Fv-Fc fusions, scFv-Fc fusions, scFv-Fv fusions, diabodies, tribodies, and tetrabodies.

[0079] In some embodiments, the targeting moiety comprises an scFv. In some embodiments, the scFv comprises, from N-terminus to C-terminus: VL-VH, where the dash is a bond or a peptide linker. In some embodiments, the scFv comprises, from N-terminus to C-terminus: VH-VL, where the dash is a bond or a peptide linker. In some embodiments, the targeting moiety is a fusion protein comprising an scFv. In some embodiments, the fusion protein is an scFv-Fc fusion protein. In some embodiments, the fusion protein is an scFv-Fv fusion protein. In some embodiments, the fusion protein is an scFv-full-length antibody fusion protein. In some embodiments, the N-terminus of the scFv is covalently fused to the C-terminus of the heavy or light chain of the full-length antibody.

[0080] In some embodiments, the targeting moiety is a Fab or Fab'. In some embodiments, the targeting moiety is a Fab-containing polypeptide that may contain part or all of a wild-type hinge sequence (usually at the carboxyl terminus of the Fab portion of the polypeptide). The Fab-containing polypeptide may be obtained or derived from any suitable immunoglobulin. In some embodiments, the Fab-containing polypeptide may be a Fab-fusion protein that combines a Fab fragment with a fusion partner, such as a target binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. In some embodiments, the targeting peptide is a domain or part of a target protein.

[0081] In some embodiments, the targeting moiety comprises a nanobody, also known as a single domain antibody (sdAb). Exemplary sdAbs include heavy chain only antibodies (e.g., VHH or V NAR), binding molecules naturally devoid of light chains, single domains (e.g., VH or VL) derived from traditional four-chain antibodies, heavy chain only humanized antibodies, human sdAbs generated by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. In some embodiments, the targeting moiety comprises a VHH.

[0082] In some embodiments, the targeting moiety comprises a multispecific antibody. In some embodiments, the targeting moiety is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody. Exemplary bispecific antibodies include full length and Fab2 constructs, as well as diabodies. In some embodiments, diabodies are non-covalent dimers of single chain Fv (scFv) fragments connected by a peptide linker. In some embodiments, another form of diabody is a single chain (Fv) in which two scFv fragments are covalently linked to each other. In some embodiments, the multispecific antibody is a trispecific antibody. Exemplary trispecific antibodies include Fab3 and triabodies, the latter being a 3scFv version of a diabody. In some embodiments, scFv-Fc is made from two linked single chain variable fragments fused to an intact Fc region. In some embodiments, minibodies are similar to scFv-Fc but contain only the CH1 domain rather than the entire Fc region. Other multivalent constructs include IgNAR and hcIgG.

[0083] In some embodiments, the targeting moiety is a chimeric, human, partially humanized, fully humanized, or semi-synthetic antibody. The antibody and / or antibody fragment may be derived from a murine antibody, a rabbit antibody, a human antibody, a fully humanized antibody, a camelid antibody variable domain and a humanized version, a shark antibody variable domain and a humanized version, and a camelid antibody variable domain. In some embodiments, the targeting moiety is an antagonist antibody or an antigen-binding fragment thereof. In some embodiments, the targeting moiety is an agonist antibody or an antigen-binding fragment thereof.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more antibody constant regions, e.g., a human antibody constant region. In some embodiments, the heavy chain constant region is of an isotype selected from IgA, IgG, IgD, IgE, and IgM. In some embodiments, the human light chain constant region is of an isotype selected from kappa and lambda. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG constant region, e.g., a human IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, if effector function is desired, an antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region may be selected. In some embodiments, if effector function is not desired, an antibody comprising a human IgG4 or IgG2 heavy chain constant region may be selected. In some embodiments, the antibody comprises a human IgG4 heavy chain constant region.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof comprises a stabilization domain. In some embodiments, the stabilization domain comprises an Fc domain. The term "Fc region", "Fc domain" or "Fc" refers to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. In some embodiments, the Fc domain is selected from the group consisting of Fc fragments of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc domain is derived from human IgG. In some embodiments, the Fc domain comprises an Fc domain of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid IgG. In some embodiments, the Fc domain has reduced effector function compared to a corresponding wild-type Fc domain (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present without affecting the structure or stability of the Fc region. In some embodiments, the targeting moiety comprises a variant Fc region having at least one amino acid substitution compared to the Fc region of a wild-type IgG or wild-type antibody.

[0086] An antibody may consist of two identical light protein chains (light chains) and two identical heavy protein chains (heavy chains), all covalently held together by interchain disulfide bonds. The N-terminal regions of the light and heavy chains may together form the antigen recognition site of the antibody. Structurally, various functions of an antibody may be constrained to distinct protein domains or regions. In some embodiments, an antibody comprises an antigen-binding portion comprising a heavy chain comprising a VH and a light chain comprising a VL. Antigen-binding antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the whole or part of the following: hinge region, CH1, CH2, and CH3 domains.

[0087] In some embodiments, the antibody is modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently accomplished by modifying the amino acid sequence to generate or remove one or more glycosylation sites. In some embodiments, the antibody is modified to increase or decrease the reactivity of a reactive functional group on the antibody. In some embodiments, the antibody is modified to add or decrease the number of reactive functional groups. In some embodiments, the antibody is modified to increase or decrease the extent to which the reactive functional group is exposed. The reactive functional group can be at the N-terminus, C-terminus, or side chain of an amino acid of the antibody. The reactive functional group can be naturally present or incorporated in the antibody. The reactive functional group can be an amine or derivative thereof, a carboxyl group or derivative thereof, a nitro, or other functional group. In some embodiments, the reactive functional group is an amine. In some embodiments, the amine is in the Fc region of the antibody.

[0088] Antibodies that specifically bind to a target molecule can be obtained using methods known in the art, for example, by immunizing a non-human mammal and obtaining hybridomas therefrom, or by cloning and subsequent selection of libraries of antibodies using molecular biology techniques known in the art, or by using phage display. Nucleic acid constructs, vectors encoding any one of the antibodies or antigen-binding fragments described herein, and host cells for preparation are also provided.

[0089] Targeting Peptides In some embodiments, the targeting moiety comprises a targeting peptide. The term "targeting peptide" refers to a non-antibody-based polypeptide that specifically binds to a target molecule, e.g., a cell surface molecule, at a targeting site. In some embodiments, the targeting moiety is a fusion protein that comprises an antibody or antigen-binding fragment thereof fused to a targeting peptide.

[0090] In some embodiments, the targeting polypeptide comprises a non-antibody scaffold. A non-antibody scaffold is an engineered protein scaffold that provides specificity for different types of targets. Compared to antibodies, engineered protein scaffolds offer a much smaller size and simpler structure that facilitates recombinant gene expression, construction of bifunctional fusion proteins, and tissue penetration. See, e.g., Skerra, Curr. Opin. Biotech. 2007, 18:298-304. More than 50 different non-antibody scaffolds have been reported. See, e.g., Vazquez-Lombardi, Rodrigo, et al. Drug discovery today 20.10(2015):1271-1283. Exemplary non-antibody scaffolds include, but are not limited to, lipocalins, anticalins (artificial antibody mimetic proteins derived from human lipocalins), "T-bodies", peptides (e.g., BICYCLE™ peptides), affibodies (antibody mimetics composed of alpha helices, e.g., three-helix bundles), peptibodies (peptide-Fc fusions), DARPins (designed ankyrin repeat proteins, engineered antibody mimetic proteins composed of a repeat motif), affimers, avimers, knottins (protein structural motifs containing three disulfide bridges), monobodies, affinity clamps, ectodomains, receptor ectodomains, receptors, cytokines, ligands, immunocytokines, and centriins. See, e.g., WO2019084060, incorporated herein by reference.

[0091] In some embodiments, the targeting moiety comprises an anticalin. Anticalins are among the more actively developed non-antibody scaffolds, with a number of lead compounds in preclinical trials directed against CTLA-4, hepcidin, hepatocyte growth factor receptor (HGFR; MET), IL-4Ra and IL-23 / IL-17. PRS-050 (ANGIOCAL®; Pieris) is an anti-angiogenic anticalin targeting VEGF-A that is currently undergoing Phase I clinical investigation. MP0112 (Molecular Partners / Allergan), a DARPin targeting VEGF-A, targeting retinal anti-angiogenic disorders, is also currently being evaluated in clinical studies. FDA-approved single non-antibody scaffolds include the plasma kallikrein inhibitors Kunitz domain KALBITOR® (ecallantide; Dyax); BERINERT® (CSL-Behring), and CINRYZE® (ViroPharma / Shire), used for the treatment of hereditary angioedema, as well as the bradykinin receptor antagonist FIRAZYR® (Shire). See, e.g., Vazquez-Lombardi, Rodrigo, et al. Drug discovery today 20.10 (2015): 1271-1283.

[0092] In some embodiments, the targeting peptide comprises a polypeptide derived from a receptor or ligand of the target molecule. In some embodiments, the targeting polypeptide is an inhibitory polypeptide that completely or partially (e.g., at least about any one of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 95%) blocks the binding of the target molecule and its ligand or receptor. In some embodiments, the targeting peptide is at least about any one of 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acids in length.

[0093] In some embodiments, the targeting peptide comprises a stabilization domain. The stabilization domain can be any suitable domain that stabilizes the targeting peptide. In some embodiments, the stabilization domain increases the half-life of the targeting peptide in vivo. In some embodiments, the stabilization domain is an Fc domain, such as any one of the Fc domains described in the "Antibody or antigen-binding fragment thereof" section. In some embodiments, the stabilization domain is an albumin domain. In some embodiments, the targeting peptide and the stabilization domain are fused to each other via a linker, such as a peptide linker.

[0094] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. The peptide linker may be of any suitable length. In some embodiments, the peptide linker tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises a substrate sequence for an enzyme reaction. In some embodiments, the peptide linker comprises a substrate sequence for an enzyme that ligates the targeting peptide and the stabilization domain.

[0095] Targeting peptides can be obtained by using methods known in the art, for example, by screening libraries of polypeptides.Polypeptides can be prepared using chemical synthesis or produced using recombinant DNA technology.Also provided are nucleic acid constructs, vectors, and host cells for preparation that code for any one of the targeting peptides described herein.

[0096] Target Molecules and Exemplary Targeting Moieties A targeting moiety specifically binds to one or more target molecules. In some embodiments, a targeting moiety is monospecific, i.e., specifically binds to a single target molecule. In some embodiments, a targeting moiety is multispecific, i.e., specifically binds to two or more different target sites in a single target molecule, or to two or more different target molecules. In some embodiments, a targeting moiety has a single target binding site. In some embodiments, a targeting moiety has two or more target binding sites.

[0097] In some embodiments, the targeting moiety specifically binds to a cell surface molecule. In some embodiments, the cell surface molecule is a cell surface molecule at the target site. In some embodiments, the target site is the site of a disease. In some embodiments, the disease is a tumor. In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is a fibrotic disease. In some embodiments, the disease is an infectious disease. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an immunodeficiency disease.

[0098] Exemplary target molecules include, but are not limited to, proteins, glycans, lipids, other small molecules, or combinations thereof. In some embodiments, the cell surface molecule is a tumor-specific marker / tumor antigen. As described herein, "tumor-specific marker" or "tumor antigen" refers to a molecular marker that may be expressed on neoplastic tumor cells and / or within the tumor microenvironment. A tumor antigen may be a tumor-specific antigen and / or a tumor-associated antigen. For example, a tumor antigen may be an antigen expressed on cells associated with a tumor, such as neoplastic cells, stromal cells, endothelial cells, fibroblasts, or tumor-infiltrating immune cells. For example, the tumor antigen Her2 / Neu may be overexpressed by certain types of breast and ovarian cancer. Tumor antigens are also ectopically expressed by tumors and contribute to cell cycle dysregulation, reduced apoptosis, metastasis, and / or escape from immune surveillance. Tumor antigens are usually proteins or polypeptides derived from them, but may be glycans, lipids, or other small organic molecules. Tumor antigens may also arise through increased or decreased post-translational processing, such as protein glycosylation, protein lipidation, protein phosphorylation, or protein acetylation, exhibited by cancer cells compared to normal cells.

[0099] In some embodiments, the tumor antigen is a cell surface carbohydrate. In some embodiments, the cell surface carbohydrate is a monosaccharide. In some embodiments, the cell surface glycan is an oligosaccharide. In some embodiments, the cell surface carbohydrate is a polysaccharide. In some embodiments, the cell surface carbohydrate is a glycan. In some embodiments, the cell surface carbohydrate is a complex carbohydrate, e.g., a glycoprotein, a glycolipid, or a proteoglycan. In some embodiments, the cell surface glycan is GD2, GD3, GM2, Le y , sLe, polysialic acid, fucosyl GMl, Tn, STn, BM3, or GloboH.

[0100] In some embodiments, the tumor antigen is a cell surface protein.In some embodiments, the cell surface protein is CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-1, PD-Ll, B7-H3, B7-DC (PD-L2), HLA-DR, carcinoembryonic antigen (CEA), TAG-72, MUC1, MUC15, MUC16, folate binding protein, A33, G250, prostate specific membrane antigen (PSMA), CA-125, CA19-9, epidermal growth factor, HER2, IL-2 receptor, EGFRvIII (de2-7 EGFR), EGFR, fibroblast activation protein (FAP), tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avβ3, WTl, LMP2, HPV E6, HPV p53 non-mutant, NY-ESO-1, GLP-3, MelanA / MARTl, Ras mutant, gpl00, p53 mutant, PRI, bcr-abl, tyrosinase, survivin, PSA, hTERT, STNI, TNC, sarcoma translocation breakpoint fusion protein, EphA2, PAP, ML-IAP, AFP, ERG, NAI 7, PAX3, ALK, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, mesothelin (MSLN), PSCA, MAGE Al, MAGE-A3, CYPIBI, PLAVI, BORIS, Tn, ETV6-AN1L, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TESI, seminal protein 17, LCK, MAGE C2, MAGE A4, GAGE, TRAILI, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, Tie3, PAGE4, ​​VEGFR2, MAD-CTI, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EPCAM, CA6, NAPI2B, TROP2, claudin-6 (CLDN6), claudin-16 (CLDN16), CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, uroplakin-IB (UPKIB), LIVI, RORI, STRA6, TMPRSS3, TIV1PRSS4, TIV1EM238, Clofl86, Fos-related antigen 1, VEGFRI, endoglin, VTCNI (B7-H4), VISTA, and fragments thereof.

[0101] In some embodiments, the tumor antigen is selected from the group consisting of PD1, PD-L1, Trop2, CTLA-4, LAG-3, TIM-3, 4-1BB, CD40, OX40, CD47, SIRPα, HER2, HER3, EGFR, VEGF, VEGR2, CD19, CD20, CD22, CD30, CD33, CD38, CD79, integrins αvβ3, αvβ6, MUC1, PMSA, uPAR, and angiopep-2. In some embodiments, the tumor antigen is Trop2. Trop-2, also known as epithelial glycoprotein-1, gastrointestinal antigen 733-1, membrane component surface marker-1, and tumor-associated calcium signaling substance-2, is the protein product of the TACSTD2 gene. Trop-2 is a transmembrane glycoprotein that is upregulated in all cancer types, independent of baseline levels of Trop-2 expression. Trop-2 is an ideal candidate for targeted therapeutics because it is a transmembrane protein with an extracellular domain that is overexpressed in a variety of tumors and is upregulated in expression compared to normal cells. See, e.g., Onco Targets Ther. 2019;12:1781-1790.

[0102] In some embodiments, the target molecule is associated with a fibrotic or inflammatory disease. In some embodiments, the target molecule is selected from the group consisting of cadherin 11, PDPN, LRRC15, integrin a, 4f37, integrin a2f31, MADCAM, nephrin, podocin, IFNARI, BDCA2, CD30, c-KIT, FAP, CD73, CD38, PDGFRf3, integrin avf31, integrin avf33, integrin avf38, GARP, endosialin, CTGF, integrin avf36, CD40, PD-1, TIM-3, TNFR2, DEC205, DCIR, CD86, CD45RB, CD45RO, MHC class II, CD25, LRRC15, MMP14, GPX8, and F2RL2.

[0103] In some embodiments, the target molecule is associated with an infectious disease or an immunodeficiency disease, hi some embodiments, the target molecule is a protein of an infectious agent, such as a pathogenic bacterium or virus.

[0104] In some embodiments, the targeting moiety specifically binds to an immune checkpoint protein. Immune checkpoint proteins control immune activation. However, some cancers may protect themselves from attack by stimulating immune checkpoint targets. See, for example, Nature Reviews.Cancer.12(4):252-64. Inhibitory checkpoint molecules are targets for cancer immunotherapy due to their potential for use in multiple types of cancer. In some embodiments, the targeting moiety comprises an immune checkpoint inhibitor. Immune checkpoint inhibitors are compounds that inhibit the activity of immune system control mechanisms. Immune system checkpoints, or immune checkpoints, are inhibitory pathways in the immune system that normally act to maintain self-tolerance or regulate the duration and magnitude of physiological immune responses to minimize collateral tissue damage. Immune checkpoint inhibitors may inhibit immune system checkpoints by stimulating the activity of stimulatory checkpoint molecules or inhibiting the activity of inhibitory checkpoint molecules in the pathway. Immune system checkpoint molecules include, but are not limited to, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 protein (PD-1), programmed cell death 1 ligand 1 (PD-L1), programmed cell death 1 ligand 2 (PD-L2), lymphocyte activation gene 3 (LAG3), B7-1, B7-H3, B7-H4, T cell membrane protein 3 (TIM3), B- and T-lymphocyte attenuator (BTLA), V-domain immunoglobulin (Ig)-containing inhibitor of T cell activation (VISTA), killer cell immunoglobulin-like receptors (KIR), and A2A adenosine receptor (A2aR). Thus, immune checkpoint inhibitors include antagonists of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, and TIM3.For example, antibodies that bind to and antagonize the function of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, or TIM3 are immune checkpoint inhibitors.

[0105] In some embodiments, the targeting moiety comprises an antibody or antigen-binding fragment that specifically binds to an immune checkpoint molecule. In some embodiments, the targeting moiety comprises an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody is derived from any anti-PD-L1 antibody known in the art. Exemplary anti-PD-L1 antibodies include atezolizumab, avelumab, durvalumab (Imfinzi), BGB-A333, SHR-1316 (HTI-1088), CK-301, BMS-936559, embafolimab (KN035, ASC22), CS1001, MDX-1105 (BMS-936559), LY3300054, STI-A1014, F AZ053, CX-072, INCB086550, GNS-1480, CA-170, CK-301, M-7824, HTI-1088(HTI-131, SHR-1316), MSB-2311, AK-106, AVA-004, BBI-801, CA-327, CBA-0710, CBT-502, FPT-155, IKT-201, IKT-703, 10 -103, JS-003, KD-033, KY-1003, MCLA-145, MT-5050, SNA-02, BCD-135, APL-502 (CBT-402 or TQB2450), IMC-001, KD-045, INBRX-105, KN-046, IMC-2102, IMC-2101, KD-005, IMM-2502, 89Zr-CX-07 2, 89Zr-DFO-6E11, KY-1055, MEDI-1109, MT-5594, SL-279252, DSP-106, Gensci-047, REMD-290, N-809, PRS-344, FS-222, GEN-1046, BH-29xx, FS-118, biosimilars thereof, and derivatives thereof. In some embodiments, antibodies that compete with any of these art-recognized antibodies for binding to PD-L1 may also be used. In some embodiments, the anti-PD-L1 antibody is a derivative of any one of the anti-PD-L1 antibodies described herein. In some embodiments, the anti-PD-L1 antibody is derived from an antibody selected from the group consisting of durvalumab, atezolizumab, and avelumab.

[0106] In some embodiments, the anti-PD-L1 antibody is derived from durvalumab. In some embodiments, the targeting moiety competitively binds to the same or substantially the same epitope as durvalumab. In some embodiments, the anti-PD-L1 antibody comprises a VH and a VL, wherein the VH comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO: 86, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 87, and a CDR H3 comprising the amino acid sequence of SEQ ID NO: 88, and the VL comprises a CDR L1 comprising the amino acid sequence of SEQ ID NO: 89, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84: and / or a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0107] In some embodiments, the anti-PD-L1 antibody is derived from atezolizumab. In some embodiments, the targeting moiety competitively binds to the same or substantially the same epitope as atezolizumab. In some embodiments, the anti-PD-L1 antibody comprises a VH and a VL, wherein the VH comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:94, a CDR H2 comprising the amino acid sequence of SEQ ID NO:95, and a CDR H3 comprising the amino acid sequence of SEQ ID NO:96, and the VL comprises a CDR L1 comprising the amino acid sequence of SEQ ID NO:97, a CDR L2 comprising the amino acid sequence of SEQ ID NO:98, and a CDR L3 comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92, and / or a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0108] In some embodiments, the targeting moiety comprises a variant of the targeting moiety described herein. For example, an antibody variant may comprise one or more modifications of the amino acid sequence of an exemplary antibody ("parent antibody") while preserving the overall molecular structure of the antibody amino acid sequence. The amino acid sequence of any region of the parent antibody chain, such as the framework region, CDR region, or constant region, may be modified. The type of modification includes substitution, insertion, deletion, or combinations thereof of one or more amino acids of the parent antibody. In some embodiments, the antibody variant comprises a CDR (e.g., CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, or CDR L3) that has at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) sequence identity with the corresponding CDR of the parent antibody. In some embodiments, the antibody variant comprises a VH that comprises an amino acid sequence at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) identical to the VH of the parent antibody. In some embodiments, the antibody variant comprises a VL that comprises an amino acid sequence at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%) identical to the VL of the parent antibody. In some particular embodiments, the antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions relative to the amino acid sequence shown in either the VH, VL, heavy chain, or light chain of the parent antibody.

[0109] In some embodiments, the targeting moiety specifically binds to calcium signaling agents. Intracellular calcium signaling agents are involved in cell signaling, migration, proliferation, and differentiation. Intracellular calcium signaling agents have been shown to have oncogenic potential through their ability to upregulate various proto-oncogenes and cell cycle-related pathways. In some embodiments, the intracellular calcium signaling agents are tumor-associated calcium signaling agents. Tumor-associated calcium signaling agents are targets for cancer immunotherapy due to their differential expression in multiple types of cancer. In some embodiments, the targeting moiety specifically binds to tumor-associated calcium signaling agent 2 (Trop-2).

[0110] Trop-2 is also known as trophoblast cell surface antigen 2, M1S1, GA733-1, EGP-1, or TACSTD2. Trop-2 is a cell surface glycoprotein originally identified in human placental trophoblasts and was subsequently found to be highly expressed in most human cancers, but showed limited or restricted expression in normal adult tissues. See, e.g., Varughese et al., Gynecologic Oncology, 122:171-177, 2011. Without being bound by any theory or hypothesis, Trop-2 is involved in several cell signaling pathways, many of which are related to tumorigenesis. For example, in thyroid cancer cell invasion, Trop-2 signaling has been identified as a downstream effect of the ERK and JNK pathways. Stoyanova et al. demonstrated that Trop-2 signaling enhances the stem cell-like properties of cancer cells, as Trop-2 controls proliferation and self-renewal via b-catenin signaling. Since the cytoplasmic domain of Trop-2 contains a PIP2 binding sequence that overlaps with a protein kinase C phosphorylation site, it has been speculated that phosphatidylinositol 4,5-bisphosphate (PIP2) may regulate Trop-2 phosphorylation and calcium signaling. See, e.g., Zaman et al, Onco Targets Ther. 2019;12:1781-1790. Trop-2 has been shown to play a role in tumor progression and metastasis. See, e.g., Lin et al., EMBO Mol Med. 2012 Jun;4(6):472-85. Elevated expression levels of Trop-2 have been shown to be predictive for cancer recurrence, including prostate cancer. See, e.g., Hsu et al., PNAS, 2020;117(4):2032-2042. Trop-2 has emerged as a promising therapeutic target due to its overexpression in multiple cancers.Sacituzumab govitecan (IMMU-132), an anti-Trop2 antibody conjugated to SN-38, a cytotoxic agent that targets DNA replication, has shown therapeutic activity in several malignancies, including triple-negative breast cancer, advanced non-small cell lung cancer, and metastatic platinum-resistant urothelial carcinoma.

[0111] In some embodiments, the targeting moiety comprises an anti-Trop-2 antibody or fragment thereof. In some embodiments, the targeting moiety comprises an anti-Trop-2 scFv. In some embodiments, the targeting moiety comprises an anti-Trop-2 scFab. In some embodiments, the targeting moiety comprises an anti-Trop-2 nanobody. The anti-Trop-2 scFv, scFab, or nanobody may be derived from any anti-Trop-2 antibody known in the art. Exemplary anti-Trop-2 antibodies include, but are not limited to, hRS7, 162-46.2, MAB650, K5-70, K5-107, K5-116-2-1, T6-16, T5-86, BR110, 3E9, 6G11, 7E6, 15E2, 18B1, 77220, KM4097, KM4590, A1, A3, 162-25.3, and antibodies produced by hybridomas AR47A6.4.2, AR52A301.5, PTA-12871, PTA-12872, PD08019, PD08020, and PD08021, their biosimilars, and their derivatives. In some embodiments, antibodies that compete with any of these art-recognized antibodies for binding to Trop-2 may also be used. In some embodiments, the anti-Trop-2 scFv, scFab, or nanobody is a derivative of any one of the anti-Trop-2 antibodies described herein. In some embodiments, the anti-Trop-2 scFv, scFab, or nanobody is derived from the group consisting of hRS7, 162-46.2, and MAB650. In some embodiments, the anti-Trop-2 scFv, scFab, or nanobody is derived from hRS7.

[0112] In some embodiments, the anti-Trop-2 antibody (e.g., scFv, scFab, or nanobody) is derived from hRS7. See, e.g., U.S. Patent No. 7,238,785. In some embodiments, the targeting moiety competitively binds to the same or substantially the same epitope as hRS7. In some embodiments, the anti-Trop-2 antibody (e.g., scFv or scFab) comprises a VH and a VL, wherein the VH comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO: 134, a CDR H2 comprising the amino acid sequence of SEQ ID NO: 135, and a CDR H3 comprising the amino acid sequence of SEQ ID NO: 136, and the VL comprises a CDR L1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR L2 comprising the amino acid sequence of SEQ ID NO: 138, and a CDR L3 comprising the amino acid sequence of SEQ ID NO: 139. In some embodiments, the anti-Trop-2 antibody (e.g., scFv or scFab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 140, and / or a VL comprising the amino acid sequence of SEQ ID NO: 141, 142, or 143. In some embodiments, the anti-Trop-2 scFv comprises SEQ ID NO: 131, 132, or 133. In some embodiments, the anti-Trop-2 scFab comprises SEQ ID NO: 130.

[0113] In some embodiments, the targeting moiety is a multispecific antibody comprising a full-length antibody fused to an antigen-binding fragment selected from the group consisting of an scFv, scFab, and nanobody. In some embodiments, the multispecific antibody comprises a full-length anti-PD-L1 antibody fused to an anti-Trop-2 scFv, scFab, or nanobody. In some embodiments, the C-terminus of the heavy chain of the anti-PD-L1 antibody is fused to the N-terminus of the anti-Trop-2 scFv, scFab, or nanobody. In some embodiments, the C-terminus of the light chain of the anti-PD-L1 antibody is fused to the N-terminus of the anti-Trop-2 scFv, scFab, or nanobody. In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies described herein.

[0114] B. Effector molecules The targeted conjugate comprises one or more effector molecules. Exemplary effector molecules include, but are not limited to, a therapeutic agent, an oligonucleotide, and a detectable label. The targeted conjugate may comprise a single type of effector molecule, or different types of effector molecules. In some embodiments, the targeted conjugate comprises one or more therapeutic agents. In some embodiments, the targeted conjugate comprises one or more oligonucleotides. In some embodiments, the targeted conjugate comprises one or more detectable labels. In some embodiments, the targeted conjugate comprises a therapeutic agent and an oligonucleotide. In some embodiments, the targeted conjugate comprises a detectable label and a therapeutic agent. In some embodiments, the targeted conjugate comprises a detectable label and an oligonucleotide.

[0115] In some embodiments, the targeted conjugate comprises a first effector molecule and a second effector molecule, in some embodiments, the ratio between the first effector molecule (e.g., a therapeutic agent) and the second effector molecule (e.g., an oligonucleotide) is at least about any one of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio between the first effector molecule (e.g., a therapeutic agent) and the second effector molecule (e.g., an oligonucleotide) is less than or equal to any one of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the ratio between the first effector molecule (e.g., a therapeutic agent) and the second effector molecule (e.g., an oligonucleotide) is any one of about 1:10 to 10:1, 1:9 to about 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:10 to 1:5, 1:5 to 1:1, 1:1 to 5:1, or 5:1 to 5:10.

[0116] Therapeutic Agents In some embodiments, the effector molecule is a therapeutic agent. Exemplary therapeutic agents include, but are not limited to, drugs, toxins, immunomodulators, hormones, hormone antagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesis inhibitors, chemotherapeutic agents, and the like. In some embodiments, the therapeutic agent is a small molecule drug. In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0117] A variety of chemotherapeutic agents can be used according to this embodiment. "Chemotherapeutic agent" refers to a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0118] Exemplary chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelanamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongiostatin; nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicins including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, and tropterin. rimethotrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergic agents such as mitotane and trilostane; folic acid supplements such as folinic acid; acegra Ton;Aldophosphamide glycosides;Aminolevulinic acid;Eniluracil;Amsacrine;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids, such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidamol;Nitraerin;Pentostatin;Fenameth;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin;Schizophyllan;Spirogermanium;Tenuazonic acid;Triaziquone;2,2',2'-trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veracrine A, roridin A and anguidine);Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitrol;Mitolactol;Pipobroman;Gacytosine;Arabinoside ("Ara-C");Cyclophosphamide ;Taxoids, such as paclitaxel and docetaxel gemcitabine;6-thioguanine;Mercaptopurine;Platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin;Vinblastine;Platinum;Etoposide (VP-16);Ifosfamide;Mitoxantrone;Vincristine;Vinorelbine;Novantrone;Teniposide;Edatrexate;Daunomycin;Aminopterin;Xeloda;Ibandronate;Irinotecan (e.g., CPT-1 l); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famecil-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0119] In some embodiments, the therapeutic agent is a kinase inhibitor. The group of kinases targeted includes receptor tyrosine kinases such as BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-a, PDGFR-beta, cKit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, ALK, cytoplasmic tyrosine kinases such as c-SRC, c-YES, Abl, JAK-2, serine / threonine kinases such as ATM, Aurora A&B, CDKs, mTOR, PKCi, PLKs, b-Raf, S6K, STK1 1 / LKB1, and lipid kinases such as PI3K, SKI. Exemplary small molecule kinase inhibitors include, for example, PHA-739358, nilotinib, dasatinib, and PD166326, NSC74341 1, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), stent (SU1 1248), sorafenib (BAY43-9006), and leflunomide (SU101). For further information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.”

[0120] In some embodiments, the chemotherapeutic agent is an anti-DNA repair agent. In some embodiments, the DNA damage repair and response inhibitor is selected from the group including topoisomerase inhibitors, PARP inhibitors, RAD51 inhibitors, and inhibitors of DNA damage response kinases selected from CHCK1, ATM, or ATR. In some embodiments, the chemotherapeutic agent comprises a topoisomerase inhibitor. In some embodiments, the therapeutic agent comprises a topoisomerase I inhibitor, such as camptothecin and related compounds. Topoisomerase I (TOP1) enzymes are essential in higher eukaryotes because they are required to relax DNA supercoiling generated by transcription, replication, and chromatin remodeling. Topoisomerases are particularly vulnerable to topoisomerase I inhibitors during their cleavage reactions and can be captured by anticancer drugs when they cleave DNA. Camptothecin, an alkaloid isolated from the Chinese tree Camptotheca acuminate, is a natural product for which TOP1 is the only cellular target. Without being bound by any theory or hypothesis, camptothecin and its derivatives function by inhibiting TOP1 activity, resulting in DNA double strand breaks and cell death. Derivatives of camptothecin have been developed for clinical use. Exemplary camptothecin derivatives include, but are not limited to, topotecan hydrochloride (Icamtin), irinotecan hydrochloride (Camptosar), SN-38 (the active form of irinotecan), 9-NC, 9-AC, DE-310, lurtotecan GI-147211 NX 211, gimatecan (ST-1481), PEG-camptothecin, BNP-1350, DB-67, BN 80915. Two camptothecin derivatives have recently been approved by the FDA: topotecan for ovarian and irinotecan for lung and colon cancer. See, e.g., Pommier, Nature Reviews Cancer 6, 789-802 (2006).

[0121] In some embodiments, the therapeutic agent is 7-ethyl-10-hydroxy-20(S)-camptothecin (SN-38). SN-38 is an active metabolite of irinotecan, but has 1000-fold higher activity than irinotecan itself. In vitro cytotoxicity assays show that the potency of SN-38 varies 2-2000-fold compared to irinotecan. Due to its low solubility and extreme toxicity, SN-38 has been developed into drug conjugates for better therapeutic efficacy. See, e.g., U.S. Patent Nos. 7,999,083 and 8,080,250. [ka]

[0122] In some embodiments, SN-38 is conjugated to the targeting moiety at its 10-hydroxyl position. Methods for selective regeneration of 10-hydroxyl group in the presence of C-20 carbonate in the preparation of drug-linker precursors containing CPT analogs, such as SN-38, are known in the art. See, for example, U.S. Pat. No. 10,266,605. In some embodiments, SN-38 is conjugated to the targeting moiety at the 20-hydroxyl position. Other protecting groups for reactive hydroxyl groups in drugs, such as phenolic hydroxyl in SN-38, such as t-butyldimethylsilyl or t-butyldiphenylsilyl, can also be used, which are deprotected by tetrabutylammonium fluoride before linking the derivatized drug to the antibody coupling site. The 10-hydroxyl group of the CPT analog is alternatively protected as an ester or carbonate. In some embodiments, SN-38 is conjugated to the targeting moiety via a linker.

[0123] In some embodiments, the therapeutic agent comprises a mitotic inhibitor. In some embodiments, the therapeutic agent comprises a tubulin disrupting agent. Microtubule / tubulin inhibitors can be classified into two major categories according to their mechanism of action: agents that promote tubulin polymerization and stabilize microtubule structures (e.g., paclitaxel) and agents that inhibit tubulin polymerization and destabilize microtubule structures (e.g., maytansinoids, auristatins, vinblastine and vincristine). See, for example, Chen et al., Molecules 22:1281, 2017. Exemplary tubulin disrupting agents include, but are not limited to, auristatins, tubulysins, colchicine, vinca alkaloids, taxanes, cryptophycins, maytansinoids, hemiasterins, and other tubulin disrupting agents. Auristatins are derivatives of the natural product dolastatins. Exemplary auristatins include dolastatin-10, MMAE (N-methylvaline-valine-dolaisoloiin-dolaproine-norephedrine) and MMAF (N-methylvaline-valine-dolaisoloiin-dolaproine-phenylalanine), and derivatives thereof. WO2015 / 057699 describes pegylated auristatins including MMAE. Additional dolastatin derivatives contemplated for use are disclosed in U.S. Pat. No. 9,345,785, which is incorporated herein by reference. Tubulysins include, but are not limited to, tubulysin D, tubulysin M, tubuphenylalanine, tubutyrosine, and derivatives thereof. Colchicines include, but are not limited to, colchicine, CA-4, and derivatives thereof. Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR), vindesine (VDS), and derivatives thereof. Taxanes include, but are not limited to, paclitaxel, docetaxel, and their derivatives. Cryptophycins include, but are not limited to, cryptophycin-1, cryptophycin-52, and their derivatives.Maytansinoids include, but are not limited to, maytansine, maytansinol, maytansine analogs, DM1, DM3, DM4, ansamatocin-2, and derivatives thereof. Exemplary maytansinoid drug moieties include those with modified aromatic rings, such as C-19-dechloro (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (prepared by acylation using acyl chloride), and derivatives thereof.

[0124] Tubulin disrupting agents have been used in antibody-drug conjugates for leukemia. For example, brentuximab vedotin is an antibody-drug conjugate composed of an anti-CD30 monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E by a protease-cleavable linker. Brentuximab vedotin has been approved for the treatment of classical Hodgkin lymphoma patients after failure of autologous stem cell transplantation (ASCT) or after failure of at least two prior multi-agent chemotherapy regimens in patients who are not ASCT candidates, and as post-ASCT stabilization for Hodgkin lymphoma patients at increased risk of relapse / progression. See US Prescribing Information for ADCETRIS® (brentuximab vedotin) and European Summary of Product Characteristics for ADCETRIS® (brentuximab vedotin). It was also approved for systemic anaplastic large cell lymphoma after failure of at least one prior multiagent chemotherapy regimen.

[0125] In some embodiments, the tubulin disrupting agent comprises an auristatin. In some embodiments, the therapeutic agent is monomethylauristatin E (MMAE). MMAE is a synthetic derivative of dolastatin 10, which functions as a highly potent antimitotic agent by inhibiting tubulin polymerization. The synthesis and structure of MMAE are described in U.S. Patent No. 6,884,869, which is incorporated herein by reference in its entirety. [ka]

[0126] In some embodiments, the therapeutic agent further comprises a drug linker. In some embodiments, the drug linker is conjugated to the therapeutic agent via a cleavable linker. In some embodiments, the drug linker is conjugated to the therapeutic agent via a non-cleavable linker. In some embodiments, the linker is an amine donor group linker. In some embodiments, the linker is a non-cleavable linker. Suitable non-cleavable linkers include, but are not limited to, NH2-R-X, NH2NH-R-X, and NH2-O-R-X, where R is an alkyl or polyethylene glycol group (also referred to as PEG) and X is an active site. A polyethylene glycol or PEG group is -(CH2CHO) n -wherein n is an integer of at least 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more).

[0127] In some embodiments, the linker is a cleavable linker. Suitable cleavable linkers include Lys-Phe-X, Lys-Val-Cit-PAB-X, NH2-(CH2CH2O) n -Val-Cit-PAB-X, and NH2-(CH2CH2O) n-(Val-Cit-PAB-X)2, where X is an active site and n is an integer of at least 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). PAB refers to p-aminobenzyloxycarbonyl. Cit refers to citrulline. Other exemplary amine donor group-linkers include, but are not limited to, Ac-Lys-Gly, aminocaproic acid, Ac-Lys-beta-Ala, amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val(valine)-Cit(citrulline)-PAB (p-aminobenzyloxycarbonyl), aminocaproyl-Val-Cit-PAB, putrescine, and Ac-Lys-putrescine. In some embodiments, the linker is (Gly) n -(PEG) m -VC-PAB-(DMAE) k (Formula II): where n, m, and k are integers, n > 1, m > 2, and k is 0 or 1. In some embodiments, the linker is (Gly) n -(PEG) m -VC-PAB, where n and m are integers, n > 1 and m > 2. In some embodiments, the linker is (Gly) n -(PEG) m -Val-Ala-PAB-(DMAE) k (wherein n, m, and k are integers, n > 1, m > 2, and k is 0 or 1). In some embodiments, the linker is (Gly) n -(PEG) m -Val-Ala-PAB, where n and m are integers, n > 1 and m > 2. In some embodiments, the linker is (Gly) n -(PEG) m -P-PAB-(DMAE) k (Formula III) (wherein n, m, and k are integers, n > 1, m > 2, k is 0 or 1, and P is a cleavage site). In some embodiments, the linker is (Gly) n-(PEG) m -P-PAB, where n and m are integers, n≧1, and m≧2.

[0128] In some embodiments, the linker is branched. In some embodiments, the linker is linear. In some embodiments, the linker has multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) binding sites for attachment of therapeutic agents. These therapeutic agents may be the same or different from one another.

[0129] In some embodiments, the therapeutic agent is glycine 3-amide-PEG-VC-PAB-DMAE-phenol linked SN38, shown as a compound of formula (1) below, where the linker is connected to position 10 of SN38 and has an 8-PEG spacer: [ka]

[0130] In some embodiments, the therapeutic agent is glycine 3-amide-PEG-VC-PAB-DMAE-α-hydroxylactone linked SN38, shown as a compound of formula (2) below, where the linker is connected to position 20 of SN38 and has an 8-PEG spacer: [ka]

[0131] In some embodiments, the therapeutic agent is (Glycine)3-(PEG)4-VC-PAB-MMAE, shown as a compound of formula (3) below, where the linker is connected to MMAE and has a 4-PEG spacer: [ka]

[0132] In some embodiments, the therapeutic agent is (glycine)3-(PEG)8-VC-PAB-MMAE, shown as a compound of formula (4) below, where the linker is connected to MMAE and has an 8-PEG spacer: [ka]

[0133] In some embodiments, the therapeutic agent is (glycine)3-amide-PEG-LGGSGRNAQVRLE-PAB-DMAE-phenol linked SN38, shown as a compound of formula (5) below: [ka] The compound of formula (5) contains a linker that can be cleaved by uPA.

[0134] In some embodiments, the therapeutic agent is (glycine)3-amide-PEG-LGGSGRNAQVRLE-PAB-DMAE-α-hydroxylactone linked SN38, shown as a compound of formula (6) below: [ka] The compound of formula 6 contains a linker that can be cleaved by uPA.

[0135] In some embodiments, the therapeutic agent is (glycine)3-amide-PEG-LGGSGRNAQVRLE-PAB-MMAE, shown as a compound of formula (7) below: [ka] The compound of formula (7) contains a linker that can be cleaved by uPA.

[0136] In some embodiments, the therapeutic agent is Glycine 3-amide-PEG-Val-Ala-PAB-DMAE-phenol linked SN38, where the linker is connected to position 10 of SN38 and has an 8-PEG spacer.

[0137] In some embodiments, the therapeutic agent is glycine 3-amide-PEG8-Val-Ala-PAB-DMAE-α-hydroxylactone linked SN38 (wherein the linker is connected to position 20 of SN38 and has an 8-PEG spacer).

[0138] In some embodiments, the therapeutic agent is (Glycine)3-(PEG)4-Val-Ala-PAB-MMAE, where the linker is connected to MMAE and has a 4-PEG spacer.

[0139] In some embodiments, the therapeutic agent is (Glycine)3-(PEG)8-Val-Ala-PAB-MMAE, where the linker is connected to MMAE and has an 8-PEG spacer.

[0140] Also provided are compounds having the structure of the compounds of formulas (1)-(7), and targeted conjugates (eg, antibody drug conjugates) comprising these compounds.

[0141] In some embodiments, the targeted conjugate comprises about 1-80 therapeutic agents conjugated to the targeting moiety. In some embodiments, the targeted conjugate comprises about any one of 1-4, 1-5, 5-10, 4-10, 10-20, 1-20, 10-20, 20-40, or 40-80 therapeutic agents conjugated to the targeting moiety. In some embodiments, the targeted conjugate comprises at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, or 80 therapeutic agents. In some embodiments, the targeted conjugate comprises no more than about any one of 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 therapeutic agent.

[0142] Oligonucleotides In some embodiments, the effector molecule is an oligonucleotide.In some embodiments, the oligonucleotide is at least about any one of 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200 or more nucleotides or base pairs in length.In some embodiments, the oligonucleotide is no more than about any one of 200, 150, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 2 nucleotides or base pairs in length. In some embodiments, the oligonucleotides are any one of about 2-10, 5-10, 10-20, 10-40, 10-60, 10-80, 10-100, 100-150, 150-200, 20-40, 40-80, 80-100, 100-200, 10-150, 10-200, 2-20, 2-50, or 2-100 nucleotides or base pairs in length.

[0143] In some embodiments, the oligonucleotide is DNA. In some embodiments, the oligonucleotide is double-stranded DNA. In some embodiments, the oligonucleotide is single-stranded DNA. In some embodiments, the oligonucleotide is synthetic DNA.

[0144] In some embodiments, the oligonucleotide is RNA.Exemplary RNA includes but is not limited to single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA) or microRNA.In some embodiments, the oligonucleotide is synthetic RNA.

[0145] In some embodiments, the oligonucleotide is an immunomodulatory polynucleotide. In some embodiments, the oligonucleotide is a pathogen-associated molecular pattern (PAMP) or other motif that can activate immune cells. PAMPs are molecules associated with various pathogens and are recognized by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) that activate innate immune responses. The ability of PAMPs to recruit the immune system in the absence of pathogens provides a strategy for treating a variety of diseases that involve cell destruction (e.g., anti-cancer therapy) through the use of innate immune system responses. One class of PAMPs that has been explored for various therapeutic applications is immunostimulatory polynucleotides, such as CpG oligodeoxynucleotides (ODNs) (e.g., agatolimod). CpG ODNs are believed to mediate TLR9 dimerization in immune cells (e.g., B cells, monocytes, and plasmacytoid dendritic cells (pDCs)) to upregulate cytokines (e.g., type I interferons and interleukins), thereby activating natural killer cells. Exemplary PAMPs include, but are not limited to, CpG oligodeoxynucleotides (CpG-ODN), herpes simplex virus (HSY) DNA, dsRNA, ssRNA. Additionally, the oligonucleotide may include one or more nucleic acid sequences that silence gene expression or induce intracellular death signaling, including, but not limited to, dsRNA, siRNA, shRNA, or microRNA. In some embodiments, the oligonucleotide is selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG oligonucleotides.

[0146] In some embodiments, the oligonucleotide is a CpG ODN. CpG ODN is a short, single-stranded synthetic DNA molecule that contains a cytosine triphosphate deoxynucleotide ("C") followed by a guanine triphosphate deoxynucleotide ("G"). The "p" refers to a phosphodiester bond between consecutive nucleotides. In some embodiments, the CpG ODN has a modified phosphorothioate (PS) backbone, e.g., all or a portion of the nucleotides in the CpG ODN have a PS backbone. When CpG motifs are not methylated, they act as immune stimulators. CpG motifs are considered pathogen-associated molecular patterns (PAMPs) because they are abundant in microbial genomes, but rare in vertebrate genomes. CpG-ODN binds to and activates Toll-like receptor 9 (TLR9), initiating an innate immune response that supports the subsequent development of adaptive immunity. CpG-ODN has been used as a vaccine adjuvant to obtain desired immunomodulatory and synergistic immune responses. CpG ODNs are generally divided into three classes: class A, class B, and class C. Class A CpG-ODNs typically contain a central palindromic sequence that includes a poly-G tail and a phosphate backbone with phosphorothioate backbones at the 3' and 5' ends. Class A CpG ODNs typically contain CpGs within the central palindromic sequence. Class B CpG-ODNs typically contain a complete phosphorothioate backbone, and the sequence at the 5' end of class B CpG ODNs is often important for TLR9 activation. Class C CpG-ODNs contain a complete phosphorothioate backbone with a 3' end sequence that allows double strand formation. In some embodiments, the CpG-ODNs are methylated. In some embodiments, the CpG-ODNs are unmethylated.

[0147] In some embodiments, the targeting site is conjugated to the oligonucleotide that comprises CpG motif.In some embodiments, the targeting site is conjugated to the oligonucleotide that comprises multiple CpG motifs.In some embodiments, the targeting site is conjugated to the oligonucleotide that comprises multiple repeats of CpG motifs with the same sequence.In some embodiments, the targeting site is conjugated to multiple CpG-ODNs with different sequences.

[0148] In some embodiments, the CpG-ODN may exhibit greater stability (e.g., stability against nucleases) than those of CpG-ODNs that contain primarily internucleoside phosphate (e.g., more than 50% internucleoside phosphate) without substantially sacrificing their immunostimulatory activity. This effect may be achieved by incorporating at least 50% (e.g., at least 70%) internucleoside phosphorothioate or phosphorodithioate or through the inclusion of internucleoside phosphotriester and / or internucleoside abasic spacer. Phosphotriester and abasic spacer are also favorable for conjugation to targeting sites. Phosphotriester and abasic spacer based on phosphate may also be used to reduce off-target activity compared to polynucleotides with a full phosphorothioate backbone. Without wishing to be bound by theory, this effect may be achieved by reducing self-delivery without interfering with targeting site-mediated delivery to target cells. Thus, an oligonucleotide can contain about 15 or less consecutive internucleoside phosphorothioates (e.g., about 14 or less, about 13 or less, about 12 or less, about 11 or less, or about 10 or less consecutive internucleoside phosphorothioates). For example, a CpG-ODN containing a total of about 12 to about 16 nucleosides can contain about 10 or less consecutive internucleoside phosphorothioates.

[0149] In some embodiments, the CpG ODN comprises one or more phosphorothioates (e.g., from about 1 to about 6 or from about 1 to about 4), e.g., at one or both termini (e.g., within the six 5'-terminal nucleosides or the six 3'-terminal nucleosides). The inclusion of one or more internucleoside phosphotriesters and / or phosphorothioates may improve the stability of the polynucleotide by decreasing the rate of exonuclease-mediated degradation.

[0150] In some embodiments, the CpG ODN further comprises one or more (e.g., 1-6, 1-12, 1-18, 1-24) auxiliary moieties (e.g., polyethylene glycol (PEG)). The auxiliary moieties may be part of a capping group, a bioreversible group, or a non-bioreversible group. The auxiliary moieties may be attached to a linker (e.g., a linker attached to a phosphate, phosphorothioate, or phosphorodithioate in an immunomodulatory (e.g., immunostimulatory) polynucleotide). Inclusion of an auxiliary moiety (e.g., PEG) in a CpG ODN may improve the pharmacokinetic and / or biodistribution properties of a targeted conjugate compared to a reference conjugate lacking such auxiliary moieties. In some embodiments, the CpG oligonucleotide is linked to a cleavable linker. In some embodiments, the CpG oligonucleotide and the attached linker are linked to a 5' aminomodifier-spacer-P h -Structure of CpG ODN (Formula IV) (wherein the spacer is (CH2) n -(PEG) m and h, n, and m are integers, h=0 or 1, n≧1, m≧0, and P is a cleavage site. In some embodiments, the CpG oligonucleotide and the associated linker include a 3′ amino modification-spacer-P h -Structure of CpG ODN (Formula V) (wherein the spacer is (CH2) n -(PEG) mwherein h, n, and m are integers, h=0 or 1, n≧1, m≧0, and P is a cleavage site. In some embodiments, the CpG oligonucleotide comprises a nucleic acid sequence selected from the group consisting of 5'-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3' (SEQ ID NO:66) and 5'-T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-3' (SEQ ID NO:67), where * represents a phosphorothioate linkage.

[0151] In some embodiments, the CpG oligonucleotide and its attached linker comprise a structure selected from the following: 5' Amino modified -(CH2) 12 -T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-3' (Sequence number 68), 5' Amino modified -(CH2) 12 -thiol (CH2)6-SST*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-3' (SEQ ID NO: 69), 5' amino modified product -(CH2)6-(PEG)6-T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-3' (SEQ ID NO: 70), 5' Amino modified -(CH2) 12 -(PEG)6-T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-3' (Sequence number 71), 5' thiol modified -(CH2)6-SST*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-3' (SEQ ID NO: 72), 5'-T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T*-SS-(CH2)6-3' thiol modification (SEQ ID NO: 73), and Formula VIII: 5' Amino Modification-LGGSGRNAQVRLEGSG (SEQ ID NO: 147)-(PEG) 12 -T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*C*G*T*T*C*G*A*A*T (array number 157), (wherein * represents phosphorothioate bond). In some embodiments, the CpG oligonucleotide has a nucleic acid sequence having at least 80% (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 66 or 67. Also provided is a CpG oligonucleotide comprising a nucleic acid sequence having at least 80% (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 66 or 67.

[0152] In some embodiments, the oligonucleotide comprises a linker, for example, a chemical linker or a peptide linker. In some embodiments, the linker is selected from the group consisting of SH2-spacer, MAL-spacer, NH2-spacer, and Osu-spacer. Spacer, as used herein, when present, directly or indirectly links the targeting moiety to the effector molecule. See, for example, US20100040637A1. In some embodiments, the linker is a poly-PEG linker.

[0153] Detectable Labels In some embodiments, the effector molecule is a detectable label. As used herein, a label is a moiety that facilitates the detection of the targeting moiety and / or the detection of the molecule to which the targeting moiety binds. Non-limiting exemplary labels include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal binding tags, etc. Those skilled in the art can select suitable labels according to intended use.

[0154] For diagnostic purposes, the label may be a radionuclide, a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photoactive agent. Such diagnostic labels are well known, and any such known label may be used.

[0155] In some embodiments, the detectable label is a radionuclide. A "radionuclide" is often referred to as a "radioactive isotope" or a "radioisotope." Exemplary radionuclides or stable isotopes that can be attached to the targeting moieties described herein include: 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C. 13 N, 15 O. 186 Re, 188 Re, 51 Mn, 52m Mn,55 Co, 72 As, 75 Br, 76 Br, 82m Rb, 83 These include, but are not limited to, Sr, or other gamma, beta, or positron emitters.

[0156] The paramagnetic ions used may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III). The metal contrast agent may include lanthanum (III), gold (III), lead (II) or bismuth (III). The radiopaque diagnostic agent may be selected from compounds, barium compounds, gallium compounds, and thallium compounds. A variety of fluorescent labels are known in the art, including, but not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde, and fluorescamine. Chemiluminescent labels used may include luminol, isoluminol, an aromatic acridinium ester, imidazole, an acridinium salt or an oxalate ester.

[0157] In some embodiments, the effector molecule comprises a chelating compound that chelates a radionuclide. In some embodiments, the chelating compound chelates a radiometal. In some embodiments, the chelating compound chelates a metal. 18 In some embodiments, the chelating compound is a hydrophilic chelating compound, which can bind to metal ions and help ensure in vivo clearance. Particularly useful metal-chelating compound combinations include: 125 I, 131 I, 123 I, 124 I, 62 Cu, 64 Cu,18 F, 111 In, 67 Ga, 68 Ga, 99 Tc, 94 Tc, 11 C. 13 N, 15 O. 76 These include 2-benzyl-DTPA (diethylenetriaminepentaacetic acid) and its monomethyl and cyclohexyl analogs, which are used with diagnostic isotopes in the general energy range of 60-4,000 keV, such as Br. The same chelates are useful for MRI when complexed with non-radioactive metals, such as manganese, iron, and gadolinium. Macrocyclic chelates, such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), TETA (bromoacetamido-benzyl-tetraethylaminetetraacetic acid), and NETA ({4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), are used with a variety of diagnostic radiometals, such as gallium, yttrium, and copper. Such metal chelate complexes can be made extremely stable by tailoring the ring size to the metal of interest.

[0158] In some embodiments, the chelating compound comprises a functional group that can be conjugated to the targeting moiety. In some embodiments, the chelating compound comprises a functional group that is reactive with the primary amine (-NH2) group in the targeting moiety. Exemplary functional groups that can be conjugated to the primary amine of the targeting moiety, for example, lysine side chain, include, but are not limited to, isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide (NHS) ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester. Most of these functional groups are conjugated to amine by either acylation or alkylation.

[0159] In some embodiments, the chelating compound contains a functional group that is reactive with cysteine ​​side chains (i.e., sulfhydryl groups) in the targeting moiety. Exemplary sulfhydryl-reactive groups include, but are not limited to, haloacetyl, maleimide, aziridine, acryloyl, arylating agents, vinylsulfone, pyridyl disulfide, TNB-thiol, and disulfide reducing agents. Most of these groups conjugate to sulfhydryls either by alkylation (usually forming a thioether bond) or by disulfide exchange (forming a disulfide bond).

[0160] C. Conjugation Sites In some embodiments, the targeting conjugate comprises one or more conjugation sites. The conjugation site allows the covalent or non-covalent binding of the effector molecule to the targeting site. The conjugation site can be part of the targeting site or can be present in a chemical site introduced to the targeting site. In some embodiments, the effector molecule is conjugated to the conjugation site located between the first targeting site and the second targeting site.

[0161] The conjugation site may contain one or more reactive groups for covalent conjugation. Common reactive groups on proteins include primary amines (-NH2), carboxyls (-COOH), sulfhydryls (-SH) and carbonyls (-CHO, which may be generated by oxidizing carbohydrate groups on glycoproteins).

[0162] In some embodiments, the conjugation site is in the polypeptide chain of the targeting site. In some embodiments, the conjugation site is in the side chain of an amino acid residue of the targeting site. In some embodiments, the conjugation site is at the N-terminus of the polypeptide chain of the targeting site. In some embodiments, the conjugation site is at the C-terminus of the polypeptide chain of the targeting site.

[0163] In some embodiments, the conjugation site is an endogenous conjugation site on the targeting site. In some embodiments, the conjugation site is an engineered conjugation site introduced into the targeting site. For example, the conjugation site may be present on a peptide, such as a tag, fused to the targeting site.

[0164] In some embodiments, the targeting moiety is bispecific or bivalent, and the targeting moiety comprises (1) a conjugation moiety C that comprises a conjugation moiety, and (2) two target binding moieties A1 and A2, where A1 is a targeting peptide or an antibody or an antigen-binding fragment thereof that recognizes a first target molecule, A2 is a targeting peptide or an antibody or an antigen-binding fragment thereof that recognizes a second target molecule, and A1 is fused to the N-terminus of C and A2 is fused to the C-terminus of C. In some embodiments, A1 and A2 are different. In some embodiments, A1 and A2 are the same.

[0165] In some embodiments, the conjugation site comprises a reactive thiol group, for example a cysteine ​​residue, of the targeting site.

[0166] In some embodiments, the conjugation site comprises a reactive amine group ("amine conjugation site") of the targeting moiety, e.g., a lysine or arginine residue. Primary amines are present at the N-terminus of each polypeptide chain and at the side chains of lysine and arginine (Lys, K) residues. These primary amines are positively charged at physiological pH; therefore, they are primarily present outside the surface of the native protein tertiary structure, where they are easily accessible to conjugation reagents introduced into aqueous media. Furthermore, among the functional groups available in typical biological or protein samples, primary amines are particularly nucleophilic; this makes them easy targets for conjugation with several reactive groups. One of the most specific and efficient reagents is one that uses N-hydroxysuccinimidyl ester (NHS ester) reactive groups. In some embodiments, sulfhydryl groups can be introduced at the site of primary amines (especially those of lysine residues) by modification with reagents such as Traut's reagent (2-iminothiolane), MBS, SPDP, SATA, and their derivatives.

[0167] Transglutaminase conjugation site In some embodiments, the effector molecule (e.g., a therapeutic agent) is conjugated to a transglutaminase conjugation site in the targeting moiety. In some embodiments, the targeting moiety and the effector molecule are conjugated to each other via an isopeptide bond. In some embodiments, the targeting moiety comprises a transglutaminase conjugation site. In some embodiments, the targeting moiety and the effector molecule are conjugated to each other by a transglutamination reaction. In some embodiments, the transglutamination reaction is catalyzed by transglutaminase.

[0168] Transglutaminase conjugation is described, for example, in WO2019057772 and WO2012059882. Transglutaminase is a protein-glutamine γ-glutamyltransferase that typically catalyzes the pH-dependent transamidation of glutamine and lysine residues. Transglutaminase can be obtained or produced from a variety of sources, or engineered to catalyze the transamidation of one or more endogenous glutamine residues with one or more lysine residues or amine donor agents that contain one or more reactive amines. Transglutaminase transfers the γ-glutaminyl of acyl donor glutamine to an acyl acceptor amine group, such as a primary amine or the ε-amino group of lysine, resulting in an isopeptide bond that connects the acyl donor glutamine and the acyl acceptor residue. The isopeptide bond formed by transglutaminase catalysis is highly stable against protease activity and is not necessarily located at the N- and C-termini. In some embodiments, transglutaminase preferentially recognizes peptide sequences that possess amino acid residues with acyl acceptor groups, referred to herein as "acyl acceptor residues" (e.g., lysine, N-terminal glycine), and peptide sequences that possess amino acid residues with acyl donor groups, referred to herein as "acyl donor residues" (e.g., glutamine).

[0169] In some embodiments, the acyl donor residue is endogenous Gln. In some embodiments, the endogenous Gln site is in the Fc region of the targeting moiety (e.g., an antibody or antigen-binding fragment thereof). In some embodiments, the Gln in the Fc of the targeting moiety is deglycosylated. In some embodiments, the endogenous Gln site is not in the Fc region. In some embodiments, there are multiple endogenous acyl donor Gln in the targeting moiety. In some embodiments, the endogenous Gln is at the N-terminus of the targeting moiety. In some embodiments, the endogenous Gln is at the C-terminus of the targeting moiety. In some embodiments, the endogenous Gln is at an internal position of the targeting moiety.

[0170] In some embodiments, the acyl donor residue is an engineered Gln residue introduced into the targeting site. In some embodiments, the non-endogenous Gln is incorporated into the targeting site through amino acid modification, such as insertion or substitution. In some embodiments, the substitution comprises replacing a wild-type amino acid with another (e.g., a non-wild-type amino acid residue). In some embodiments, the insertion comprises inserting one or more amino acid(s) (e.g., inserting 1, 2, 3 or more amino acids).

[0171] In some embodiments, the non-endogenous Gln is in the tag. In some embodiments, the acyl donor glutamine-containing tag comprises at least one Gln. In some embodiments, the tag comprises multiple copies of the same sequence. In some embodiments, there are multiple Gln-containing tags linked to the targeting site. In some embodiments, the tag is linked to the targeting site via a peptide linker. In some embodiments, the peptide linker is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids long. In some embodiments, the tag may further comprise a purification tag. Exemplary purification tags include, but are not limited to, FLAG, GST, HA, poly-histidine, Myc, T7, AU1 epitope, AU5 epitope, ABP, streptavidin / biotin, calmodulin binding peptide, MBP, chloramphenicol acetyltransferase, chitin binding domain, galactose binding protein, EE-tag, histidine affinity tag, and the like.

[0172] In some embodiments, the acyl donor glutamine-containing tag has the amino acid sequence (X r Q s X t -L p ) q (Formula VI, SEQ ID NO:154) where r≧0, t≧0, s≧1, p=0 or 1, q≧1, L is a linker, and X is any amino acid (e.g., the conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp or an unconventional amino acid). In some embodiments, the acyl donor glutamine-containing tag comprises the amino acid sequence (XQXX-L p ) q(Formula VII, SEQ ID NO:155) where p=0 or 1, q≧1, L is an amino acid linker, and X is any amino acid (e.g., the conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp or an unconventional amino acid).In some embodiments, the acyl donor glutamine-containing tag is Q, LQG, LLQ, LQSP (SEQ ID NO: 153), LLQGG (SEQ ID NO: 13), LLQG (SEQ ID NO: 14), GLLQG (SEQ ID NO: 15), LSLSQG (SEQ ID NO: 16), GGGLLQGG (SEQ ID NO: 17), GSPLAQSHGG (SEQ ID NO: 18), GLLQGGG (SEQ ID NO: 19), GLLQGG (SEQ ID NO: 20), GLLQ (SEQ ID NO: 21), LLQLLQGA (SEQ ID NO: 22), LLQGA (SEQ ID NO: 23), LLQYQGA (SEQ ID NO: 24), LLQG SG (SEQ ID NO: 25), LLQYQG (SEQ ID NO: 26), LLQLLQG (SEQ ID NO: 27), SLLQG (SEQ ID NO: 28), LLQLQ (SEQ ID NO: 29), LLQLLQ (SEQ ID NO: 30), LLQGR (SEQ ID NO: 31), LLQGPP (SEQ ID NO: 32), LLQGPA (SEQ ID NO: 33), GGLLQGPP (SEQ ID NO: 34), GGLLQGA (SEQ ID NO: 35), LLQGPGK (SEQ ID NO: 36), LLQGPG (SEQ ID NO: 37), LLQGP (SEQ ID NO: 38), LLQP (SEQ ID NO: 39), LLQPGK (SEQ ID NO: 40), LLQAPG K (SEQ ID NO: 41), LLQGAPG (SEQ ID NO: 42), LLQGAP (SEQ ID NO: 43), LLQLQG (SEQ ID NO: 44), QVQLKE (SEQ ID NO: 45), VQLKE (SEQ ID NO: 46), LQQP (SEQ ID NO: 47), PQQF (SEQ ID NO: 48), and GQQQL (SEQ ID NO: 49), LLQGLLQGLLQG (SEQ ID NO: 1), LLQGGSGLLQGGSGLLQG (SEQ ID NO: 2), LQSPLQSPLQSP (SEQ ID NO: 3), LQSPGSGLQSPGSGLQSP (SEQ ID NO: 4), PNPQLPFPNPQLPFPNPQLPF (SEQ ID NO: 5), PNPQLPFGSGPNPQLPFGSGPNPQLPF (SEQ ID NO: 6), PKPQQFMPKPQQFMPKPQQFM (SEQ ID NO: 7), PKPQQFMGSGPKPQQFMGSGPKPQQFM (SEQ ID NO: 8), GQQQLGGQQQLGGQQQLG (SEQ ID NO: 9), GQQQLGGSGGQQQLGGSGGQQQLG (SEQ ID NO: 10), RLQQPRLQQPRLQQP (SEQ ID NO: 11), RLQQPGSGRLQQPGSGRLQQP (SEQ ID NO: 12).

[0173] In some embodiments, a transglutaminase conjugation peptide is provided that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. In some embodiments, a targeted conjugate is provided that comprises a targeting moiety and an effector molecule, wherein the effector molecule is conjugated to the targeting moiety via a transglutaminase conjugation peptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0174] In some embodiments, the Gln-containing tag is at the N-terminus of the targeting moiety. In some embodiments, the Gln-containing tag is at the C-terminus of the targeting moiety. In some embodiments, the Gln-containing tag is at an internal position of the targeting moiety.

[0175] In some embodiments, the targeting site comprises at least one endogenous glutamine residue that has been made reactive in a transamidation reaction by antibody engineering. In some embodiments, the antibody engineering is an amino acid modification, including antibody deglycosylation (e.g., enzymatic deglycosylation); or an amino acid deletion, insertion, substitution, mutation, or any combination thereof in the antibody. For example, the wild type amino acid Asn (N) at position 297 in the antibody can be substituted or replaced with the amino acid Ala (A), resulting in aglycosylation at position 297 and a reactive endogenous glutamine (Q) at position 295. In another example, the amino acid modification in the antibody is an amino acid substitution from N to Q at position 297, resulting in aglycosylation at position 297, a reactive endogenous Q at position 295, and site-specific conjugation at these two sites between N297Q and Q295 and one or more amine donor agents in the presence of transglutaminase. In some embodiments, the antibody can be engineered to remove glycosylation sites.

[0176] In some embodiments, the targeting site comprises an endogenous glutamine residue that is reactive for transglutaminase conjugation.

[0177] In some embodiments, the targeting moiety comprises a transglutaminase conjugation site having multiple glutamine residues that are reactive for conjugation to amine groups on the effector molecule. In some embodiments, the transglutaminase conjugation site comprises a tag sequence that contains multiple glutamines in tandem, and multiple (e.g., 2, 3, 4, 5, 6, or more) effector molecules are conjugated to the transglutaminase conjugation site.

[0178] Oligonucleotide-binding Polypeptides (OBPs) In some embodiments, the oligonucleotide is non-covalently conjugated to the targeting site via the oligonucleotide binding polypeptide introduced to the targeting site. In some embodiments, the oligonucleotide is conjugated to the oligonucleotide binding polypeptide via charge-charge interaction. In some embodiments, the oligonucleotide binding polypeptide is a cationic peptide. In some embodiments, the oligonucleotide binding polypeptide is a neutrally charged peptide.

[0179] Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which can have either specific sequences or specific conformations.It has been shown that peptides containing alternating lysines bind to poly(dG-d5meC) in Z conformation and stabilize the higher energy form.See, for example, H.Takeuchi et al.,(1991)FEBS Lett.,279,253-255 and H.Takeuchi et al.,(1994)J.Mol.Biol.,236,610-617.

[0180] In some embodiments, the CpG binding polypeptide is positively charged. In some embodiments, the CpG binding polypeptide comprises one or more positively charged amino acid residues. In some embodiments, the CpG binding polypeptide comprises a polar amino acid residue. In some embodiments, the CpG binding polypeptide comprises a short motif comprising multiple positively charged amino acid residues. In some embodiments, the short motif comprises tandem positively charged amino acid residues. In some embodiments, the CpG binding peptide further comprises a modification at the N-terminus. In some embodiments, the CpG binding peptide comprises an aminobenzoic acid at the N-terminus.

[0181] In some embodiments, the CpG binding polypeptide is selected from the group consisting of RSQSRSRYYRQRQRSRRRRRRS (SEQ ID NO:56); RRRLHRIHRRQHRSCRRRKRR (SEQ ID NO:57); MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR (SEQ ID NO:58); KKSAKKTPKKAKKPKKSAKKTPKKAKKP (SEQ ID NO:59); AKKAKSPKKAKAAKPKKAPKSPAKAK (SEQ ID NO:60); MRRAHHRRRRASHRRMRGG (SEQ ID NO:61); KHKHKHKHKKKHKHKHKHKKKHKHKHKK (SEQ ID NO:62); KGKGKGKKKKGKGKGKKGKKKKGKGKKK (SEQ ID NO:63); KKALLALALHHLAHLALHLALALKKA (SEQ ID NO:64); and YSPTSPSYSPTSPSYSPTSPSY (SEQ ID NO:65). Also provided is a CpG binding polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-65. In some embodiments, a targeted conjugate is provided comprising a targeting moiety, a CpG oligonucleotide, and a CpG binding polypeptide, wherein the CpG oligonucleotide is conjugated to the targeting moiety via a CpG binding polypeptide, and the CpG binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-65.

[0182] In some embodiments, the CpG binding polypeptide is fused to the targeting moiety. In some embodiments, the CpG binding polypeptide is fused to one or more N-terminus of the polypeptide chain of the targeting moiety. In some embodiments, the CpG binding polypeptide is fused to one or more C-terminus of the polypeptide chain of the targeting moiety. In some embodiments, the CpG binding polypeptide is fused to one or more internal positions of the polypeptide chain of the targeting moiety. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment, the CpG binding polypeptide is fused to the C-terminus of the heavy chain of the antibody or antigen-binding fragment, for example, via a single peptide bond or linker. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment, the CpG binding polypeptide is fused to the C-terminus of the light chain of the antibody or antigen-binding fragment, for example, via a single peptide bond or linker.

[0183] D. Cutting site In some embodiments, the targeting conjugate comprises one or more cleavage sites. In some embodiments, the targeting conjugate comprises one or more cleavage sites in or linked to the targeting moiety. In some embodiments, the targeting moiety does not comprise one or more cleavage sites. In some embodiments, the targeting conjugate comprises one or more cleavage sites in or linked to the effector molecule. In some embodiments, the targeting conjugate comprises one or more cleavage sites in or linked to the oligonucleotide binding polypeptide.

[0184] In some embodiments, one or more cleavage sites in the targeting conjugate are cleaved at the target site, e.g., diseased site. In some embodiments, cleavage is caused by conditions at the target site, e.g., proteases, changes in pH, redox levels, hypoxia, oxidative stress, hyperthermia, and / or extracellular ATP concentration. In some embodiments, one or more cleavage sites in the targeting conjugate are not cleaved at non-target sites, e.g., in normal or healthy tissue. In some embodiments, the conditions that cause cleavage of the cleavage site at the target site are not found at the non-target site. In some embodiments, the conditions that cause cleavage of the cleavage site are present at the target site at a level at least about any one of 1.5, 2, 5, 10, 20, 50, 100, 200, 500, 1000 times or more higher than the conditions at the non-target site. For example, the protease (e.g., uPA) that cleaves the cleavage site is present at a concentration at the target site that is at least about 1.5, 2, 5, 10, 20, 50, 100, 200, 500, 1000 times or more higher than the concentration of the protease at the non-target site. In some embodiments, the extracellular ATP concentration at the target site is at least about 1.5, 2, 5, 10, 20, 50, 100, 200, 500, 1000 times or more higher than the extracellular ATP concentration at the non-target site. In some embodiments, the pH at the target site is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, or 3 times lower than the pH at the non-target site. In some embodiments, the pH at the target site is at least about any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, or 3 higher than the pH at the non-target site.

[0185] In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is cleavable by a disease-specific protease, e.g., a tumor-specific protease. In some embodiments, the tumor-specific protease is expressed at elevated levels in diseased tissue, e.g., tumor tissue, compared to normal tissue. For example, studies have shown that tumor tissue exhibits increased activity of certain proteases and decreased activity of opposing endogenous inhibitors (Sevenich L and Joyce JA. Genes & Dev. 2014.28:2331-2347. 2014). In some embodiments, the tumor-specific protease is selected from the group consisting of matriptase (MTSP1), urinary plasminogen activator (uPA), legumain, PSA (KLK3, also called kallikrein-related peptidase-3), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP9), human neutrophil elastase (HNE), proteinase 3 (Pr3), cathepsin B, and cathepsin K. In some embodiments, cleavage occurs at the tumor site, e.g., in the tumor microenvironment. In some embodiments, cleavage occurs outside the cell, e.g., outside the tumor cell in the tumor microenvironment.

[0186] In some embodiments, the cleavage site is a substrate for an enzyme selected from the group consisting of legumain, plasmin, TMPRSS-3 / 4, MMP-9, MTl-MMP, cathepsin, caspase, human neutrophil elastase, beta-beta secretase, uPA, and PSA.

[0187] In some embodiments, the cleavage site is a uPA substrate peptide. Urokinase plasminogen activator (uPA) is a serine protease that is part of the urokinase plasminogen activation system (uPAS). uPA converts the proenzyme plasminogen in the serine protease plasmin, which is involved in numerous physiopathological processes that require basement membrane (BM) and / or extracellular matrix (ECM) remodeling, including tumor progression and metastasis. In some embodiments, the uPA cleavage site comprises an amino acid sequence selected from the group consisting of LSGRSDNH (SEQ ID NO: 50), SGRSA (SEQ ID NO: 51), LGGSGRSANAILE (SEQ ID NO: 52), LGGSGRNAQVRLE (SEQ ID NO: 53), GSGRNAQV (SEQ ID NO: 54), and SGR (SEQ ID NO: 55). Also provided are uPA cleavage peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-55.

[0188] In some embodiments, the targeted conjugate comprises a single peptide substrate cleavable by a disease-specific protease, e.g., uPA, e.g., any one of SEQ ID NOs: 50-55. In some embodiments, the targeted conjugate comprises two or more copies (e.g., any one of 2, 3, 4, 5, or more) of a peptide substrate cleavable by a disease-specific protease. In some embodiments, the targeted conjugate comprises a peptide substrate that can be cleaved by multiple (e.g., any one of 2, 3, 4, or more) disease-specific proteases. In some embodiments, the targeted conjugate comprises two or more (e.g., any one of 2, 3, 4, or more) peptide substrates cleavable by one or more (e.g., any one of 2, 3, 4, or more) disease-specific proteases. Any one of the protease peptide substrate sequences (e.g., SEQ ID NOs: 50-55) disclosed herein can be mixed and matched to provide a disease-sensing releasable site with optimal mechanism and kinetics for release of the effector molecule at the target (e.g., disease) site. Different protease substrate sequences or copies thereof can be fused to each other via peptide linkers to provide suitable cleavage sites.

[0189] E. Exemplary Targeting Conjugates In some embodiments, a targeted conjugate is provided that includes a targeting moiety, one or more effector molecules conjugated to the targeting moiety via a conjugation moiety. In some embodiments, each effector molecule is conjugated to the conjugation moiety via one or more linkers, and / or one or more protease cleavage sites. In some embodiments, the effector molecule is covalently conjugated to the targeting moiety. In some embodiments, the effector molecule is non-covalently conjugated to the targeting moiety. In some embodiments, the targeting moiety includes two or more polypeptide chains. In some embodiments, the targeting moiety includes a protease cleavage site. In some embodiments, the targeted conjugate includes a first targeting moiety, a second targeting moiety, and a conjugation moiety that links the first targeting moiety and the second targeting moiety, and one or more effector molecules are conjugated to the conjugation moiety. In some embodiments, the protease cleavage is performed outside the cell. In some embodiments, the targeted conjugate has a structure of formula I.

[0190] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker, and a first antibody heavy chain and a first antibody light chain. A targeted conjugate is provided, in which: the first antibody heavy chain and the second antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first effector molecule is conjugated to the first conjugation site via a first linker; the second effector molecule is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first effector molecule is released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 1A. An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO:112 and an antibody light chain comprising SEQ ID NO:113; and a therapeutic agent selected from the group consisting of a compound of Formula (1), a compound of Formula (2), a compound of Formula (3), and a compound of Formula (4).

[0191] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), a first conjugation site, and a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), a second conjugation site, and a second antigen binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker, wherein the first antibody heavy chain and the first antibody light chain are linked together by a first a first antigen-binding site that specifically binds to a third epitope; a second antibody heavy chain and a second antibody light chain that specifically bind to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; a first effector molecule is conjugated to the first conjugation site via a first linker; a second effector molecule is conjugated to the second conjugation site via a second linker; upon cleavage of the first protease cleavage site, the first effector molecule and the first antigen-binding fragment are released from the targeted conjugate; and upon cleavage of the second protease cleavage site, the second effector molecule and the second antigen-binding fragment are released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in FIG. 1B. An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 124 and an antibody light chain comprising SEQ ID NO: 125; and a therapeutic agent selected from the group consisting of a compound of formula (1), a compound of formula (2), a compound of formula (3), and a compound of formula (4).

[0192] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, a first conjugation site, a second protease cleavage site, and a first antigen-binding fragment (e.g., scFv / scFab / nanobody); (B) from N-terminus to C-terminus: a second antibody heavy chain, a third protease cleavage site, a second conjugation site, a fourth protease cleavage site, and (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a first antigen-binding site that specifically binds to a first epitope; a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; a first effector molecule is conjugated to the first conjugation site via a first linker; a second effector molecule is conjugated to the second conjugation site via a second linker; and after cleavage of the first protease cleavage site, a fourth effector molecule is conjugated to the first conjugation site via a second linker. A targeted conjugate is provided, in which one effector molecule and a first antigen-binding fragment are released from the targeted conjugate; after cleavage of the second protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; after cleavage of the third protease cleavage site, the second effector molecule and a second antigen-binding fragment are released from the targeted conjugate; after cleavage of the fourth protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 1C.

[0193] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker and a third cleavage site; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker and a fourth cleavage site, and the first antibody heavy chain and the first antibody light chain. A targeted conjugate is provided, wherein the first antibody heavy chain forms a first antigen-binding site that specifically binds to a first epitope; the second antibody light chain forms a second antigen-binding site that specifically binds to a second epitope; the first effector molecule is conjugated to the first conjugation site via a first linker; the second effector molecule is conjugated to the second conjugation site via a second linker; after cleavage of the first and / or third protease cleavage sites, the first effector molecule is released from the targeted conjugate; after cleavage of the second and / or fourth protease cleavage sites, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in Figure 2A. An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 112 and an antibody light chain comprising SEQ ID NO: 113; and a therapeutic agent selected from the group consisting of a compound of formula (1), a compound of formula (2), a compound of formula (3), and a compound of formula (4). An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 112 and an antibody light chain comprising SEQ ID NO: 113; and a therapeutic agent selected from the group consisting of a compound of formula (5), a compound of formula (6), and a compound of formula (7).

[0194] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), a first conjugation site, and a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), a second conjugation site, and a second (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker and a third cleavage site; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker and a fourth cleavage site, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; The heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first effector molecule is conjugated to the first conjugation site via a first linker; the second effector molecule is conjugated to the second conjugation site via a second linker; and the first protease. The targeted conjugate is provided in which, after cleavage of the protease cleavage site, the first effector molecule and the first antigen-binding fragment are released from the targeted conjugate; after cleavage of the second protease cleavage site, the second effector molecule and the second antigen-binding fragment are released from the targeted conjugate; after cleavage of the third cleavage site, the first effector molecule is released from the targeted conjugate; after cleavage of the fourth cleavage site, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 2B.An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 124 and an antibody light chain comprising SEQ ID NO: 125; and a therapeutic agent selected from the group consisting of a compound of formula (1), a compound of formula (2), a compound of formula (3), and a compound of formula (4). An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 124 and an antibody light chain comprising SEQ ID NO: 125; and a therapeutic agent selected from the group consisting of a compound of formula (5), a compound of formula (6), and a compound of formula (7).

[0195] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, a first conjugation site, a second protease cleavage site, and a first antigen binding fragment (e.g., scFv / scFab / nanobody); (B) a second antibody heavy chain, a third protease cleavage site, a second conjugation site, a fourth protease cleavage site, and a second antigen binding fragment (e.g., scFv / scFab / nanobody). (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker and a fifth cleavage site; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker and a sixth cleavage site, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding site. the conjugated fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first effector molecule is conjugated to the first conjugation site via a first linker; the second effector molecule is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first effector molecule and the first antigen-binding fragment are released from the targeting conjugate; after cleavage of the second protease cleavage site, the first antigen-binding fragment is released from the targeting conjugate; A targeted conjugate is provided in which, after cleavage of the third protease cleavage site, the second effector molecule and the second antigen-binding fragment are released from the targeted conjugate; after cleavage of the fourth protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate; after cleavage of the fifth protease cleavage site, the first effector molecule is released from the targeted conjugate; and after cleavage of the sixth cleavage site, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site.In some embodiments, two or more effector molecules are conjugated to the second conjugation moiety. An exemplary targeting conjugate is shown in Figure 2C.

[0196] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker. ), wherein a first antibody heavy chain and a first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; a second antibody heavy chain and a second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; a first effector molecule is conjugated to the first conjugation site via a first linker; and a second effector molecule is conjugated to the second conjugation site via a second linker. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 3A. An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 104 and an antibody light chain comprising SEQ ID NO: 105; and a therapeutic agent selected from the group consisting of a compound of Formula (1), a compound of Formula (2), a compound of Formula (3), and a compound of Formula (4).

[0197] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first conjugation site, and a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second conjugation site, and a second antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker. A targeted conjugate is provided, comprising a first antibody heavy chain and a first antibody light chain forming a first antigen-binding site that specifically binds to a first epitope; a second antibody heavy chain and a second antibody light chain forming a second antigen-binding site that specifically binds to a second epitope; a first antigen-binding fragment that specifically binds to a third epitope; a second antigen-binding fragment that specifically binds to a fourth epitope; a first effector molecule conjugated to a first conjugation site via a first linker; and a second effector molecule conjugated to a second conjugation site via a second linker. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 3B. An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 114 or 122 and an antibody light chain comprising SEQ ID NO: 115 or 123; and a therapeutic agent selected from the group consisting of a compound of Formula (1), a compound of Formula (2), a compound of Formula (3), and a compound of Formula (4).

[0198] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first conjugation site, a first protease cleavage site, a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second conjugation site, a second protease cleavage site, and a second antigen binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker, wherein the first antibody heavy chain and the first antibody light chain are linked together by a first conjugation site, a first conjugation site, a first protease cleavage site, a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); A targeted conjugate is provided, wherein the first antigen-binding site specifically binds to a third epitope; the second antibody heavy chain and the second antibody light chain specifically bind to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first effector molecule is conjugated to the first conjugation site via a first linker; the second effector molecule is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation moiety. An exemplary targeting conjugate is shown in Figure 3C.

[0199] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker and a first cleavage site; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker and a second cleavage site, wherein the first antibody heavy chain and the first antibody light chain are specific for a first epitope. A targeted conjugate is provided, in which the first antibody heavy chain and the second antibody light chain form a first antigen-binding site that specifically binds to a second epitope; the first effector molecule is conjugated to the first conjugation site via a first linker; the second effector molecule is conjugated to the second conjugation site via a second linker; after cleavage of the first cleavage site, the first effector molecule is released from the targeted conjugate; after cleavage of the second cleavage site, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 4A. An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 104 and an antibody light chain comprising SEQ ID NO: 105; and a therapeutic agent selected from the group consisting of a compound of formula (1), a compound of formula (2), a compound of formula (3), and a compound of formula (4). An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 104 and an antibody light chain comprising SEQ ID NO: 105; and a therapeutic agent selected from the group consisting of a compound of formula (5), a compound of formula (6), and a compound of formula (7).

[0200] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first antigen-binding fragment (e.g., scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second antigen-binding fragment (e.g., scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker and a first cleavage site; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker and a second cleavage site, wherein the first antibody heavy chain and the first antibody light chain are linked to a first polypeptide that specifically binds to a first epitope. a first antigen-binding site; a second antibody heavy chain and a second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; a first antigen-binding fragment specifically binds to a third epitope; a second antigen-binding fragment specifically binds to a fourth epitope; a first effector molecule is conjugated to the first conjugation site via a first linker; a second effector molecule is conjugated to the second conjugation site via a second linker; after cleavage of the first cleavage site, the first effector molecule is released from the targeted conjugate; and after cleavage of the second cleavage site, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation moiety. An exemplary targeting conjugate is shown in FIG. 4B. An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 114 or 122 and an antibody light chain comprising SEQ ID NO: 115 or 123; and a therapeutic agent selected from the group consisting of a compound of formula (1), a compound of formula (2), a compound of formula (3), and a compound of formula (4).An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 114 or 122 and an antibody light chain comprising SEQ ID NO: 115 or 123; and a therapeutic agent selected from the group consisting of a compound of Formula (5), a compound of Formula (6), and a compound of Formula (7).

[0201] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first conjugation site, a first protease cleavage site, a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second antibody heavy chain, a second conjugation site, a second protease cleavage site, and a second antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first effector molecule (e.g., a small molecule drug) comprising a first linker and a third cleavage site; (F) a second effector molecule (e.g., a small molecule drug) comprising a second linker and a fourth cleavage site, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; The heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; a first effector molecule is conjugated to the first conjugation site via a first linker; and a second effector molecule is conjugated to the second conjugation site via a second linker. A targeted conjugate is provided in which the first protease cleavage site is cleaved, the first antigen-binding fragment is released from the targeted conjugate; the second protease cleavage site is cleaved, the second antigen-binding fragment is released from the targeted conjugate; the third cleavage site is cleaved, the first effector molecule is released from the targeted conjugate; the fourth cleavage site is cleaved, the second effector molecule is released from the targeted conjugate. In some embodiments, two or more effector molecules are conjugated to the first conjugation site. In some embodiments, two or more effector molecules are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 4C.

[0202] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide comprising a first linker (e.g., a CpG oligonucleotide); (F) a second oligonucleotide comprising a second linker (e.g., a CpG oligonucleotide), and a first antibody heavy chain and A targeted conjugate is provided, in which one antibody light chain forms a first antigen-binding site that specifically binds to a first epitope; a second antibody heavy chain and a second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; a first oligonucleotide is conjugated to a first conjugation site via a first linker; a second oligonucleotide is conjugated to a second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first oligonucleotide is released from the targeted conjugate; after cleavage of the second protease cleavage site, the second oligonucleotide is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 5A. An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 112 and an antibody light chain comprising SEQ ID NO: 113; and a CpG ODN comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 68-73.

[0203] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), a first conjugation site, and a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), a second conjugation site, and a second antigen binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide comprising a first linker (e.g., a CpG oligonucleotide); (F) a second oligonucleotide comprising a second linker (e.g., a CpG oligonucleotide), and the first antibody heavy chain and the first antibody light chain. A targeted conjugate is provided, wherein the first and second antibody heavy and light chains form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is conjugated to the first conjugation site via a first linker; the second oligonucleotide is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first oligonucleotide and the first antigen-binding fragment are released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second oligonucleotide and the second antigen-binding fragment are released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation moiety. An exemplary targeting conjugate is shown in FIG. 5B. An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 124 and an antibody light chain comprising SEQ ID NO: 125, and a CpG ODN comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 68-73.

[0204] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, a first conjugation site, a second protease cleavage site, and a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second antibody heavy chain, a third protease cleavage site, a second conjugation site, a fourth protease cleavage site, and a second antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker; (F) a second oligonucleotide (e.g., a CpG oligonucleotide) comprising a second linker, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the antibody light chain of said antibody forms a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is conjugated to the first conjugation site via a first linker; the second oligonucleotide is conjugated to the second conjugation site via a second linker; and the first protease cleavage site is conjugated to said conjugation site via a second linker. A targeted conjugate is provided in which, after cleavage of the second protease cleavage site, the first oligonucleotide and the first antigen-binding fragment are released from the targeted conjugate; after cleavage of the third protease cleavage site, the second oligonucleotide and the second antigen-binding fragment are released from the targeted conjugate; after cleavage of the fourth protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 5C.

[0205] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker and a third cleavage site; (F) a second oligonucleotide (e.g., a CpG oligonucleotide) comprising a second linker and a fourth cleavage site, wherein the first antibody heavy chain and the first antibody light chain comprise a first antigen binding site that specifically binds to a first epitope. a second antibody heavy chain and a second antibody light chain form a second antigen binding site that specifically binds to a second epitope; a first oligonucleotide is conjugated to the first conjugation site via a first linker; a second oligonucleotide is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first oligonucleotide is released from the targeted conjugate; after cleavage of the second protease cleavage site, the second oligonucleotide is released from the targeted conjugate; after cleavage of the third cleavage site, the first oligonucleotide is released from the targeted conjugate; after cleavage of the fourth cleavage site, the second oligonucleotide is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in Figure 6A. An exemplary targeting conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 112 and an antibody light chain comprising SEQ ID NO: 113, a CpG ODN comprising the nucleic acid sequence of SEQ ID NO: 157, and a cleavage site comprising the amino acid sequence of SEQ ID NO: 147, wherein the cleavage site is disposed between the antibody heavy chain and the CpG ODN.

[0206] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site (e.g., a uPA cleavage site), a first conjugation site, and a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second antibody heavy chain, a second protease cleavage site (e.g., a uPA cleavage site), a second conjugation site, and a second antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker and a third cleavage site; (F) a second oligonucleotide (e.g., a CpG oligonucleotide) comprising a second linker and a fourth cleavage site, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; The second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is conjugated to the first conjugation site via a first linker; the second oligonucleotide is conjugated to the second conjugation site via a second linker; and the first protein is conjugated to the first conjugation site via a second linker. The targeted conjugate is provided in which, after cleavage of the protease cleavage site, the first oligonucleotide and the first antigen-binding fragment are released from the targeted conjugate; after cleavage of the second protease cleavage site, the second oligonucleotide and the second antigen-binding fragment are released from the targeted conjugate; after cleavage of the third cleavage site, the first oligonucleotide is released from the targeted conjugate; after cleavage of the fourth cleavage site, the second oligonucleotide is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site.An exemplary targeting conjugate is shown in Figure 6B. An exemplary targeting conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 124 and an antibody light chain comprising SEQ ID NO: 125, a CpG ODN comprising the nucleic acid sequence of SEQ ID NO: 157, and a cleavage site comprising the amino acid sequence of SEQ ID NO: 147, wherein the cleavage site is disposed between the antibody heavy chain and the CpG ODN.

[0207] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, a first conjugation site, a second protease cleavage site, and a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second antibody heavy chain, a third protease cleavage site, a second conjugation site, a fourth protease cleavage site, and a second antigen binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker and a fifth cleavage site; (F) a second oligonucleotide (e.g., a CpG oligonucleotide) comprising a second linker and a sixth cleavage site, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; and the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope. the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is conjugated to a first conjugation site via a first linker; the second oligonucleotide is conjugated to a second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first oligonucleotide and the first antigen-binding fragment are released from the targeting conjugate; A targeted conjugate is provided in which, after cleavage, the first antigen-binding fragment is released from the targeted conjugate; after cleavage of the third protease cleavage site, the second oligonucleotide and the second antigen-binding fragment are released from the targeted conjugate; after cleavage of the fourth protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate; after cleavage of the fifth cleavage site, the first oligonucleotide is released from the targeted conjugate; and after cleavage of the sixth cleavage site, the second oligonucleotide is released from the targeted conjugate.In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in Figure 6C.

[0208] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide comprising a first linker (e.g., a CpG oligonucleotide); (F) a second oligonucleotide comprising a second linker (e.g., a CpG oligonucleotide). A targeted conjugate is provided, the targeted conjugate comprising a first oligonucleotide and a first antibody heavy chain, the first antibody light chain forming a first antigen binding site that specifically binds to a first epitope; a second antibody heavy chain and a second antibody light chain forming a second antigen binding site that specifically binds to a second epitope; the first oligonucleotide is conjugated to a first conjugation site via a first linker; and the second oligonucleotide is conjugated to a second conjugation site via a second linker. In some embodiments, two or more oligonucleotides are conjugated to a first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to a second conjugation site. An exemplary targeted conjugate is shown in FIG. 7A. An exemplary targeted conjugate may comprise a targeting site comprising an antibody heavy chain comprising SEQ ID NO: 104 and an antibody light chain comprising SEQ ID NO: 105, and a CpG ODN comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 68-73.

[0209] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first antigen-binding fragment (e.g., scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second antigen-binding fragment (e.g., scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide comprising a first linker (e.g., a CpG oligonucleotide); (F) a second oligonucleotide comprising a second linker (e.g., a CpG oligonucleotide). A targeted conjugate is provided, comprising: a first antibody heavy chain and a first antibody light chain forming a first antigen-binding site that specifically binds to a first epitope; a second antibody heavy chain and a second antibody light chain forming a second antigen-binding site that specifically binds to a second epitope; a first antigen-binding fragment specifically binds to a third epitope; a second antigen-binding fragment specifically binds to a fourth epitope; a first oligonucleotide is conjugated to a first conjugation site via a first linker; and a second oligonucleotide is conjugated to a second conjugation site via a second linker. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 7B. An exemplary targeting conjugate may include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 114 or 122 and an antibody light chain comprising SEQ ID NO: 115 or 123, and a CpG ODN comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 68-73.

[0210] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first conjugation site, a first protease cleavage site, and a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain comprising a second conjugation site, a second protease cleavage site, and a second antigen binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide comprising a first linker (e.g., a CpG oligonucleotide); (F) a second effector molecule comprising a second linker (e.g., a CpG oligonucleotide), and the first antibody heavy chain and the first conjugation site; A targeted conjugate is provided, in which the antibody light chain forms a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is conjugated to the first conjugation site via a first linker; the second oligonucleotide is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in Figure 7C.

[0211] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first conjugation site; (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second conjugation site; (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker and a first cleavage site; (F) a second oligonucleotide (e.g., a CpG oligonucleotide) comprising a second linker and a second cleavage site, wherein the first antibody heavy chain and the first antibody light chain are linked to a first epitope. A targeted conjugate is provided, in which the first antigen-binding site specifically binds to the second epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site specifically binds to the second epitope; the first oligonucleotide is conjugated to the first conjugation site via a first linker; the second oligonucleotide is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 8A. An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 104 and an antibody light chain comprising SEQ ID NO: 105, a CpG ODN comprising the nucleic acid sequence of SEQ ID NO: 157, and a cleavage site comprising the amino acid sequence of SEQ ID NO: 147, wherein the cleavage site is disposed between the antibody heavy chain and the CpG ODN.

[0212] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain and a first antigen-binding fragment (e.g., scFv / scFab / nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain and a second antigen-binding fragment (e.g., scFv / scFab / nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker and a first cleavage site; (F) a second oligonucleotide (e.g., a CpG oligonucleotide) comprising a second linker and a second cleavage site, wherein the first antibody heavy chain and the first antibody light chain are a first oligonucleotide that specifically binds to a first epitope. a first antigen-binding site; a second antibody heavy chain and a second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; a first antigen-binding fragment specifically binds to a third epitope; a second antigen-binding fragment specifically binds to a fourth epitope; a first oligonucleotide is conjugated to a first conjugation site via a first linker; a second oligonucleotide is conjugated to a second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; after cleavage of the second protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in Figure 8B. An exemplary targeting conjugate can include a targeting moiety comprising an antibody heavy chain comprising SEQ ID NO: 114 or 122 and an antibody light chain comprising SEQ ID NO: 115 or 123, a CpG ODN comprising the nucleic acid sequence of SEQ ID NO: 157, and a cleavage site comprising the amino acid sequence of SEQ ID NO: 147, wherein the cleavage site is disposed between the antibody heavy chain and the CpG ODN.

[0213] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first conjugation site, a first protease cleavage site, and a first antigen binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second conjugation site, a second protease cleavage site, and a second antigen binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide) comprising a first linker and a third cleavage; (F) a second effector molecule (e.g., a CpG oligonucleotide) comprising a second linker and a fourth cleavage, wherein the first antibody heavy chain and the first antibody light chain form a first antigen binding site that specifically binds to a first epitope; A targeted conjugate is provided, wherein the two antibody heavy chains and the second antibody light chain form a second antigen binding site that specifically binds to a second epitope; the first antigen binding fragment specifically binds to a third epitope; the second antigen binding fragment specifically binds to a fourth epitope; the first oligonucleotide is conjugated to the first conjugation site via a first linker; the second oligonucleotide is conjugated to the second conjugation site via a second linker; after cleavage of the first protease cleavage site, the first antigen binding fragment is released from the targeted conjugate; after cleavage of the second protease cleavage site, the second antigen binding fragment is released from the targeted conjugate; after cleavage of the third cleavage site, the first oligonucleotide is released from the targeted conjugate; and after cleavage of the fourth cleavage site, the second oligonucleotide is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in Figure 8C.

[0214] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, and a first non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site, and a second non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide); (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide (e.g., a CpG oligonucleotide); (F) a second oligonucleotide (e.g., a CpG oligonucleotide). A targeted conjugate is provided, in which the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first oligonucleotide is non-covalently conjugated to the first conjugation site; the second oligonucleotide is non-covalently conjugated to the second conjugation site; after cleavage of the first protease cleavage site, the first oligonucleotide is released from the targeted conjugate; after cleavage of the second protease cleavage site, the second oligonucleotide is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 9A. An exemplary targeted conjugate may comprise a targeting moiety comprising an antibody heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 106, 108, and 110, and an antibody light chain comprising a sequence selected from the group consisting of SEQ ID NOs: 107, 109, and 111, and a CpG ODN comprising the nucleic acid sequence of SEQ ID NO: 67.

[0215] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, a first non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), and a first antigen binding fragment (e.g., an scFv / scFab / nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second protease cleavage site, a second non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), and a second antigen binding fragment (e.g., an scFv / scFab / nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide); (F) a second oligonucleotide (e.g., a CpG oligonucleotide), and a first antibody heavy chain. A targeted conjugate is provided, wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is non-covalently conjugated to the first conjugation site; the second oligonucleotide is non-covalently conjugated to the second conjugation site; after cleavage of the first protease cleavage site, the first oligonucleotide and the first antigen-binding fragment are released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second oligonucleotide and the second antigen-binding fragment are released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in Figure 9B.

[0216] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first protease cleavage site, a first non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), a second protease cleavage site, and a first antigen-binding fragment (e.g., an scFv / scFab / nanobody); (B) a second antibody heavy chain, a third protease cleavage site, a second non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), a first protease cleavage site, and a first antigen-binding fragment (e.g., an scFv / scFab / nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide); (F) a second oligonucleotide (e.g., a CpG oligonucleotide), wherein the first antibody heavy chain and the first antibody light chain are a first polypeptide that specifically binds to a first epitope. forming an antigen-binding site; the second antibody heavy chain and the second antibody light chain forming a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is non-covalently conjugated to the first conjugation site; the second oligonucleotide is non-covalently conjugated to the second conjugation site; and the first protease. A targeted conjugate is provided, in which after cleavage of the cleavage site, the first oligonucleotide and the first antigen-binding fragment are released from the targeted conjugate; after cleavage of the second protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; after cleavage of the third protease cleavage site, the second oligonucleotide and the second antigen-binding fragment are released from the targeted conjugate; after cleavage of the fourth protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site.An exemplary targeting conjugate is shown in FIG. 9C.

[0217] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, and a first non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, and a second non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide); (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide (e.g., a CpG oligonucleotide); (F) a second oligonucleotide (e.g., a CpG oligonucleotide), wherein the first antibody heavy chain and the first antibody light chain form a first antigen binding site that specifically binds to a first epitope; and the second antibody heavy chain and the second antibody light chain A targeting conjugate is provided, which forms a second antigen binding site that specifically binds to a second epitope; the first oligonucleotide is non-covalently conjugated to the first conjugation site; and the second oligonucleotide is non-covalently conjugated to the second conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeting conjugate is shown in FIG. 10A. An exemplary targeting conjugate may include a targeting site that includes an antibody heavy chain that includes SEQ ID NO: 100 and an antibody light chain that includes SEQ ID NO: 101, and a CpG ODN that includes the nucleic acid sequence of SEQ ID NO: 67.

[0218] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), and a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), and a second antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain; (E) a first oligonucleotide (e.g., a CpG oligonucleotide). (F) a second oligonucleotide (e.g., a CpG oligonucleotide), wherein the first antibody heavy chain and the first antibody light chain form a first antigen-binding site that specifically binds to a first epitope; the second antibody heavy chain and the second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is non-covalently conjugated to the first conjugation site; and the second oligonucleotide is non-covalently conjugated to the second conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in FIG. 10B.

[0219] In some embodiments, a targeted conjugate comprises: (A) a first polypeptide comprising, from N-terminus to C-terminus: a first antibody heavy chain, a first non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), a first protease cleavage site, and a first antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (B) a second polypeptide comprising, from N-terminus to C-terminus: a second antibody heavy chain, a second non-covalent conjugation site (e.g., an oligonucleotide binding polypeptide), a second protease cleavage site, and a second antigen-binding fragment (e.g., an scFv, scFab, or nanobody); (C) a first antibody light chain; (D) a second antibody light chain, (E) a first oligonucleotide (e.g., a CpG oligonucleotide); (F) a second oligonucleotide (e.g., a CpG oligonucleotide); and a second antibody heavy chain and a second antibody light chain form a first antigen-binding site that specifically binds to a first epitope; a second antibody heavy chain and a second antibody light chain form a second antigen-binding site that specifically binds to a second epitope; the first antigen-binding fragment specifically binds to a third epitope; the second antigen-binding fragment specifically binds to a fourth epitope; the first oligonucleotide is non-covalently conjugated to the first conjugation site; the second oligonucleotide is non-covalently conjugated to the second conjugation site; after cleavage of the first protease cleavage site, the first antigen-binding fragment is released from the targeted conjugate; and after cleavage of the second protease cleavage site, the second antigen-binding fragment is released from the targeted conjugate. In some embodiments, two or more oligonucleotides are conjugated to the first conjugation site. In some embodiments, two or more oligonucleotides are conjugated to the second conjugation site. An exemplary targeted conjugate is shown in Figure 10C.

[0220] In some embodiments of any one of the targeted conjugates described in this section, the first and second effector molecules are the same. In some embodiments, the first and second effector molecules are different. In some embodiments, the targeting moiety is monospecific. In some embodiments, the targeting moiety is multispecific, for example, bispecific, or trispecific.

[0221] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain and a transglutaminase conjugation site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 100; (b) a third polypeptide chain comprising an antibody light chain; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from, for example, a compound of Formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, for example, a CpG oligonucleotide, for example, an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0222] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain and a transglutaminase conjugation site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 104; (b) a third polypeptide chain comprising an antibody light chain; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from the compounds of formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide, e.g., an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0223] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a transglutaminase conjugation site, a protease cleavage site, and an oligonucleotide binding site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 106; (b) a third polypeptide chain and a fourth polypeptide chain comprising an antibody light chain, the third polypeptide chain and the fourth polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or any one of 100%) sequence identity to SEQ ID NO: 107; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from, for example, a compound of formula (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, for example, a CpG oligonucleotide, for example, an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0224] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a transglutaminase conjugation site, a protease cleavage site, and an oligonucleotide binding site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 108; (b) a third polypeptide chain and a fourth polypeptide chain comprising an antibody light chain, the third polypeptide chain and the fourth polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or any one of 100%) sequence identity to SEQ ID NO: 109; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from, for example, a compound of formula (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, for example, a CpG oligonucleotide, for example, an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0225] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a transglutaminase conjugation site, a protease cleavage site, and an oligonucleotide binding site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO:110; (b) a third polypeptide chain and a fourth polypeptide chain comprising an antibody light chain, the third polypeptide chain and the fourth polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or any one of 100%) sequence identity to SEQ ID NO: 111; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from, for example, a compound of formula (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, for example, a CpG oligonucleotide, for example, an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0226] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a protease cleavage site, and a transglutaminase conjugation site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO:112; (b) an antibody light chain and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from the compounds of formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide, e.g., an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0227] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a transglutaminase conjugation site, and an scFab, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO:114; (b) a second polypeptide chain comprising an antibody light chain; A targeted conjugate is provided, comprising: (a) a third polypeptide chain and a fourth polypeptide chain, the third polypeptide chain and the fourth polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or any one of 100%) sequence identity to SEQ ID NO: 115; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent, e.g., selected from the compounds of formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide, e.g., an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0228] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a protease cleavage site, a transglutaminase conjugation site, and an scFab, wherein the first polypeptide chain and the second polypeptide chain comprise an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO:116; (b) an antibody light chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a protease cleavage site, a transglutaminase conjugation site, and an scFab, wherein the first polypeptide chain and the second polypeptide chain comprise an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO:116; and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from the compounds of formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide, e.g., an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0229] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a transglutaminase conjugation site, and an scFv, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 122; (b) an antibody light chain and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from the compounds of formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide, e.g., an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0230] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a protease cleavage site, a transglutaminase conjugation site, and an scFv, wherein the first polypeptide chain and the second polypeptide chain comprise an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 124; (b) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain, a protease cleavage site, a transglutaminase conjugation site, and an scFv, wherein the first polypeptide chain and the second polypeptide chain comprise an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 124; (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from the compounds of formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, e.g., a CpG oligonucleotide, e.g., an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0231] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain and an scFv, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 126; (b) a third polypeptide chain comprising an antibody light chain. and (c) an effector molecule, the effector molecule being conjugated to a transglutaminase conjugation site. In some embodiments, the effector molecule is a therapeutic agent selected from, for example, a compound of Formulae (1)-(7). In some embodiments, the effector molecule is an oligonucleotide, for example, a CpG oligonucleotide, for example, an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-73 and 157.

[0232] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain and a transglutaminase conjugation site, the first polypeptide chain and the second polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 104. (b) a third and fourth polypeptide chain comprising an antibody light chain, wherein the third and fourth polypeptide chains comprise an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 105; and (c) a CpG oligonucleotide of SEQ ID NO: 70. In some embodiments, the targeted conjugate further comprises one or more uPA cleavage sites comprising one or more amino acid sequences selected from SEQ ID NOs: 50-55.

[0233] In any of the embodiments described herein, a targeted conjugate is provided that comprises an antibody heavy chain comprising SEQ ID NO: 104 and an antibody light chain comprising SEQ ID NO: 105; and a CpG ODN comprising the nucleic acid sequence of SEQ ID NO: 70. In some embodiments, the targeted conjugate further comprises one or more uPA cleavage sites comprising one or more amino acid sequences selected from SEQ ID NOs: 50-55.

[0234] In some embodiments, a targeted conjugate comprises: (a) a first polypeptide chain and a second polypeptide chain comprising, from N-terminus to C-terminus: an antibody heavy chain and a transglutaminase conjugation site, wherein the first polypeptide chain and the second polypeptide chain comprise an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 124. and a second polypeptide chain; (b) a third polypeptide chain and a fourth polypeptide chain comprising an antibody light chain, the third polypeptide chain and the fourth polypeptide chain comprising an amino acid sequence having at least 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%; or 100%) sequence identity to SEQ ID NO: 125; and (c) a therapeutic agent of the compound of Formula (1). In some embodiments, the targeted conjugate further comprises one or more uPA cleavage sites comprising one or more amino acid sequences selected from SEQ ID NOs: 50-55.

[0235] In any of the embodiments described herein, there is provided a targeted conjugate comprising an antibody heavy chain comprising SEQ ID NO: 124 and an antibody light chain comprising SEQ ID NO: 125; and a therapeutic agent of a compound of Formula (1). In some embodiments, the targeted conjugate further comprises one or more uPA cleavage sites comprising one or more amino acid sequences selected from SEQ ID NOs: 50-55.

[0236] III.How to use The targeted conjugates and compositions thereof (e.g., pharmaceutical compositions) described herein can be used to treat or diagnose a variety of diseases, such as cancer, including solid tumors or hematological cancers, infectious diseases, inflammatory diseases, autoimmune diseases, and immunodeficiency diseases.

[0237] Thus, in some embodiments, a method of treating a disease in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of any one of the targeted conjugates described herein, wherein the targeted conjugate comprises an effector molecule selected from the group consisting of a therapeutic agent and an oligonucleotide. In some embodiments, the disease is selected from the group consisting of a tumor, an infectious disease, an inflammatory disease, an autoimmune disease, and an immunodeficiency disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, and uterine cancer.

[0238] In some embodiments, a method of treating a disease (e.g., cancer) in an individual includes administering an effective amount of a compound of the structure of Formula I: [ka] wherein A1 is a first targeting moiety that specifically binds to a first target molecule associated with the disease; A2 is a second targeting moiety that specifically binds to a second target molecule associated with the disease; P1 is a first cleavage site; P2 is a second cleavage site; P3 is a third cleavage site; C is a conjugation site; L is a linker; D is a therapeutic agent or an oligonucleotide; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20) to an individual. In some embodiments, the disease is selected from the group consisting of a tumor, an infectious disease, an inflammatory disease, an autoimmune disease, and an immunodeficiency disease. In some embodiments, the affected site is a tumor site. In some embodiments, cleavage occurs at the tumor site, e.g., at the tumor microenvironment. In some embodiments, cleavage occurs outside the cell, for example outside a tumor cell in the tumor microenvironment.

[0239] In some embodiments, a method of diagnosing a disease in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of any one of the targeted conjugates described herein, wherein the targeted conjugate comprises a detectable label, and detection of the detectable label indicates the presence of the disease. In some embodiments, the disease is selected from the group consisting of a tumor, an infectious disease, an inflammatory disease, an autoimmune disease, and an immunodeficiency disease. In some embodiments, the disease is a cancer. In some embodiments, the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, and uterine cancer.

[0240] In some embodiments, a method of diagnosing a disease (e.g., cancer) in an individual comprises administering an effective amount of a compound of the structure of Formula I: [ka] wherein A1 is a first targeting moiety that specifically binds to a first target molecule associated with the disease; A2 is a second targeting moiety that specifically binds to a second target molecule associated with the disease; P1 is a first cleavage site; P2 is a second cleavage site; P3 is a third cleavage site; C is a conjugation site; L is a linker; D is a detectable label; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; b=1-20), and detection of the detectable label indicates the presence of the disease. In some embodiments, the disease is selected from the group consisting of a tumor, an infectious disease, an inflammatory disease, an autoimmune disease, and an immunodeficiency disease. In some embodiments, the affected site is a tumor site. In some embodiments, cleavage occurs at the tumor site, e.g., at the tumor microenvironment. In some embodiments, cleavage occurs outside the cell, for example outside a tumor cell in the tumor microenvironment.

[0241] In some embodiments, a method is provided for preferentially delivering an effector molecule (e.g., a therapeutic agent, an oligonucleotide, and / or a detectable label) to a diseased site in an individual in need of treatment or diagnosis with the effector molecule, comprising administering to the individual an effective amount of a targeted conjugate comprising a structure of Formula I, wherein A1 is a first targeting moiety that specifically binds to a first target molecule associated with the disease; A2 is a second targeting moiety that specifically binds to a second target molecule associated with the disease; P1 is a first cleavage site; P2 is a second cleavage site; P3 is a third cleavage site; C is a conjugation site; L is a linker; D is an effector molecule; x=0 or 1; y=0 or 1; z=0 or 1; u=0 or 1; v=0 or 1; a=1-20; and b=1-20. In some embodiments, the disease is selected from the group consisting of a tumor, an infectious disease, an inflammatory disease, an autoimmune disease, and an immunodeficiency disease.

[0242] In some embodiments, the method increases the effective concentration of the effector molecule at an affected site in an individual in need of treatment with the effector molecule, hi some embodiments, the effective concentration of the effector molecule is increased by any of about 10%, 20%, 50%, 1×, 2×, 5×, 10× or more at the affected site compared to administration of the effector molecule to an individual in the same effective amount.

[0243] In some embodiments, the method reduces binding of an effector molecule to a target molecule in normal tissues in an individual having a disease requiring treatment with the effector molecule, hi some embodiments, administration of the targeted conjugate reduces binding of the effector molecule to its target molecule on normal cells by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to administration of the effector molecule to the individual.

[0244] In some embodiments, the method reduces the toxicity (e.g., on-target extracellular toxicity) of the effector molecule to an individual having a disease requiring treatment with the effector molecule. In some embodiments, administration of the targeted conjugate reduces the toxicity of the effector molecule by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to administration of the effector molecule in the same effective amount to the individual.

[0245] Also provided is any one of the targeted conjugates described herein for use in treating or diagnosing a disease in an individual in need thereof, or the use of any one of the targeted conjugates described herein in the preparation of a medicament for treating or diagnosing a disease in an individual in need thereof.

[0246] The targeted conjugates described herein can be administered to an individual via a variety of routes, for example, via the same route of administration as the targeted site in the targeted conjugate. Exemplary routes of administration include, but are not limited to, parenteral, intravenous, intramuscular, and subcutaneous administration.

[0247] The effective amount, suitable dose, and dosing schedule of the targeted conjugate administered to an individual can vary depending on the particular composition, route of administration, and the particular type of disease being treated. In some embodiments, the effective amount of the targeted conjugate is based on the effective amount of the targeting moiety and / or effector molecule (e.g., therapeutic agent, oligonucleotide, and / or detectable label).

[0248] In some embodiments, the individual is a mammal, such as a human, a rodent, or a primate. In some embodiments, the individual is a human.

[0249] In some embodiments, the targeted conjugates are used alone or in combination with other anti-cancer treatments, such as chemotherapeutic agents, ionizing radiation, hormone therapy, cytokines, immunotherapy, cell therapy, vaccines, monoclonal antibodies, anti-angiogenic agents, targeted therapeutic agents (small molecule drugs), or biological therapies. For example, chemotherapeutic agents include anti-tumor alkylating agents, such as mustards (mechlorethamine HCl, melphalan, chlorambucil, cyclophosphamide, ifosfamide, busulfan), nitrosoureas (BCNU / canustine, CCNU / lomustine, MeCCNU / semustine, fotemustine treptozotocin), tetrazines (dacarbazine, mitozolomide, temozolomide), aziridines (thiotepa, mitomycin C, AZQ / diaziquone), procarbazine HCl, Hexamethylmelamine, Adzelesin; Cisplatin and its analogs, Cisplatin, Carboplatin, Oxaliplatin; Antimetabolites, Methotrexate, Other antifolates, 5-Fluoropyrimidines (5-Fluorouracil / 5-FU), Cytarabine, Azacitidine, Gemcitabine, 6-Thiopurines (6-Mercaptopurine, Thioguanine), Hydroxyurea; Topoisomerase interactors Epipodophyllotoxins (Etoposide, Teniposide), Camptothecin analogs (Topo camptothecin HCl, irinotecan, 9-aminocamptothecin), anthracyclines and related compounds (doxorubicin HCl, liposomal epirubicin, daunorubicin HCl, liposomal daunorubicin HCl citrate, epirubicin, idarubicin), mitoxantrone, rosoxantrone, actinomycin-D, amsacrine, pyrazoloacridine; antimicrotubule agents vinca alkaloids (vindesine, vincristine, vinblastine, vinorelbine), taxanes (paclitaxel cel, docetaxel), estramustine; fludarabine, 2-chlorodeoxyadenosine, 2'-deoxycoformycin, homoharringtonine, suramin, bleomycin, L-asparaginase, floxuridine, capecitabine, cladribine, leucovorin, pentostatin, retinoids (all-trans retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, isotretinoin, tretinoin), pamidronate, thalidomide, cyclosporine;Hormonal TherapyAntiestrogens (tamoxifen, toremifene, medroxyprogesterone acetate, megestrol acetate), aromatase inhibitors (aminoglutethimide, letrozole / femara, anastrozole / arirnidex, exemestane / aromasin, vorozole), gonadotropin-releasing hormone analogues, antiandrogens (flutamide, casodex), fluoxymethorone, diethylstilbestrol, octreotide, leuprolide acetate, zoladex; steroidal and nonsteroidal anti-inflammatory drugs (dexamethasone, prednisone); monoclonal antibodies, including but not limited to anti-HER2 / neu antibodies (Herceptin / Trastuzumab), anti-EGFR antibodies (Cetuximab / Erbitux, ABX-EGF / Panitumumab, Nimotuzurnab), anti-CD20 antibodies (Rituxan / Rituximab, Ibritumomab / Zevalin, Tositumomab / Bexar), anti-CD33 antibodies (Gemtuzumab / Mylotarg), Alemtuzumab / Campus, Bevacizumab / Avastin; as well as small molecule inhibitors;

[0250] IV. PHARMACEUTICAL COMPOSITIONS, KITS AND ARTICLES OF MANUFACTURE The present application further provides compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any one of the targeted conjugates disclosed herein. In some embodiments, pharmaceutical compositions are provided comprising any one of the targeted conjugates described herein and a pharma- ceutically acceptable carrier. In some embodiments, the compositions (e.g., pharmaceutical compositions) comprise a carrier, diluent, or excipient that may facilitate administration of the composition to an individual in need thereof. Examples of carriers, diluents, and excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, such as lactose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents.

[0251] In some embodiments, the pharmaceutical composition comprises a plurality of targeting conjugates. In some embodiments, the targeting conjugate molecules in the pharmaceutical composition do not have the same number of effector molecules. In some embodiments, at least two of the targeting conjugates in the pharmaceutical composition comprise different numbers of effector molecules. The ratio between the effector molecule and the targeting moiety in the pharmaceutical composition is within a predetermined range for the pharmaceutical composition to receive regulatory approval.

[0252] In some embodiments, the average drug loading of the pharmaceutical compositions is at least about any one of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1 or more. In some embodiments, the average drug loading of the pharmaceutical compositions is less than or equal to about any one of 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. In some embodiments, the average drug loading of the pharmaceutical compositions is any one of about 2:1 to 4:1, 2:1 to 8:1, 2:1 to 10:1, 2:1 to 16:1, 4:1 to 20:1, 10:1 to 20:1, 20:1 to 40:1, 40:1 to 100:1, 2:1 to 20:1, 2:1 to 40:1, or 10:1 to 40:1.

[0253] In some embodiments, the pharmaceutical composition has an average ratio of effector molecule to targeting moiety (e.g., first targeting moiety and / or second targeting moiety) of at least about any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 17:1, 18:1, 19:1, or 20:1. In some embodiments, the pharmaceutical composition has an average ratio of effector molecule to targeting moiety (e.g., the first targeting moiety and / or the second targeting moiety) of no more than about any one of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, pharmaceutical compositions have an average ratio of effector molecule to targeting moiety (e.g., first targeting moiety and / or second targeting moiety) of about any one of 1:1 to 2:1, 2:1 to 4:1, 4:1 to 8:1, 1:1 to 10:1, 1:1 to 16:1, 4:1 to 20:1, 10:1 to 20:1, 1:1 to 20:1, or 2:1 to 20:1.

[0254] The pharmaceutical compositions described herein can be prepared by mixing the ingredients according to generally accepted procedures. For example, selected ingredients can be simply mixed in a blender or other standard device to produce a concentrated mixture, which can then be adjusted to a final concentration and viscosity by adding water or a thickening agent and, optionally, a buffer to control pH or additional solutes to control tonicity.

[0255] Other pharma- ceutically acceptable carriers and their formulations are described in standard formulation literature, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Y. J. and Hanson, M. A. "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S (1988).

[0256] In some embodiments, the pharmaceutical composition is a liquid suspension, hi some embodiments, the pharmaceutical composition is a sterile composition.

[0257] Also provided is a kit comprising any one of the targeting conjugates described herein. The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags). The kit may optionally provide additional components, such as buffers and instruction information. Thus, the present application provides articles of manufacture including vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like.

[0258] The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. Typically, the container holds a composition effective for treating or diagnosing a disease described herein and may have a sterile access port. The label or package insert indicates that the composition is used to treat or diagnose a disease in an individual. The label or package insert further includes instructions for administering the composition to an individual.

[0259] Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic or diagnostic products. In some embodiments, the package insert indicates that the composition is used for treating or diagnosing a disease (e.g., cancer).

[0260] The article of manufacture may also further comprise a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. EXAMPLES

[0261] The following examples are intended to be purely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0262] Example 1 Design and screening of uPA substrate peptides 1. Peptide cleavage efficiency Urokinase-type plasminogen activator (uPA) substrate peptides (SEQ ID NOs: 50-54) were screened for the efficiency of substrate cleavage by uPA, while the resistance to tissue-type plasminogen activator (tPA) digestion. Substrate peptides and PBS were added to a 384-well plate and mixed with uPA or tPA. The efficiency of digestion was measured by fluorescence resonance energy transfer (FRET). All peptide solutions were diluted to 50 μL with 20 mM PBS (pH=7.4). The experimental design is shown in Table 2. The results are shown in Tables 3-5. [Table 2]

[0263] uPA efficiently cleaved peptides of SEQ ID NOs:50-54, as shown in Table 3 below: [Table 3]

[0264] The shorter the time it takes to reach a plateau in fluorescence intensity, the faster the uPA digestion. The higher the fold increase in fluorescence intensity, the more efficient the uPA digestion. Thus, the ranking of uPA digestion rates for peptides was SEQ ID NO:52, 54>SEQ ID NO:53>SEQ ID NO:50>SEQ ID NO:51. The ranking of reaction efficiency was SEQ ID NO:51>SEQ ID NO:50>SEQ ID NO:54>SEQ ID NO:52>SEQ ID NO:53.

[0265] 7.5×10 -3 At mg / mL tPA concentrations, only SEQ ID NOs:51 and 52 were lightly digested, as shown in Table 4: [Table 4]

[0266] 2.25×10 -2 At mg / mL tPA concentrations, SEQ ID NOs: 51, 52 and 54 were digested as shown in Table 5: [Table 5]

[0267] 2. Stability of substrate peptide in plasma measured by FRET Plasma was mixed with antibiotics (penicillin-streptomycin) and then peptides of SEQ ID NOs: 50-54 were added to the plasma mixture. The reaction mixture was incubated at room temperature. Samples were taken at 0 h, 8 h, 24 h, 32 h, 48 h, 72 h, 96 h, 120 h and 144 h for FRET measurements. The experimental design is shown in Table 6. [Table 6]

[0268] The fold increase in fluorescence intensity for each of the substrate peptides in plasma is shown in Table 7 below: [Table 7]

[0269] The ranking of peptide stability in plasma is approximately SEQ ID NO:53>SEQ ID NO:51, 52>SEQ ID NO:50>SEQ ID NO:54.

[0270] 3. Stability of substrate peptide in plasma from kinetic measurements The experiment was designed as shown in Table 8. The plasma-peptide mixture was incubated at room temperature for 12 hours and the fluorescence intensity was measured continuously. The results are shown in Table 9. PS: Penicillin-Streptomycin. [Table 8] [Table 9]

[0271] The rank order of peptide stability in plasma was approximately SEQ ID NO:52,53>SEQ ID NO:50>SEQ ID NO:51,54. For SEQ ID NO:52 and 53, the fluorescence intensity increased 2.0-fold and 1.7-fold, respectively, over time, but did not reach a plateau within 12 hours. SEQ ID NO:50 reached a plateau at 4.8 hours, with a maximum fold increase in fluorescence intensity of 2.8-fold. The fluorescence intensity of SEQ ID NO:51 and 54 increased 2.3-fold and 2.7-fold, respectively, within 3.6 hours.

[0272] 4. uPA Digestion in Antibodies Antibodies Ab3C (heavy chain sequence SEQ ID NO:108, light chain sequence SEQ ID NO:109), Ab3E (heavy chain sequence SEQ ID NO:110, light chain sequence SEQ ID NO:111) and Ab5D (heavy chain sequence SEQ ID NO:124, light chain sequence SEQ ID NO:125) containing uPA substrate sequence in their heavy chains were used to test the uPA digestion efficiency in antibodies. Antibody Ab5C (heavy chain sequence SEQ ID NO:122, light chain sequence SEQ ID NO:123) not containing uPA substrate sequence was used as a control. The experimental design is shown in Table 10. The reaction mixture was incubated at 37° C. for 24 hours. [Table 10]

[0273] 1 The initial concentrations of Ab3C, Ab3E, Ab5C, and Ab5D were 2.12 mg / mL, 1.07 mg / mL, 9.2 mg / mL, and 7.8 mg / mL, respectively. 2 The initial concentration of uPA was 0.25 mg / mL. 3 The concentration of EDTA was 1 mM.

[0274] The uPA digestion products were analyzed by LC-MS. The heavy chains of Ab3C, Ab3E, and Ab5D were cleaved by uPA to generate fragments of the expected molecular weights. Ab5C was not cleaved by uPA.

[0275] Example 2. Drug-Linker Design, Synthesis, and Analysis 1. Purpose of the experiment (1) Design MMAE drug-linkers containing PEG spacers of different molecular weights, investigate the coupling efficiency of TGase enzyme-catalyzed conjugation of MMAE drug-linkers to polypeptides and antibodies, and screen for PEG spacers and drug-linkers with high coupling efficiency. (2) Design linkers for connecting to different positions of SN-38, and screen for SN-38 drug-linkers with high coupling efficiency, stability, and high biological activity.

[0276] 2. Drug-Linker Synthesis Compounds of formulas (1)-(7) were chemically synthesized and tested by HPLC, LC-MS, and H-NMR. The drug linkers were used in subsequent experiments after meeting the quality standards.

[0277] 3. Restriction enzyme digestion of drug-linker with cathepsin B To simulate the release of the drug under physiological conditions, the drug-linker was subjected to cleavage by cathepsin B, followed by an elimination reaction to release the unmodified drug.

[0278] 3.1 Reaction mechanism (1) Digestion of a compound of formula (1) [ka]

[0279] (2) Digestion of the compound of formula (2) [ka]

[0280] 3.2 Experimental Method Lyophilized cathepsin B enzyme powder was dissolved in 130 μL of pure water and divided into 13 tubes of 10 μL each. The concentration of each tube was 0.15 mg / ml (4.15 μM). Activation buffer (20 mM sodium acetate, 30 mM DTT, 15 mM EDTA, pH 5.5) was then added to the enzyme tube to activate the enzyme for 1 hour at room temperature. The activated enzyme was diluted to 125 nM with enzyme digestion buffer (25 mM sodium acetate, 1 mM EDTA, pH 5.5). Drug-linker was added to the activated enzyme to a final concentration of 62.5 μM and incubated at 37°C for 3 hours.

[0281] 3.3 Results and conclusions Both compounds of formula (1) and (2) were digested effectively. Compound (1) was digested to release SN38, and compound (2) was digested to release an SN38 derivative.

[0282] 4. Stability study The stability of the drugs was tested by measuring the lactone and lactone ring-opening products in the compound of formula (1), the compound of formula (2) and SN-38 in buffers of different pH, plasma and dynamic pH solutions.

[0283] 4.1 Stability of SN-38 A hydrochloric acid solution with a pH of 4 and a sodium hydroxide solution with a pH of 10 were prepared, respectively. 5 mg of SN-38 was dissolved in 5 mL of acetonitrile. The stability of SN-38 in solutions with varying pH (from pH=10 to pH=4) was measured by shaking the SN-38 at room temperature for 10 minutes at pH=10, then adjusting the pH back to 4 and shaking at room temperature for 10 minutes before sampling. The SN-38 was then incubated on a shaker at room temperature overnight (16 hours) and then assayed. [Table 11]

[0284] 4.2 Stability of compounds of formula (1) and (2) in buffer solutions Preparation of 20 mM phosphate buffer (1 L) with pH=7.4: 8.0 g of sodium chloride, 0.2 g of potassium chloride, 7.3 g of disodium hydrogen phosphate dodecahydrate, and 0.48 g of potassium dihydrogen phosphate were dissolved in water and diluted to 1 L at pH 7.4.

[0285] The buffer solutions were adjusted to pH 9.0, 7.0 and 5.0 with saturated phosphate and sodium hydroxide solutions. When investigating the stability of the drug in solutions of varying pH (9 to 5), the compounds of formula (1) / (2) were incubated at pH 9 for 10 minutes on a shaker at room temperature. The solution pH was adjusted back to 5 and shaken overnight at room temperature (16 h) before testing. [Table 12]

[0286] 4.3 Stability of compounds of formula (1) and (2) in plasma Preparation of protein precipitation solution: 1 g of NaCl was added to 3 mL of methanol and 5 mL of acetonitrile, stirred for 1 hour, then left at room temperature for 1 hour before use.

[0287] Then 200 μL of protein precipitation solution was added to the reaction solution and centrifuged at 8000 g for 10 min. The supernatant was collected for analysis. [Table 13]

[0288] 4.4 Results and conclusions SN-38 existed as the lactone under acidic conditions (94%) and as the lactone ring-opened form under alkaline conditions (92%). After subjecting SN-38 to alkaline conditions for 10 minutes and then acidic conditions and then testing, 56% of SN-38 was present in the lactone form and 42% of the lactone ring-opened product was present. After overnight incubation, 86% was in the lactone form and 13% was present in the lactone ring-opened product form.

[0289] The compound of formula (1) exists mainly in the lactone form under acidic conditions (88%), and under alkaline conditions it exists mainly as the ring-opened form of the lactone (92%). After subjecting the compound of formula (1) to alkaline conditions for 10 minutes and then to acidic conditions and then testing, 98% was in the lactone form and 0.21% was in the form of the lactone-opened product. The compound of formula (1) was partially hydrolyzed in plasma and mainly in the lactone-opened form;

[0290] The compound of formula (2) was not significantly affected by pH and was present in the lactone form in buffer at 98%. The stability of the compound of formula (2) in plasma was also better than that of the compound of formula (2). The compound of formula (2) was mainly in the lactone form in plasma, and the ring-opened product was essentially invisible.

[0291] Example 3. Design and synthesis of CpG-ODN-linkers CpG ODNs (cytosine-phosphodiester-guanine oligodeoxynucleotides) and linkers of SEQ ID NOs: 66-73, as well as formula VIII, were designed, synthesized, and tested for their coupling efficiency to the TGase substrate sequence during TGase catalysis. CpG-ODNs were synthesized by phosphoramidite chemistry and characterized by electrophoresis and mass spectrometry. The formula of the CpG ODN linker is 5'amino-modified-spacer-P h -CpG ODN (Formula IV) and 3' amino modified-spacer-P h -CpG ODN (Formula V) (wherein the spacer is (CH2) n -(PEG) m where h, n, and m are integers, h=0 or 1, n≧1, m≧0, and P is a cleavage site.

[0292] Example 4. Design and screening of TGase conjugation peptides 1. Purpose of the experiment The coupling efficiency between substrate peptides (SEQ ID NOs: 1-12) and small molecule toxins (drug-linker, Formulas II-V) using TGase (transglutaminase) was investigated. Substrate peptides with high coupling efficiency were selected for the development of peptide conjugates, protein conjugates and antibody conjugates. [Table 14]

[0293] 2. Peptide synthesis and analysis Peptides were synthesized by conventional solid phase synthesis and analyzed by HPLC and LC-MS.

[0294] 3. Peptide synthesis and analysis Microbial / bacterial transglutaminase (MTG / BTG) was used herein. The enzymatic activity of MTG / BTG was determined by conventional methods in the art. TGase was then used to crosslink the drug linker with the peptides of SEQ ID NOs: 1-12. The reactivity between the peptides and the drug linker was determined by the HPLC peak area of ​​the product.

[0295] The peptide was diluted to 10 mg / mL in PBS at pH=7.4. The drug linker (compound of formula (1)-(4)) was diluted to 31 mg / mL in DMSO. The reaction mixture contained peptide at a final concentration of 1 mg / mL, DMSO at a final concentration of 20%, and the molar ratio of peptide:drug linker was 1:5. TGase was added to the reaction mixture to a final concentration of 25 U / mL. The reaction was measured by RP-HPLC after 16 h at room temperature. The results are shown in Table 15. [Table 15]

[0296] 4. Conclusion Between the SN-38 drugs, the compound of formula (1), which reacts with the linker at the hydroxyl group at position 10, had higher reactivity compared to the compound of formula (2), which reacts with the linker at position 20. Between the two MMAE drugs, the compound of formula (4) (8 PEG spacer) had higher reactivity compared to the compound of formula (3) (4 PEG spacer).

[0297] The compound of formula (1) could be crosslinked to SEQ ID NOs:7-10 at DPR=4 and to SEQ ID NOs:1-6 and 11-12 at DPR=3 (with some products at DPR=2). The overall order of crosslinking reactivity of the peptides was approximately: SEQ ID NO:9 ≒ SEQ ID NO:10> SEQ ID NO:7 ≒ SEQ ID NO:8> SEQ ID NO:5 ≒ SEQ ID NO:4 ≒ SEQ ID NO:3> SEQ ID NO:2> SEQ ID NO:6> SEQ ID NO:12 ≒ SEQ ID NO:11> SEQ ID NO:1.

[0298] The compound of formula (2) was able to crosslink to SEQ ID NO:1 at DPR=3, to SEQ ID NOs:2, 5, and 6 at DPR=2, and to the other peptides tested at DPR=1. The overall order of crosslinking reactivity of the peptides was approximately: SEQ ID NO:1>SEQ ID NO:5>SEQ ID NO:6>SEQ ID NO:4>SEQ ID NO:2>SEQ ID NO:12>SEQ ID NO:3>SEQ ID NOs:8, 9, 10, 11>SEQ ID NO:7.

[0299] Most of the compounds of formula (3) cross-linked to peptides with DPR = 1. The overall order of cross-linking reactivity of the peptides was approximately SEQ ID NO:8, 9, 10, 12 > SEQ ID NO:5, 7 > SEQ ID NO:4, 6 > SEQ ID NO:11 > SEQ ID NO:3 > SEQ ID NO:1, 2.

[0300] The compound of formula (4) could be crosslinked to SEQ ID NOs:2,3,4, and 7 at DPR=3, and to SEQ ID NOs:6,8,11 at DPR=2. The overall order of crosslinking reactivity of the peptides was approximately SEQ ID NOs:2,3,4,7>SEQ ID NO:8>SEQ ID NOs:6,11>SEQ ID NO:1>SEQ ID NOs:5,10>SEQ ID NOs:9,12.

[0301] Example 5. Characterization of antibodies The monospecific PD-L1 antibodies Ab2B (heavy chain SEQ ID NO:100, light chain SEQ ID NO:101), Ab3A (heavy chain SEQ ID NO:104, light chain SEQ ID NO:105), Ab3B (heavy chain SEQ ID NO:106, light chain SEQ ID NO:107), Ab3C (heavy chain SEQ ID NO:108, light chain SEQ ID NO:109), Ab3E (heavy chain SEQ ID NO:110, light chain SEQ ID NO:111), Ab3F (heavy chain SEQ ID NO:112, light chain SEQ ID NO:113), and the bispecific PD-L1 / Trop-2 antibodies Ab4A (heavy chain SEQ ID NO:114, light chain SEQ ID NO:115), Ab4B (heavy chain SEQ ID NO:116, light chain SEQ ID NO:117), Ab4C (heavy chain SEQ ID NO:118, light chain SEQ ID NO:119), Ab4D (heavy chain SEQ ID NO:120, light chain SEQ ID NO:121), Ab4E (heavy chain SEQ ID NO:122, light chain SEQ ID NO:123), Ab4F (heavy chain SEQ ID NO:124, light chain SEQ ID NO:125), Ab4G (heavy chain SEQ ID NO:126, light chain SEQ ID NO:127), Ab4G (heavy chain SEQ ID NO:128, light chain SEQ ID NO:129), Ab4H (heavy chain SEQ ID NO:130, light chain SEQ ID NO:131), Ab4H (heavy chain SEQ ID NO:132, light chain SEQ ID NO:133), and Ab4H (heavy chain SEQ ID NO:134, light chain SEQ ID NO:135), Ab4H (heavy chain SEQ Ab5A (heavy chain SEQ ID NO:118, light chain SEQ ID NO:119), Ab5B (heavy chain SEQ ID NO:120, light chain SEQ ID NO:121), Ab5C (heavy chain SEQ ID NO:122, light chain SEQ ID NO:123), Ab5D (heavy chain SEQ ID NO:124, light chain SEQ ID NO:125), Ab5E (heavy chain SEQ ID NO:126, light chain SEQ ID NO:127), and Ab5F (heavy chain SEQ ID NO:128, light chain SEQ ID NO:129) were expressed and tested for affinity to human PD-L1 / B7-H1 / CD274 and also for the bispecific antibodies to human Trop-2 / TACSTD2 protein. The affinity results to PD-L1 are shown in Table 20. The affinity results to Trop-2 are shown in Table 21. The results show that both the monospecific and bispecific antibodies maintained high affinity to the antigen(s). [Table 16] [Table 17]

[0302] Example 6. Synthesis and characterization of antibody drug conjugates (ADCs) 1. Antibody-drug conjugation of monospecific antibodies The drug-linker (compounds of Formulas (1) and (2)) was conjugated to anti-PD-L1 antibody Ab3A of SEQ ID NO: 104 (comprising a heavy chain and a TGase substrate sequence) and 105 (light chain), or anti-PD-L1 antibody Ab3F of SEQ ID NO: 112 (comprising a heavy chain, a uPA substrate peptide, and a TGase substrate sequence) and 113 (light chain). The experimental design of the conjugation experiments is shown in Table 18. The reaction mixtures were incubated at room temperature for 16 hours and the drug-to-antibody ratios (DARs) of the conjugation products were determined by mass spectrometry. [Table 18]

[0303] For ADC Ab3A-CD-1, the DAR value was 3.93 and for Ab3A-CD-2, the DAR value was 3.77. For ADC Ab3F-CD-1, the DAR value was 4.98 and for Ab3F-CD-2, the DAR value was 4.85.

[0304] 2. Antibody-drug Conjugation of Bispecific Antibodies The drug linkers (compounds of formula (1) and (2)) were conjugated to anti-PD-L1 and anti-Trop-2 bispecific antibodies Ab5C (heavy chain SEQ ID NO: 122, light chain SEQ ID NO: 123) and Ab5D (heavy chain SEQ ID NO: 124, light chain SEQ ID NO: 125). The drug-to-antibody ratios (DAR) of the conjugation products were determined by mass spectrometry. The reaction mixtures were incubated at room temperature for 16 hours. The experimental design of the conjugation experiments and the DAR values ​​of the ADCs are shown in Table 19. [Table 19]

[0305] 3. Antigen-binding ability of ADC The ADCs in this example were tested for their affinity to human PD-L1 / B7-H1 / CD274, and the bispecific antibody-drug linker conjugates were further tested for their affinity to human Trop-2 / TACSTD2. The results are shown in Table 20. [Table 20]

[0306] Example 7. Synthesis and characterization of antibody-oligonucleotide conjugates (AOCs) 1. Non-covalent Antibody-CpG ODN Conjugation CpG ODN of SEQ ID NO:67 was non-covalently conjugated to anti-PD-L1 antibodies Ab3B (heavy chain SEQ ID NO:106 and light chain SEQ ID NO:107), Ab3C (heavy chain SEQ ID NO:108 and light chain SEQ ID NO:109), and Ab3E (heavy chain SEQ ID NO:110 and light chain SEQ ID NO:111). The conjugation experimental design is shown in Table 21. The reaction mixture was incubated at room temperature for 16 hours and the drug-to-antibody ratio (DAR) of the conjugation product was determined by gel shift assay. The results are shown in Figure 11. [Table 21]

[0307] Figure 11 shows that SEQ ID NO:67 was effectively non-covalently bound to the antibody. The order of binding efficiency was Ab3C>Ab3B>Ab3E. Also, when the molar ratio of mAb:CpG was greater than 2:1, all CpG ODN was bound to Ab3C.

[0308] 2. Covalent Antibody-CpG ODN Conjugation CpG ODN-linkers containing the CpG sequences of SEQ ID NO: 68, 69 and 71 were covalently conjugated to Ab3A (heavy chain SEQ ID NO: 104, light chain SEQ ID NO: 105) by TGase, respectively. The CpG ODN-linkers were mixed with Ab3A at a molar ratio of 40:1. TGase was added to the reaction mixture to a final concentration of 5 U / mL, and the reaction mixture was incubated at room temperature for 16 hours. The conjugation products were analyzed by gel shift assay. The results are shown in Figures 12 and 13.

[0309] Figures 12 and 13 show that TGase can catalyze the conjugation between CpG ODN-linker and antibody with TGase substrate sequence. The CpG ODN-linker containing the sequence of SEQ ID NO: 71 showed the highest conjugation efficiency, followed by the CpG ODNs of SEQ ID NO: 68 and 69. These results suggested that the length of the linker affected the conjugation efficiency, and the longest of the three had the highest coupling efficiency.

[0310] Example 8. Immune Checkpoint Bioassay for PD-1 / PD-L1 Blockade A luciferase reporter assay was used to analyze the mechanism of blocking PD-1 / PD-L1 on the activation of the NFAT pathway. The bioassay consisted of two genetically engineered cell lines: PD-1 effector cells, which were Jurkat T cells expressing human PD-1 and a luciferase reporter gene under the control of the NFAT promoter (NFAT-Luc2); PD-L1 aAPC / 293T-OS8 cells, which were 293T-OS8 cells expressing human PD-L1. Briefly, Jurkat-NFAT-Luc2-PD1 stable effector cells and 293T-OS8-PD-L1 stable cell lines were co-cultured in triplicate in the presence of serially diluted antibodies at a 1:1 ratio for 6 hours at 37°C, 5% CO2. Luminescence was measured using the ONE-Step™ Luciferase Assay System (BPS Bioscience). Results and Conclusions [Table 22]

[0311] ICs obtained from PD-1 / PD-L1 immune blockade reporter assay 50 The values ​​are summarized in Table 22. The dose-response curves of the drugs are shown in Figure 14. The results revealed that the fusion antibody Ab3A, the bispecific antibodies Ab5C and Ab5D (all constructed based on the antibody Ab2B) have similar immune blocking potency as Ab2B, which is also similar to the reference antibody Atezolizumab.

[0312] Example 9. In vitro cytotoxicity study Cell line construction and culture conditions A human colon adenocarcinoma HCT15 cell line stably expressing exogenous human PDL1 gene was generated using a lentiviral plasmid carrying the human PDL1 gene (NM_014143) sequence. The cell line was named HCT15-hPDL1. The cells were cultured at 37°C with 5% CO2 using RPMI1640 medium supplemented with 10% FBS plus 1 μg / ml puromycin.

[0313] Cell injury research In vitro antitumor activity was determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Briefly, cells were plated at 2 × 10 per well in 100 μL of complete medium. 3 Cells were seeded in 96-well plates and then incubated overnight at 37°C with 5% CO2. Untreated cells served as controls. ADC, antibody, or payload in 100 μL of medium was added in duplicate at various concentrations using a 2-fold dilution series starting at a concentration of 1 uM, respectively. Cells were exposed to the test articles for 5 days. Absorbance was measured by a microplate reader at 450 nm. Cell viability (%) was calculated using the following formula: Sampl...

Claims

1. 1. A targeted conjugate comprising a targeting moiety and an effector molecule, wherein the targeting moiety specifically binds to a target molecule, and the effector molecule is conjugated to the targeting moiety via a conjugation site, and wherein the effector molecule can be released from the targeted conjugate via cleavage.

2. The targeted conjugate has the following structure: 【Chemistry 17】 (In the formula, A1 is a first targeting moiety; A2 is a second targeting moiety; P1 is the first cleavage site; P2 is the second cleavage site; P3 is the third cleavage site; C is the conjugation site; L is a linker; D is the effector molecule; x=0 or 1; y=0 or 1; z=0 or 1; v=0 or 1; u=0 or 1; a=1 to 20; b = 1 to 20) The targeting conjugate of claim 1 , comprising:

3. The targeting conjugate of claim 1 or 2, wherein the targeting moiety comprises a targeting peptide, or an antibody or antigen-binding fragment thereof.

4. The targeted conjugate of any one of claims 1 to 3, wherein the cleavage is triggered by a condition at a target site for the targeting moiety.

5. 5. The targeted conjugate of claim 4, wherein the condition at the target site is selected from the group consisting of proteases, pH changes, redox changes, hypoxia, oxidative stress, hyperthermia, and extracellular ATP concentration, and / or the target site is a site of disease.

6. The targeted conjugate of any one of claims 1 to 5, wherein the cleavage is by a protease.

7. The targeted conjugate of claim 6, wherein the protease is uPA and / or the cleavage site comprises an amino acid sequence of any of SEQ ID NOs: 50 to 55.

8. The targeted conjugate of any one of claims 1 to 7, wherein the effector molecule is selected from the group consisting of a therapeutic agent, an oligonucleotide, and a detectable label.

9. The targeted conjugate of claim 8, wherein the oligonucleotide is a CpG oligonucleotide, and the CpG oligonucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 66-67 and 157.

10. The targeting conjugate comprises a 5' amino modification-spacer-P ｈ -CpG ODN or 3' amino modified-spacer-P h -CpG ODN structure (wherein the spacer is (CH ２ ) ｎ -(PEG) ｍ and P is a cleavage site, and h, n, and m are integers, h=0 or 1, n≧1, and m≧0.

11. The targeted conjugate described in any one of claims 1 to 10, wherein the target molecule is an immune checkpoint molecule or a tumor antigen.

12. 12. The targeting conjugate of any one of claims 2 to 11, wherein A1 is a first targeting peptide or a first antibody or antigen-binding fragment thereof that recognizes a first target molecule, and A2 is a second targeting peptide or a second antibody or antigen-binding fragment thereof that recognizes a second target molecule. (i) the first target molecule is a first immune checkpoint molecule and the second target molecule is a second immune checkpoint molecule; (ii) the first target molecule is a first tumor antigen and the second target molecule is a second tumor antigen; (iii) the first target molecule is an immune checkpoint molecule and the second target molecule is a tumor antigen; or (iv) the first target molecule is a tumor antigen and the second target molecule is an immune checkpoint molecule; The targeted conjugate of claim 12.

14. The targeted conjugate of claim 13, wherein the immune checkpoint molecule is PD-L1 and / or the tumor antigen is Trop-2. (i) u=1, A1 is a full length anti-PD-L1 antibody, A2 is an anti-Trop-2 scFv, sdAb or scFab, and any of P1, C and any of P2 are fused to the C-terminus of one or both heavy chains of the full length anti-PD-L1 antibody, and A2 is fused to the C-terminus of any of P2 at the C-terminus of one or both heavy chains of the full length anti-PD-L1 antibody; or (ii) u=0, A1 is a full length anti-PD-L1 antibody, and said optional P1, C and said optional P2 are fused to the C-terminus of said one or both heavy chains of said full length anti-PD-L1 antibody. The targeting conjugate according to any one of claims 2 to 14.

16. The targeted conjugate of any one of claims 1 to 15, wherein the conjugation site is a transglutaminase conjugation site.

17. 17. The targeted conjugate of any one of claims 1 to 16, wherein the conjugation site comprises multiple glutamine-containing tags fused in tandem to each other and / or the transglutaminase conjugation site comprises an amino acid sequence of any of SEQ ID NOs: 1-49, 145 and 153-155.

18. 16. The targeted conjugate of any one of claims 1 to 15, wherein the conjugation site comprises an oligonucleotide-binding polypeptide and / or the conjugation site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-65.

19. L is a group of the formula: SH ２ -spacer, MAL-spacer, NH ２ 19. The targeted conjugate of any one of claims 2 to 18, wherein L is represented by a N-spacer, an Osu-spacer, or an Osu-spacer, and / or L is represented by the formula (Gly)n-(PEG)m-P-PAB-(DMAE)k, (Gly)n-(PEG)m-VC-PAB-(DMAE)k, or (Gly)n-(PEG)m-Val-Ala-PAB-(DMAE)k, where n, m and k are integers, n > 1, m > 2, k is 0 or 1, and P is a cleavage site.

20. The targeted conjugate of claim 8, wherein the therapeutic agent is selected from the group consisting of compounds of formulas (1) to (7).

21. A targeted conjugate comprising a targeting moiety conjugated to an effector molecule having a formula selected from the group consisting of the compounds of formulas (1)-(7).

22. A composition comprising a plurality of the targeted conjugates of any one of claims 1 to 21.

23. 23. The composition of claim 22, wherein the average ratio of the effector molecule and the targeting moiety in the composition is at least about 1:1, and / or at least two of the targeted conjugates in the composition comprise different numbers of effector molecules.

24. 24. The composition of claim 22 or 23 for treating a disease in an individual, wherein the effector molecule is a therapeutic agent or an oligonucleotide, the disease expresses a target molecule, and the targeted conjugate specifically binds to the target molecule.

25. 24. The composition of claim 22 or 23 for diagnosing a disease in an individual, wherein the effector molecule is a detectable label, the disease expresses a target molecule, the targeted conjugate specifically binds to the target molecule, and detection of the detectable label indicates the presence of the disease.

26. 22. A method of making a targeted conjugate according to any one of claims 1 to 21, said method comprising conjugating said effector molecule to said targeting moiety.

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