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WO2023044016A2 - Antibody immune-stimulant conjugates and use thereof - Google Patents

Antibody immune-stimulant conjugates and use thereof Download PDF

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Publication number
WO2023044016A2
WO2023044016A2 PCT/US2022/043820 US2022043820W WO2023044016A2 WO 2023044016 A2 WO2023044016 A2 WO 2023044016A2 US 2022043820 W US2022043820 W US 2022043820W WO 2023044016 A2 WO2023044016 A2 WO 2023044016A2
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Prior art keywords
antibody
cancer
compound
formula
seq
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French (fr)
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WO2023044016A3 (en
Inventor
Wen Zhang
Zhaopeng SUN
Xixin HU
Xiwu HUI
Mo DAN
Can YUAN
Lu LV
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CSPC Dophen Corp
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CSPC Dophen Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present disclosure relates in general to methods and compositions for cancer immunotherapy, in particular antibody conjugates.
  • tumor growth necessitates the acquisition of mutations that facilitate immune evasion. Even so, tumorigenesis results in the accumulation of mutated antigens, or neoantigens, that are readily recognized by the host immune system following ex vivo stimulation. Why and how the immune system fails to recognize neoantigens are beginning to be elucidated. Groundbreaking studies by Carmi et al. (Nature, 521 : 99-104 (2015)) have indicated that immune ignorance can be overcome by delivering neoantigens to activated dendritic cells via antibody-tumor immune complexes.
  • An object of the present invention is to provide novel immune stimulating antibody conjugates with anti -tumor efficacy.
  • a further object of the present invention is to provide stable and homogeneous antibody-drug conjugates by using stable linkers and enzyme- catalyzed site-specific conjugation.
  • a compound having Formula Al or a pharmaceutically acceptable salt or an isotopic derivative thereof
  • n 1-20 (or 1-18, 1-15, 1-12, 1-10, 1-8, 1-6, or 1-4, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • a further aspect relates to a compound having Formula A2, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
  • Another aspect relates to a compound having Formula A3, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
  • n 1-20 (or 1-18, 1-15, 1-12, 1-10, 1-8, 1-6, or 1-4, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • an antibody conjugate comprising the compound of any of Formulae A1-A4 or a salt or an isotopic derivative thereof conjugated to an antibody or an antigen-binding fragment thereof.
  • each antibody or fragment thereof attaches to 1-4 compounds of any one of Formulae A1-A4 conjugated thereon.
  • the compound is conjugated via Cysteine conjugation.
  • the compound is conjugated via site specific microbial transglutaminase (mTGase) catalyzed conjugation.
  • mTGase site specific microbial transglutaminase
  • the antibody is anti-HER.2 antibody (for example, Trastuzumab or trastuzumab biosimilar DP001), anti-CEACAM5 antibody, anti-CAIX antibody, anti-Nectin-4 antibody, anti-PD-Ll antibody, and/or anti-Claudin 18.2 antibody.
  • anti-HER.2 antibody for example, Trastuzumab or trastuzumab biosimilar DP001
  • anti-CEACAM5 antibody for example, trastuzumab or trastuzumab biosimilar DP001
  • anti-CAIX antibody anti-CAIX antibody
  • anti-Nectin-4 antibody anti-PD-Ll antibody
  • anti-Claudin 18.2 antibody anti-HER.2 antibody
  • DP001 is an anti-HER2 monoclonal antibody, which has the same amino acid sequence as trastuzumab (HERCEPTIN®). Specifically, it contains 1328 amino acids with two heavy chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO: 1), and two light chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 2).
  • HC heavy chains
  • LC light chains
  • DP001 is a heterotetramer of two HCs of the IgGl subclass, and two LCs of the kappa subclass linked by 16 disulfide bonds (12 intra and 4 inter chain).
  • FIG. 1 A schematic structure of DP001 is depicted in FIG. 1.
  • the antibody conjugate can include the compound of Formula A2 or salt or an isotopic derivative thereof and have the structure of Formula 2:
  • the antibody conjugate can include the compound of Formula A3 or salt or an isotopic derivative thereof and have the structure of Formula 3:
  • the antibody conjugate can include the compound of Formula
  • A4 or salt or an isotopic derivative thereof have the structure of Formula 4:
  • the antibody conjugate can include the compound of Formula A4 or salt or an isotopic derivative thereof and have the structure of Formula 5:
  • n an integer
  • n 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • the antibody conjugate can include the compound of Formula
  • A4 or salt or an isotopic derivative thereof have the structure of Formula 6:
  • n an integer
  • n 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)
  • d is a number
  • d 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
  • composition comprising the antibody conjugate of any one of Formulae 1-6, and a pharmaceutically acceptable excipient.
  • the antibody conjugate can be provided for use in the manufacture of a medicament in the treatment of cancer.
  • a further aspect relates to a method for treating cancer, comprising administering a therapeutically effective amount of the antibody conjugate or pharmaceutical composition disclosed herein to a subject in need thereof.
  • Fig. 1 depicts a schematic structure of DP001
  • Fig. 2 HIC, SEC and MS spectra of DP001-TM1
  • Fig. 3 HIC, SEC and MS spectra of DP001 -TM2
  • Fig. 4 HIC and SEC spectra of DP001-TM3
  • Fig. 5 HIC, SEC and MS spectra of DP001 -TM5
  • Fig. 6 HIC, SEC and MS spectra of DP001-TM6
  • Fig. 7 HIC, SEC and MS spectra of DP001-TM7
  • Fig. 8 HIC, SEC and MS spectra of DP001-TM3-HIC
  • Fig. 9 HIC and SEC of DP001-TM8
  • Fig. 10 HIC and SEC of DP001-TM9
  • Fig. 11 HIC and SEC ofDPOOl-TMIO
  • Fig. 12 hTLR 7 activation of TM3, TM5, TM9, TM10
  • Fig. 13 hTLR 7 activation of TM6, TM7, TM8
  • Fig. 14 hTLR 8 activation of TM5
  • Fig. 15 in vitro human myeloid cell activation by DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, DP001-TM7
  • Fig. 16 Antibody-TLR7/8 agonist conjugates of DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, and DP001-TM7 elicit antitumor immunity in Calu-3 tumor xenograft model
  • Fig. 17 Antibody-TLR7/8 agonist conjugates of mDP001-TM5, mDP001-TM8, and mDPOOl-TMIO elicit antitumor immunity in Calu-3 tumor xenograft model (mDPOOl is DP001 with mouse IgG2a)
  • Fig. 18 Antibody-TLR7/8 agonist conjugates of DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, and DP001-TM7 elicit antitumor immunity in HCC1954 tumor xenograft model
  • Fig. 19 Stability of the antibody conjugates of DP001-TM5 in buffer, human, or mouse plasma.
  • TLR agonists are a well-characterized class of immune stimulants that can activate APCs, leading to direct tumor cell killing and enhanced T-cell-mediated immunity against tumor neoantigens. It has been demonstrated that antitumor immune responses can be greatly augmented when immune stimulants such as TLR agonists are conjugated with tumortargeting antibody. This combination strategy results in enhanced tumor antigen uptake by tumor-associated APCs leading to T-cell activation, immunological memory and the eradication of both primary and metastatic tumors. Systemic administration of the antibody conjugate activated localized APC in the TME while circumventing the typical toxicities associated with systemic delivery of TLR agonists.
  • the present disclosure provides antibody immune-stimulant conjugates for cancer treatment.
  • the conjugates described herein can include a tumortargeting monoclonal antibody conjugated to an TLR7/8 agonist via a protease cleavable or non-cleavable linker.
  • the articles “a” and “an” refer to one or more than one, e.g., to at least one, of the grammatical object of the article.
  • the use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of "one or more,” “at least one,” and “one or more than one.”
  • “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.
  • the term “substantially” means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are present in a given embodiment, yet open to the inclusion of unspecified elements.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.
  • compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • an "antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • conjugate refers to an antibody construct, or antibody, that is covalently bonded to a non-naturally occurring chemical moiety as described herein.
  • conjugate refers to an antibody construct, or antibody, that is covalently bonded to a non-naturally occurring chemical moiety as described herein.
  • conjugate refers to an antibody construct, or antibody, that is covalently bonded to a non-naturally occurring chemical moiety as described herein.
  • conjugate refers to an antibody construct, or antibody, that is covalently bonded to a non-naturally occurring chemical moiety as described herein.
  • immunoconjugate immunoconjugate
  • antibody immune-stimulant conjugate and “AISC” are used interchangeably herein.
  • antibody construct refers to polypeptide comprising an antigen binding fragment and an Fc domain.
  • An antibody construct can comprise an antibody.
  • biological product in reference to a biological product, means that the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.
  • immune-stimulant refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant, such as the TLR agonists known in the art or disclosed herein.
  • TLR Toll-like receptor
  • TLR polypeptides share a characteristic structure that includes an extracellular domain that has leucine-rich repeats, a transmembrane domain, and an intracellular domain that is involved in TLR signaling.
  • Toll-like receptor 7 and “TLR7” refer to nucleic acids or polypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a publicly-available TLR7 sequence, e.g., GenBank accession number AAZ99026 for human TLR7 polypeptide, or GenBank accession number AAK62676 for murine TLR7 polypeptide.
  • Toll-like receptor 8 and “TLR8” refer to nucleic acids or polypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a publicly-available TLR8 sequence, e.g., GenBank accession number AAZ95441 for human TLR8 polypeptide, or GenBank accession number AAK62677 for murine TLR8 polypeptide.
  • Toll-like receptor 7/8 and “TLR7/8" refer to nucleic acids or polypeptides that are both TLR7 agonists and TLR8 agonists.
  • a "TLR agonist” is a substance that binds, directly or indirectly, to a TLR (e.g., TLR7 and/or TLR8) to induce TLR signaling. Any detectable difference in TLR signaling can indicate that an agonist stimulates or activates a TLR. Signaling differences can be manifested, for example, as changes in the expression of target genes, in the phosphorylation of signal transduction components, in the intracellular localization of downstream elements such as NK-KB, in the association of certain components (such as IRAK) with other proteins or intracellular structures, or in the biochemical activity of components such as kinases (such as MAPK).
  • Immune checkpoint inhibitors refers to any modulator that inhibits the activity of the immune checkpoint molecule.
  • Immune checkpoint inhibitors can include, but are not limited to, immune checkpoint molecule binding proteins, small molecule inhibitors, antibodies, antibody-derivatives (including Fc fusions, Fab fragments and scFvs), antibody-drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides and peptide mimetics.
  • linker refers to a functional group that covalently bonds two or more moi eties in a compound or material.
  • the linker can serve to covalently bond an immune-stimulant to an antibody construct in an immunoconjugate.
  • the terms “treat,” “treatment,” and “treating” refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., cognitive impairment), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom.
  • the treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.
  • cancer refers to conditions including solid cancers, lymphomas, and leukemias.
  • examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer (e.g., melanoma), choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, gliom
  • lung cancer e.g., non
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • a particular cancer may be characterized by a solid mass tumor.
  • the solid tumor mass if present, may be a primary tumor mass.
  • a primary tumor mass refers to a growth of cancer cells in a tissue resulting from the transformation of a normal cell of that tissue. In most cases, the primary tumor mass is identified by the presence of a cyst, which can be found through visual or palpation methods, or by irregularity in shape, texture or weight of the tissue.
  • tumors are not palpable and can be detected only through medical imaging techniques such as X-rays (e.g., mammography), or by needle aspirations. The use of these latter techniques is more common in early detection. Molecular and phenotypic analysis of cancer cells within a tissue will usually confirm if the cancer is endogenous to the tissue or if the lesion is due to metastasis from another site.
  • the term "tumor" is inclusive of solid tumors and non-solid tumors.
  • the conjugates and compositions of the present disclosure can be administered locally at the site of a tumor (e.g., by direct injection) or remotely. In some embodiments, the conjugate or composition is administered to the subject systemically, e.g., intravascular, such as intravenous administration.
  • the terms "effective amount” and “therapeutically effective amount” refer to a dose of a substance such as an immunoconjugate that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.
  • the term "subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.
  • administering refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to the subject.
  • a slow-release device e.g., a mini-osmotic pump
  • phrases "pharmaceutically acceptable salt” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention.
  • Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, -toluenesulfonate, and pamoate (i.e., l,l'-methylene
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion.
  • the counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.
  • the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanes
  • an inorganic acid such as hydrochloric acid
  • Acids which are generally considered suitable for the formation of pharmaceutically useful or acceptable salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1 19; P. Gould, International J. of Pharmaceutics (1986) 33 201 217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; Remington's Pharmaceutical Sciences, 18 th ed., (1995) Mack Publishing Co., Easton PA; and in The Orange Book (Food & Drug Administration, Washington, D. C. on their website). These disclosures are incorporated herein by reference thereto.
  • the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
  • an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
  • suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
  • amino acids such as glycine and arginine
  • ammonia such as glycine and arginine
  • primary, secondary, and tertiary amines such as piperidine, morpholine and piperazine
  • inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
  • isotopic derivative refers to a derivative of a compound in which one or more atoms are isotopically enriched or labelled.
  • an isotopic derivative of a compound is isotopically enriched with regard to, or labelled with, one or more isotopes as compared to the corresponding compound.
  • the isotopic derivative is enriched with regard to, or labelled with, one or more atoms selected from 2 H, 13 C, 14 C, 15 N, 18 O, 29 Si, 31 P, and 34 S.
  • the isotopic derivative is a deuterium labeled compound (i.e., being enriched with 2 H with regard to one or more atoms thereof).
  • the compound is a 18 F labeled compound. In some embodiments, the compound is a 123 I labeled compound, a 124 I labeled compound, a 125 I labeled compound, a 129 I labeled compound, a 134 I labeled compound, a 135 I labeled compound, or any combination thereof. In some embodiments, the compound is a 33 S labeled compound, a 34 S labeled compound, a 35 S labeled compound, a 36 S labeled compound, or any combination thereof.
  • the 18 F, 123 I, 124 I, 125 I, 129 I, 131 I, 135 I, 32 S, 34 S, 35 S, and/or 36 S labeled compound can be prepared using any of a variety of art-recognized techniques.
  • the deuterium labeled compound can generally be prepared by substituting a 18 F, 123 I, 124 I, 125 I, 129 I, 131 I, 135 I, 3 S, 34 S, 35 S, and/or 36 S labeled reagent for a non-isotope labeled reagent.
  • a compound of the invention or a pharmaceutically acceptable salt or solvate thereof that contains one or more of the aforementioned 18 F, 123 I, 124 I, 125 I, 129 I, 131 I, 135 I, 32 S, 34 S, 35 S, and 36 S atom(s) is within the scope of the invention. Further, substitution with isotope (e.g., 18 F, 123 I, 124 I, 125 I, 129 I, 131 I, 135 I, 3 S, 34 S, 35 S, and/or 36 S) may afford certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo halflife or reduced dosage requirements.
  • immune stimulants such as TLR7/8 agonists, also referred to as “payloads” that can be used to construct the compositions and conjugates of the present disclosure.
  • payloads also referred to as “payloads” that can be used to construct the compositions and conjugates of the present disclosure.
  • Exemplary immune stimulants are described as follows.
  • TLR Toll-like receptor
  • TLRs are type-I transmembrane proteins that are responsible for initiation of innate immune responses in vertebrates. TLRs recognize a variety of pathogen-associated molecular patterns from bacteria, viruses, and fungi and act as a first line of defense against invading pathogens. TLRs elicit overlapping yet distinct biological responses due to differences in cellular expression and in the signaling pathways that they initiate.
  • TLRs Once engaged (e.g., by a natural stimulus or a synthetic TLR agonist) TLRs initiate a signal transduction cascade leading to activation of NF-KB via the adapter protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF -receptor associated factor 6 (TRAF6), which results in the phosphorylation of the NF-KB inhibitor I-KB.
  • TNF-KB enters the cell nucleus and initiates transcription of genes whose promoters contain NF-KB binding sites, such as cytokines.
  • TLR signaling Additional modes of regulation for TLR signaling include TIR-domain containing adapter-inducing interferon-P (TRIF)-dependent induction of TRAF6 and activation of MyD88 independent pathways via TRIF and TRAF3, leading to the phosphorylation of interferon response factor three (IRF3).
  • TIR TIR-domain containing adapter-inducing interferon-P
  • MyD88 dependent pathway also activates several IRF family members, including IRF5 and IRF7 whereas the TRIF dependent pathway also activates the NF-KB pathway.
  • any ligands capable of activating TLR7 and/or TLR8 can be installed in the immunoconjugates of the present disclosure.
  • TLR7 agonists and TLR8 agonists are described, e.g., by Vacchelli et al. (Oncolmmunology, 2: 8, e25238, DOI: 10.4161/onci.25238 (2013)) and Patinote et al. (Agonist and antagonist ligands of tolllike receptors 7 and 8: Ingenious tools for therapeutic purposes. Eur J Med Chem. 2020; 193: 112238. doi: 10.1016/j.ejmech.2020.112238), each of which is hereby incorporated by reference in its entirety.
  • TLR7 and TLR8 are both expressed in monocytes and dendritic cells. In humans, TLR7 is also expressed in plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is expressed mostly in cells of myeloid origin, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 are capable of detecting the presence of "foreign" single-stranded RNA within a cell, as a means to respond to viral invasion. Treatment of TLR8-expressing cells, with TLR8 agonists can result in production of high levels of IL-12, IFN-y, IL-1, TNF-a, IL-6, and other inflammatory cytokines.
  • TLR8 agonists can result in production of high levels of IL-12, IFN-y, IL-1, TNF-a, IL-6, and other inflammatory cytokines.
  • TLR7-expressing cells such as pDCs
  • TLR7 agonists can result in production of high levels of IFN-a and other inflammatory cytokines.
  • TLR7/TLR8 engagement and resulting cytokine production can activate dendritic cells and other antigen-presenting cells, driving diverse innate and acquired immune response mechanisms leading to tumor destruction.
  • TLR7, TLR8 or TLR7/8 agonists include but are not limited to: Gardiquimod (l-(4-amino-2-ethylaminomethylimidazo[4,5-c]quinolin-l-yl)-2-methylpropan- 2-ol), Imiquimod (R837) (agonist for TLR7), loxoribine (agonist for TLR7), IRM1 (l-(2- amino-2-methylpropyl)-2-(ethoxymethyl)-lH-imidazo-[4,5-c]quinolin-4-amine), IRM2 (2- methyl-l-[2-(3-pyridin-3-ylpropoxy)ethyl]-lH-imidazo[4,5-c]quinolin-4-amine) (agonist for TLR8), IRM3 (N-(2-[2-[4-amino-2-(2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l- yl]e
  • TLR-1 (l-(4-(aminomethyl)benzyl)-2-butyl-U/-imidazo[4,5- c]quinolin-4-amine), a commercially available TLR 7/8 agonist, was used as a payload for antibody conjugates with both cleavable linker and covalently modified to form new compounds for antibody conjugates with non-cleavable linkers.
  • TLR-2 (A-(4-((4-amino-2-butyl-U/-imidazo[4,5-c]quinolin-l- yl)methyl)benzyl)-2-hydroxyacetamide), a novel TLR7/8 agonist can be used.
  • This compound was used as a payload for antibody conjugates with cleavable linkers
  • TLR-3 (l-(4-aminobutyl)-2-butyl-U/-imidazo[4,5-c]quinolin- 4-amine), a commercially available TLR 7/8 agonist, was covalently modified to form new compounds for antibody conjugates with non-cleavable linkers.
  • linkers disclosed herein can be reacted with various linkers via known chemistry.
  • linkers disclosed in Anami et al., Nature Communications 2018, 9, 2512 can be used, which is incorporated herein by reference in its entirety.
  • Exemplary linker-payload constructs are represented by Formulae A1-A4 as described herein. Additional linker-payload constructs are shown as follows.
  • TLR-1 is covalently modified with non-cleavable linkers.
  • TLR-3 is covalently modified with non-cleavable linkers.
  • n 1-20 (e.g. 1-8, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • TLR-1 is covalently modified with non-cleavable linkers.
  • Suitable coupling methods can be used to conjugate the linkerpayload constructs disclosed herein to an antibody or antibody construct. Examples include: 1. Cysteine conjugation; 2. Site specific microbial transglutaminase (mTGase) catalyzed conjugation. Suitable antibodies can include immune checkpoint inhibitors.
  • the immune checkpoint inhibitor is cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD 152), T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced TNFR-related protein (GITR, also known as TNFRSF18), inducible T cell costimulatory (ICOS, also known as CD278), CD96, poliovirus receptor-related 2 (PVRL2, also known as CD112R), programmed cell death protein 1 (PD-1, also known as CD279), programmed cell death 1 ligand 1 (PD-L1, also known as B7-H3 and CD274), programmed cell death ligand 2 (PD-L2, also known as B7-DC and CD273), lymphocyte activation gene-3 (LAG-3, also known as CD223), B7-H4, killer immunoglobulin receptor (KIR), Tumor Necrosis Factor Receptor superfamily member 4 (TNFRSF4, also known as 0X40 and
  • the antibody is selected from: ipilimumab (also known as Yervoy®) pembrolizumab (also known as Keytruda®), nivolumab (also known as Opdivo®), atezolizumab (also known as Tecentrig®), avelumab (also known as Bavencio®), and durvalumab (also known as ImfinziTM).
  • the antibody is selected from: ipilimumab (also known as Yervoy®), pembrolizumab (also known as Keytruda®), nivolumab (also known as Opdivo®), and atezolizumab (also known as Tecentrig®).
  • the antibody is selected from anti-HER2 antibody (e.g., Trastuzumab or trastuzumab biosimilar DP001), and antibodies targeting CEACAM5, CAIX, Nectin-4, PD-L1, Claudin 18.2, etc.
  • anti-HER2 antibody e.g., Trastuzumab or trastuzumab biosimilar DP001
  • the antibody is anti-HER2 antibody.
  • the antibody is anti-HER2 antibody, which contains a heavy chain and a light chain, wherein the heavy chain contains:
  • the antibody is anti-HER2 antibody, which contains a heavy chain variable domain and a light chain variable domain; wherein the sequence of the heavy chain variable domain contains an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 11); wherein the sequence of the light chain variable domain contains an amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 12).
  • the antibody is anti-HER2 antibody, which comprises a heavy chain having an amino acid sequence of
  • the antibody is anti-HER2 antibody, which comprises a heavy chain having an amino acid sequence of
  • the compound of the present invention for example those of formulae A1-A4 or pharmaceutically acceptable salts or an isotopic derivative thereofs, are conjugated to Q295 of the antibody, via site specific microbial transglutaminase (mTGase) catalyzed conjugation, and the N297 of the antibody is glycosylated.
  • mTGase site specific microbial transglutaminase
  • Suitable mTGase include, but is not limited to, bacterial transglutaminase (BTG).
  • the transglutaminase is wildtype, for example the TGase is from Strep Ladakanum (TG SL, SEQ ID NO: 13) or from Strep Mobaraensis (TG-SM, SEQ ID NO: 14).
  • the transglutaminase is an engineered transglutaminase, such as an engineered transglutaminase comprising an amino acid sequence having at least about 80% (for example, at least about 85%, 90%, 95%, or 99%) identity to SEQ ID NO: 13 or SEQ ID NO: 14.
  • Exemplary antibody conjugates are represented by Formulae 1-4 disclosed herein. Additional examples are as follows.
  • d 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
  • the structure formula does not mean that the conjugation only occurs on one heavy chain, conjugation can happen on both of the heavy chains. This also applies to other formulae herein, if appropriate.
  • the conjugation to the antibody can occur on each heavy chain of the antibody, and a small molecule (or one compound according to the present invention, i.e. one compound of formulae A-I to A-X) can conjugate onto either heavy chain of the antibody, preferably at the Q295 of the heavy chains of the antibody, Q295 is numbered by EU index.
  • the actual number of the linker-drug molecule(s) conjugated to the antibody or antigen-binding fragment thereof is represented by the number d, which can be an individual value or an average value.
  • the individual value stands for the actual number of the linker-drug molecule(s) conjugated to the antibody or antigen-binding fragment thereof in one antibody-conjugate; and the average value stands for the average of the actual numbers of the linker-drug molecule(s) conjugated to the antibody or antigen-binding fragment thereof in a mixture of antibody-conjugates.
  • conjugates of the present disclosure can be administered as isolated compounds, these compounds can also be administered as part of a pharmaceutical composition.
  • the subject disclosure thus further provides compositions comprising one or more conjugates disclosed herein in association with at least one pharmaceutically acceptable carrier.
  • Conjugates and compositions containing them can be administered to a subject locally at or adjacent to a site of intended action (e.g., a tumor or lesion), or systemically (e.g., intravascularly).
  • the conjugate and pharmaceutical composition can be adapted for various routes of administration, such as enteral, parenteral, intravenous, intramuscular, topical, subcutaneous, and so forth. Administration can be continuous or at distinct intervals, as can be determined by a person of ordinary skill in the art.
  • the antagonists and/or immune effector molecules of the conjugates, and suitable bioactive agents that are optionally administered with the conjugates separately or within the same formulation can be formulated as pharmaceutically acceptable salts or solvates.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like (See, for example, Handbook of Pharmaceutical Excipients, Arthur H. Kibbe (ed., 2000, which is incorporated by reference herein in its entirety), Am. Pharmaceutical Association, Washington, D.C.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • Such compositions contain a therapeutically effective amount of a therapeutic agent preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.
  • the pharmaceutical compositions may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • an implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the conjugate does not absorb.
  • the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • a liposome see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574).
  • polymeric materials can be used to achieve controlled or sustained release of the antibodies of the disclosure or fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and poly orthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). [0135] Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the disclosure or fragments thereof.
  • a pharmaceutical composition can be formulated to be compatible with its intended route of administration.
  • routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings.
  • a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection.
  • compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts of compounds may be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • the ingredients of the compositions disclosed herein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the disclosure provides that the conjugates, or pharmaceutical compositions thereof, can be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent.
  • the conjugates, or pharmaceutical compositions thereof is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject.
  • the conjugates, or pharmaceutical compositions thereof is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.
  • the lyophilized agents, or pharmaceutical compositions herein should be stored at between 2 °C and 8 °C in its original container and the therapeutic agents, or pharmaceutical compositions of the disclosure should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
  • the pharmaceutical composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent.
  • the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml.
  • the liquid form should be stored at between 2 °C and 8 °C in its original container.
  • the disclosure provides that the composition of the disclosure is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the conjugate.
  • compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the amount of the conjugate or composition comprising the conjugate which is effective in the treatment of one or more symptoms associated with ancer can be determined by standard clinical techniques.
  • the precise dose to be employed in the formulation can also depend on the route of administration and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the conjugates and compositions can be used to treat different types of cancer, which can include, but are not limited to, lung cancer (e.g., nonsmall cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer (e.g., melanoma), choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphom
  • lung cancer e.g
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • mAb monoclonal antibodies
  • DP001 has the same amino acid sequence as trastuzumab
  • mDPOOl is DP001 in which some amino acids replaced with mouse IgG2a.
  • DP001 and mDPOOl were made by recombinant DNA methods, their amino acid sequences are showed as below:
  • the mAh was exchanged into PBS (10 mM PB (disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 1 : 1), pH 6.0, 5 mM EDTA (disodium edetate), 137mM NaCl) via molecular weight cut-off centrifugal filtration (Millipore, 30 kDa).
  • PBS 10 mM PB (disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 1 : 1), pH 6.0, 5 mM EDTA (disodium edetate), 137mM NaCl)
  • the resultant mAh solution was transferred to a 50 mL conical tube.
  • the mAb concentration was determined to be 10 mg/mL by A280 (280nm ultraviolet absorption).
  • TCEP 1.1 eq-1.2eq, 7.48 pL-8.16 pL, 10 mM stock
  • the resultant mixture was incubated at 37 °C for 1 hr, with gentle shaking.
  • the corresponding TM compound i.e. the payload-linker compound
  • the resultant reaction mixture was allowed to be stirred at ambient temperature for 30 minutes, at which point L-NAC (10.0 eq, 6.8 pL,100 mM) was added.
  • the crude conjugate was then purified by Protein A chromatography (Mab selectsure) using 5 mM citric acid and 50 mM sodium citrate solution.
  • mTgase Transglutaminase
  • the mAh was exchanged into Tris-HCl (25 mM, pH 7.5) via molecular weight cutoff centrifugal filtration (Millipore, 30 kDa). The resultant mAh solution was transferred to a 50 mL conical tube. The mAb concentration was determined to be 30 mg/mL by A280. To the 333.3 pL mAb solution was added lOOuL reaction buffer (500 mM Tris-OAc, 20 mM EDTA, 0.2% Tween-20), 100 pL mTgase (15 mg/mL, SEQ ID NO: 13, wild type), 26 pL the corresponding TM compound (i.e.
  • the payload-linker compound (50 eq, 92mg/mL dissolved in DMSO) and 441 pL purified water, at room temperature and the resultant mixture was incubated at 30°C for no more than 120 hr, until modification rate >95%, with gentle shaking. At which point, the crude conjugate was then purified by Protein A chromatography (MabSelect Sure) using 5 mM citric acid and 50 mM sodium citrate solution.
  • Example 13 General procedure for the determination of the drug-antibody-ratios by hydrophobic interaction chromatography (HIC)
  • Sample (20 pg) was directly injected onto a TSKgel Butyl-NPR analytical column (Tosoh, 4.6 mm* 100 mm) at 30°C.
  • Solvent A consisted of 1.5 M ammonium sulfate, 25 mM potassium phosphate, pH 7.0 and solvent B consisted of 25mM potassium phosphate (pH 7.0) and 25% isopropanol.
  • the analytes were separated with a gradient from 0 % to 50 % solvent B from 0 to 20 minutes.
  • the column was maintained at 30°C with a flow rate of 0.8 mL/min.
  • the elution profile was monitored at 214 nm.
  • Sample (30 pg) was directly injected onto a XBridge®BEH200A analytical column (Waters, 7.8*300mm, 3.5pm) at ambient temperature.
  • a mobile phase consisting of 100 mM potassium phosphate with 100 mM sodium chloride, pH 6.7, and 10% isopropanol, was used.
  • the flow rate was 0.8 mL/min, run for 20 minutes.
  • the elution profile was monitored at 280 nm.
  • Example 15 Analytical data for mAh (Trastuzumab biosimilar DP001)-TLR conjugates
  • Fig. 2 shows the HIC, SEC and MS spectra of DP001-TM1.
  • Fig. 6 shows the HIC, SEC and MS spectra of DP001-TM6.
  • Digested peptides were monitored on HPLC by UV absorbance at 214 nm (/.max of TM5, TM8 and TM10). Only one peak at 214 nm was identified in DAR2 samples, whereas no peak was detected in control antibody.
  • MALDI-TOF analysis identified that peak as a single charged peptide EEQYNSTYR from trypsin digestion or NAKTKPREEQY from chymotrypsin digestion containing Glutamine 295 with exactly one TM5, TM8 or TM10.
  • Fig. 12 hTLR 7 activation: R848 (a known TLR7/8 Agonist); TM3, TM5, TM9, TM10
  • Fig. 13 hTLR 7 activation: TM6, TM7, TM8
  • Fig. 14 hTLR 8 activation: R848; TM5
  • Example 17 Activation of TLR7 and TLR8 signal transduction pathway of the antibody conjugates.
  • Fig. 15 shows in vitro human myeloid cell activation by DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, DP001-TM7.
  • Example 18 In vivo antitumor activity [0210] Calu-3 tumor cells were implanted into NU/NU mice subcutaneously for the xenograft studies.
  • Tumor size was recorded twice a week. Once tumors reached 80- 120mm 3 , treatments were initiated. Mice were randomized by tumor size into treatment groups before initial treatment. Her2 antibody and its TLR 7/8 agonist conjugates were administered at 3-6 mg/kg at qw *4 schedule or 5-10 mg/kg at qw x 2 schedule.
  • HCC1954 tumor cells were implanted into NU/NU mice subcutaneously for the xenograft studies.
  • Tumor size was recorded twice a week. Once tumors reached 80- 120mm 3 , treatments were initiated. Mice were randomized by tumor size into treatment groups before initial treatment. Her2 antibody and its TLR 7/8 agonist conjugates were administered at 5-10 mg/kg at q2w*2 schedule.
  • Example 19 Stability of the conjugates in buffer, human, or mouse plasma
  • mAb conjugates were incubated in buffer, human and mouse plasma at 37 °C for 120 hours.
  • the test samples were analyzed by LC-MS to measure released payload.
  • Fig. 19 shows stability of the non-cleavable antibody conjugate of DP001-TM5 in buffer, human, or mouse plasma.
  • PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300
  • DSDERVTPPA EPLDRMPDPY RPSYGRAETI VNNYIRKWQQ VYSHRDGRKQ QMTEEQREWL 60 SYGCVGVTWV NSGQYPTNRL AFAFFDEDKY KNELKNGRPR SGETRAEFEG RVAKDSFDEA 120 KGFQRARDVA SVMNKALENA HDEGAYLDNL KKELANGNDA LRNEDARSPF YSALRNTPSF 180 KDRNGGNHDP SKMKAVIYSK HFWSGQDRSG SSDKRKYGDP EAFRPDRGTG LVDMSRDRNI 240
  • DSDDRVTPPA EPLDRMPDPY RPSYGRAETV VNNYIRKWQQ VYSHRDGRKQ QMTEEQREWL 60 SYGCVGVTWV NSGQYPTNRL AFASFDEDRF KNELKNGRPR SGETRAEFEG RVAKESFDEE 120 KGFQRAREVA SVMNRALENA HDESAYLDNL KKELANGNDA LRNEDARSPF YSALRNTPSF 180 KERNGGNHDP SRMKAVIYSK HFWSGQDRSS SADKRKYGDP DAFRPAPGTG LVDMSRDRNI 240

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Abstract

The present disclosure provides, in some embodiments, antibody-immune stimulant conjugates for cancer immunotherapy. Methods for making and using the antibody-immune stimulant conjugates are also provided herein.

Description

Antibody Immune-stimulant Conjugates and Use Thereof
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of Unites States Provisional Application No. 63/245,331, filed on September 17, 2021, which is hereby incorporated by reference in its entirety.
FIELD
[0002] The present disclosure relates in general to methods and compositions for cancer immunotherapy, in particular antibody conjugates.
BACKGROUND
[0003] It is now well appreciated that tumor growth necessitates the acquisition of mutations that facilitate immune evasion. Even so, tumorigenesis results in the accumulation of mutated antigens, or neoantigens, that are readily recognized by the host immune system following ex vivo stimulation. Why and how the immune system fails to recognize neoantigens are beginning to be elucidated. Groundbreaking studies by Carmi et al. (Nature, 521 : 99-104 (2015)) have indicated that immune ignorance can be overcome by delivering neoantigens to activated dendritic cells via antibody-tumor immune complexes. In these studies, simultaneous delivery of tumor binding antibodies and dendritic cell adjuvants via intratumoral injections resulted in robust anti -tumor immunity. New compositions and methods for the delivery of antibodies and immune-stimulants are needed in order to reach inaccessible tumors and to expand treatment options for cancer patients and other subjects.
SUMMARY
[0004] An object of the present invention is to provide novel immune stimulating antibody conjugates with anti -tumor efficacy. A further object of the present invention is to provide stable and homogeneous antibody-drug conjugates by using stable linkers and enzyme- catalyzed site-specific conjugation.
[0005] In one aspect, provided herein is a compound having Formula Al, or a pharmaceutically acceptable salt or an isotopic derivative thereof
Figure imgf000003_0001
Formula Al wherein m and n are integers, m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 (or 1-18, 1-15, 1-12, 1-10, 1-8, 1-6, or 1-4, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0006] A further aspect relates to a compound having Formula A2, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000003_0002
Formula A2 wherein m and n are integers, m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 (or 1-18, 1-15,
1-12, 1-10, 1-8, 1-6, or 1-4, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0007] Another aspect relates to a compound having Formula A3, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000003_0003
Formula A3 wherein m and n are integers, m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 (or 1-18, 1-15, 1-12, 1-10, 1-8, 1-6, or 1-4, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0008] A further aspect relates to a compound having Formula A4, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000004_0001
wherein m and n are integers, m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 (or 1-18, 1-15,
1-12, 1-10, 1-8, 1-6, or 1-4, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0009] In a further aspect, provided herein is an antibody conjugate, comprising the compound of any of Formulae A1-A4 or a salt or an isotopic derivative thereof conjugated to an antibody or an antigen-binding fragment thereof.
[0010] In some embodiments, each antibody or fragment thereof attaches to 1-4 compounds of any one of Formulae A1-A4 conjugated thereon.
[0011] In certain embodiments, the compound is conjugated via Cysteine conjugation.
[0012] In some embodiments, the compound is conjugated via site specific microbial transglutaminase (mTGase) catalyzed conjugation.
[0013] In various embodiments, the antibody is anti-HER.2 antibody (for example, Trastuzumab or trastuzumab biosimilar DP001), anti-CEACAM5 antibody, anti-CAIX antibody, anti-Nectin-4 antibody, anti-PD-Ll antibody, and/or anti-Claudin 18.2 antibody.
[0014] DP001 is an anti-HER2 monoclonal antibody, which has the same amino acid sequence as trastuzumab (HERCEPTIN®). Specifically, it contains 1328 amino acids with two heavy chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO: 1), and two light chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 2). DP001 is a heterotetramer of two HCs of the IgGl subclass, and two LCs of the kappa subclass linked by 16 disulfide bonds (12 intra and 4 inter chain). A schematic structure of DP001 is depicted in FIG. 1.
[0015] In some embodiments, the antibody conjugate can include the compound of Formula Al or salt or an isotopic derivative thereof and have the structure of Formula 1 :
Figure imgf000004_0002
Formula 1 wherein m and n are integers, and m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7), n = 1-20 (e.g. 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0016] In some embodiments, the antibody conjugate can include the compound of Formula A2 or salt or an isotopic derivative thereof and have the structure of Formula 2:
Figure imgf000005_0001
Formula 2 wherein m and n are integers, and m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7), n = 1-20 (e.g. 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0017] In some embodiments, the antibody conjugate can include the compound of Formula A3 or salt or an isotopic derivative thereof and have the structure of Formula 3:
Figure imgf000005_0002
Formula 3 wherein m and n are integers, and m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7), n = 1-20 (e.g. 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0018] In some embodiments, the antibody conjugate can include the compound of Formula
A4 or salt or an isotopic derivative thereof and have the structure of Formula 4:
Figure imgf000005_0003
Formula 4 wherein m and n are integers, and m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7), n = 1-20 (e.g. 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0019] In some embodiments, the antibody conjugate can include the compound of Formula A4 or salt or an isotopic derivative thereof and have the structure of Formula 5:
Figure imgf000006_0001
Formula 5 wherein n is an integer, and n = 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0020] In some embodiments, the antibody conjugate can include the compound of Formula
A4 or salt or an isotopic derivative thereof and have the structure of Formula 6:
Figure imgf000006_0002
Formula 6 wherein n is an integer, and n = 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0021] Also provided herein is a pharmaceutical composition, comprising the antibody conjugate of any one of Formulae 1-6, and a pharmaceutically acceptable excipient.
[0022] The antibody conjugate can be provided for use in the manufacture of a medicament in the treatment of cancer.
[0023] A further aspect relates to a method for treating cancer, comprising administering a therapeutically effective amount of the antibody conjugate or pharmaceutical composition disclosed herein to a subject in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS [0024] Fig. 1 depicts a schematic structure of DP001
[0025] Fig. 2: HIC, SEC and MS spectra of DP001-TM1
[0026] Fig. 3 : HIC, SEC and MS spectra of DP001 -TM2
[0027] Fig. 4: HIC and SEC spectra of DP001-TM3
[0028] Fig. 5 : HIC, SEC and MS spectra of DP001 -TM5
[0029] Fig. 6: HIC, SEC and MS spectra of DP001-TM6
[0030] Fig. 7: HIC, SEC and MS spectra of DP001-TM7
[0031] Fig. 8: HIC, SEC and MS spectra of DP001-TM3-HIC
[0032] Fig. 9: HIC and SEC of DP001-TM8
[0033] Fig. 10: HIC and SEC of DP001-TM9
[0034] Fig. 11 : HIC and SEC ofDPOOl-TMIO
[0035] Fig. 12: hTLR 7 activation of TM3, TM5, TM9, TM10
[0036] Fig. 13 : hTLR 7 activation of TM6, TM7, TM8
[0037] Fig. 14: hTLR 8 activation of TM5
[0038] Fig. 15: in vitro human myeloid cell activation by DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, DP001-TM7
[0039] Fig. 16: Antibody-TLR7/8 agonist conjugates of DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, and DP001-TM7 elicit antitumor immunity in Calu-3 tumor xenograft model
[0040] Fig. 17: Antibody-TLR7/8 agonist conjugates of mDP001-TM5, mDP001-TM8, and mDPOOl-TMIO elicit antitumor immunity in Calu-3 tumor xenograft model (mDPOOl is DP001 with mouse IgG2a)
[0041] Fig. 18: Antibody-TLR7/8 agonist conjugates of DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, and DP001-TM7 elicit antitumor immunity in HCC1954 tumor xenograft model
[0042] Fig. 19: Stability of the antibody conjugates of DP001-TM5 in buffer, human, or mouse plasma.
DETAILED DESCRIPTION
[0043] The widespread adoption of checkpoint inhibitors greatly benefited cancer patients. Unfortunately, patients with “cold” tumors are less responsive to checkpoint blockade and have worse prognoses than patients with ‘hot’ tumors where immunosuppressed but potentially tumor-reactive T cells are present within the tumor microenvironment (TME). Antigen-presenting cells (APCs) can elicit widespread T-cell immunity and are more frequent within the TME than T cells. Therefore, APCs have become an attractive target for therapeutic intervention.
[0044] TLR agonists are a well-characterized class of immune stimulants that can activate APCs, leading to direct tumor cell killing and enhanced T-cell-mediated immunity against tumor neoantigens. It has been demonstrated that antitumor immune responses can be greatly augmented when immune stimulants such as TLR agonists are conjugated with tumortargeting antibody. This combination strategy results in enhanced tumor antigen uptake by tumor-associated APCs leading to T-cell activation, immunological memory and the eradication of both primary and metastatic tumors. Systemic administration of the antibody conjugate activated localized APC in the TME while circumventing the typical toxicities associated with systemic delivery of TLR agonists.
[0045] The present disclosure provides antibody immune-stimulant conjugates for cancer treatment. In some embodiments, the conjugates described herein can include a tumortargeting monoclonal antibody conjugated to an TLR7/8 agonist via a protease cleavable or non-cleavable linker.
Definitions
[0046] Certain terms are defined herein below. Additional definitions are provided throughout the application.
[0047] As used herein, the articles “a” and “an” refer to one or more than one, e.g., to at least one, of the grammatical object of the article. The use of the words "a" or "an" when used in conjunction with the term "comprising" herein may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." [0048] As used herein, “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values. The term “substantially” means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.
[0049] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are present in a given embodiment, yet open to the inclusion of unspecified elements.
[0050] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.
[0051] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[0052] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
[0053] An "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
[0054] In the context,
Figure imgf000009_0001
represents an antibody or an antigen-binding fragment thereof.
[0055] As used herein, the term “conjugate” refers to an antibody construct, or antibody, that is covalently bonded to a non-naturally occurring chemical moiety as described herein. The terms “conjugate,” "immunoconjugate," "antibody immune-stimulant conjugate," and "AISC" are used interchangeably herein. As used herein, the phrase "antibody construct" refers to polypeptide comprising an antigen binding fragment and an Fc domain. An antibody construct can comprise an antibody.
[0056] As used herein, the term "biosimilar" in reference to a biological product, means that the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.
As used herein, the term "immune-stimulant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant, such as the TLR agonists known in the art or disclosed herein.
[0057] As used herein, the terms "Toll-like receptor" and "TLR" refer to any member of a family of highly-conserved mammalian proteins which recognize pathogen-associated molecular patterns and act as key signaling elements in innate immunity. TLR polypeptides share a characteristic structure that includes an extracellular domain that has leucine-rich repeats, a transmembrane domain, and an intracellular domain that is involved in TLR signaling.
[0058] The terms "Toll-like receptor 7" and "TLR7" refer to nucleic acids or polypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a publicly-available TLR7 sequence, e.g., GenBank accession number AAZ99026 for human TLR7 polypeptide, or GenBank accession number AAK62676 for murine TLR7 polypeptide. [0059] The terms "Toll-like receptor 8" and "TLR8" refer to nucleic acids or polypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a publicly-available TLR8 sequence, e.g., GenBank accession number AAZ95441 for human TLR8 polypeptide, or GenBank accession number AAK62677 for murine TLR8 polypeptide.
[0060] The terms "Toll-like receptor 7/8" and "TLR7/8" refer to nucleic acids or polypeptides that are both TLR7 agonists and TLR8 agonists.
[0061] A "TLR agonist" is a substance that binds, directly or indirectly, to a TLR (e.g., TLR7 and/or TLR8) to induce TLR signaling. Any detectable difference in TLR signaling can indicate that an agonist stimulates or activates a TLR. Signaling differences can be manifested, for example, as changes in the expression of target genes, in the phosphorylation of signal transduction components, in the intracellular localization of downstream elements such as NK-KB, in the association of certain components (such as IRAK) with other proteins or intracellular structures, or in the biochemical activity of components such as kinases (such as MAPK).
[0062] As used herein, the term "immune checkpoint inhibitors" refers to any modulator that inhibits the activity of the immune checkpoint molecule. Immune checkpoint inhibitors can include, but are not limited to, immune checkpoint molecule binding proteins, small molecule inhibitors, antibodies, antibody-derivatives (including Fc fusions, Fab fragments and scFvs), antibody-drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides and peptide mimetics.
[0063] As used herein, the term "linker" refers to a functional group that covalently bonds two or more moi eties in a compound or material. For example, the linker can serve to covalently bond an immune-stimulant to an antibody construct in an immunoconjugate.
[0064] As used herein, the terms "treat," "treatment," and "treating" refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., cognitive impairment), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.
[0065] As used herein, the term "cancer" refers to conditions including solid cancers, lymphomas, and leukemias. Examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer (e.g., melanoma), choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, and multiple myeloma. [0066] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. For example, a particular cancer may be characterized by a solid mass tumor. The solid tumor mass, if present, may be a primary tumor mass. A primary tumor mass refers to a growth of cancer cells in a tissue resulting from the transformation of a normal cell of that tissue. In most cases, the primary tumor mass is identified by the presence of a cyst, which can be found through visual or palpation methods, or by irregularity in shape, texture or weight of the tissue. However, some primary tumors are not palpable and can be detected only through medical imaging techniques such as X-rays (e.g., mammography), or by needle aspirations. The use of these latter techniques is more common in early detection. Molecular and phenotypic analysis of cancer cells within a tissue will usually confirm if the cancer is endogenous to the tissue or if the lesion is due to metastasis from another site. The term "tumor" is inclusive of solid tumors and non-solid tumors. The conjugates and compositions of the present disclosure can be administered locally at the site of a tumor (e.g., by direct injection) or remotely. In some embodiments, the conjugate or composition is administered to the subject systemically, e.g., intravascular, such as intravenous administration.
[0067] As used herein the terms "effective amount" and "therapeutically effective amount" refer to a dose of a substance such as an immunoconjugate that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Oilman's The Pharmacological Basis of Therapeutics, 11th Edition, 2006, Brunton, Ed., McGraw-Hill; and Remington: The Science and Practice of Pharmacy, 215* Edition, 2005, Hendrickson, Ed., Lippincott, Williams & Wilkins).
[0068] As used herein, the term "subject" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.
[0069] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to the subject.
[0070] Various aspects of the disclosure are described in further detail below. Additional definitions are set out throughout the specification.
[0071] The phrase "pharmaceutically acceptable salt" as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, -toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.
[0072] If the compound of the invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like. Acids which are generally considered suitable for the formation of pharmaceutically useful or acceptable salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1 19; P. Gould, International J. of Pharmaceutics (1986) 33 201 217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; Remington's Pharmaceutical Sciences, 18th ed., (1995) Mack Publishing Co., Easton PA; and in The Orange Book (Food & Drug Administration, Washington, D. C. on their website). These disclosures are incorporated herein by reference thereto.
[0073] If the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
[0074] The term "isotopic derivative", as used herein, refers to a derivative of a compound in which one or more atoms are isotopically enriched or labelled. For example, an isotopic derivative of a compound is isotopically enriched with regard to, or labelled with, one or more isotopes as compared to the corresponding compound. In some embodiments, the isotopic derivative is enriched with regard to, or labelled with, one or more atoms selected from 2H, 13C, 14C, 15N, 18O, 29Si, 31P, and 34S. In some embodiments, the isotopic derivative is a deuterium labeled compound (i.e., being enriched with 2H with regard to one or more atoms thereof). In some embodiments, the compound is a 18F labeled compound. In some embodiments, the compound is a 123I labeled compound, a 124I labeled compound, a 125I labeled compound, a 129I labeled compound, a 134I labeled compound, a 135I labeled compound, or any combination thereof. In some embodiments, the compound is a 33S labeled compound, a 34S labeled compound, a 35S labeled compound, a 36S labeled compound, or any combination thereof.
[0075] It is understood that the 18F, 123I, 124I, 125I, 129I, 131I, 135I, 32S, 34S, 35S, and/or 36S labeled compound, can be prepared using any of a variety of art-recognized techniques. For example, the deuterium labeled compound can generally be prepared by substituting a 18F, 123I, 124I, 125I, 129I, 131I, 135I, 3S, 34S, 35S, and/or 36S labeled reagent for a non-isotope labeled reagent.
[0076] A compound of the invention or a pharmaceutically acceptable salt or solvate thereof that contains one or more of the aforementioned 18F, 123I, 124I, 125I, 129I, 131I, 135I, 32S, 34S, 35S, and 36S atom(s) is within the scope of the invention. Further, substitution with isotope (e.g., 18F, 123I, 124I, 125I, 129I, 131I, 135I, 3S, 34S, 35S, and/or 36S) may afford certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo halflife or reduced dosage requirements.
Payloads
[0077] Disclosed herein, in some embodiments, are immune stimulants such as TLR7/8 agonists, also referred to as “payloads” that can be used to construct the compositions and conjugates of the present disclosure. Exemplary immune stimulants are described as follows.
[0078] Any ligands capable of activating a Toll-like receptor (TLR) can be installed in the immunoconjugates of the present disclosure. Toll-like receptors (TLRs) are type-I transmembrane proteins that are responsible for initiation of innate immune responses in vertebrates. TLRs recognize a variety of pathogen-associated molecular patterns from bacteria, viruses, and fungi and act as a first line of defense against invading pathogens. TLRs elicit overlapping yet distinct biological responses due to differences in cellular expression and in the signaling pathways that they initiate. Once engaged (e.g., by a natural stimulus or a synthetic TLR agonist) TLRs initiate a signal transduction cascade leading to activation of NF-KB via the adapter protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF -receptor associated factor 6 (TRAF6), which results in the phosphorylation of the NF-KB inhibitor I-KB. AS a result, NF-KB enters the cell nucleus and initiates transcription of genes whose promoters contain NF-KB binding sites, such as cytokines. Additional modes of regulation for TLR signaling include TIR-domain containing adapter-inducing interferon-P (TRIF)-dependent induction of TRAF6 and activation of MyD88 independent pathways via TRIF and TRAF3, leading to the phosphorylation of interferon response factor three (IRF3). Similarly, the MyD88 dependent pathway also activates several IRF family members, including IRF5 and IRF7 whereas the TRIF dependent pathway also activates the NF-KB pathway.
[0079] Any ligands capable of activating TLR7 and/or TLR8 can be installed in the immunoconjugates of the present disclosure. Examples of TLR7 agonists and TLR8 agonists are described, e.g., by Vacchelli et al. (Oncolmmunology, 2: 8, e25238, DOI: 10.4161/onci.25238 (2013)) and Patinote et al. (Agonist and antagonist ligands of tolllike receptors 7 and 8: Ingenious tools for therapeutic purposes. Eur J Med Chem. 2020; 193: 112238. doi: 10.1016/j.ejmech.2020.112238), each of which is hereby incorporated by reference in its entirety. TLR7 and TLR8 are both expressed in monocytes and dendritic cells. In humans, TLR7 is also expressed in plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is expressed mostly in cells of myeloid origin, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 are capable of detecting the presence of "foreign" single-stranded RNA within a cell, as a means to respond to viral invasion. Treatment of TLR8-expressing cells, with TLR8 agonists can result in production of high levels of IL-12, IFN-y, IL-1, TNF-a, IL-6, and other inflammatory cytokines. Similarly, stimulation of TLR7- expressing cells, such as pDCs, with TLR7 agonists can result in production of high levels of IFN-a and other inflammatory cytokines. TLR7/TLR8 engagement and resulting cytokine production can activate dendritic cells and other antigen-presenting cells, driving diverse innate and acquired immune response mechanisms leading to tumor destruction.
[0080] Examples of TLR7, TLR8 or TLR7/8 agonists include but are not limited to: Gardiquimod (l-(4-amino-2-ethylaminomethylimidazo[4,5-c]quinolin-l-yl)-2-methylpropan- 2-ol), Imiquimod (R837) (agonist for TLR7), loxoribine (agonist for TLR7), IRM1 (l-(2- amino-2-methylpropyl)-2-(ethoxymethyl)-lH-imidazo-[4,5-c]quinolin-4-amine), IRM2 (2- methyl-l-[2-(3-pyridin-3-ylpropoxy)ethyl]-lH-imidazo[4,5-c]quinolin-4-amine) (agonist for TLR8), IRM3 (N-(2-[2-[4-amino-2-(2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l- yl]ethoxy]ethyl)-N-methylcyclo hexanecarboxamide) (agonist for TLR8), CL097 (2- (ethoxymethyl)-lH-imidazo[4,5-c]quinolin-4-amine) (agonist for TLR7/8), CL307 (agonist for TLR7), CL264 (agonist for TLR7), Resiquimod (agonist for TLR7/8), 3M- 052/MEDI9197 (agonist for TLR7/8), SD-101 (N-[(4S)-2,5-dioxo-4-imidazolidinyl]-urea) (agonist for TLR7/8), motolimod (2-amino-N,N-dipropyl-8-[4-(pyrrolidine-l- carbonyl)phenyl]-3H-l-benzazepine-4-carboxamide) (agonist for TLR8), CL075 (3M002, 2- propylthiazolo[4,5-c]quinolin-4-amine) (agonist for TLR7/8), and TL8-506 (3H-1- benzazepine-4-carboxylic acid, 2-amino-8-(3 -cyanophenyl)-, ethyl ester) (agonist for TLR8). [0081] Antibody conjugates of TLR 7, TLR8 or TLR7/8 have been described, for example, in US 2017/0121421 Al, US2020/0113912 Al, and US 10,675,358 B2, each of which is hereby incorporated by reference in its entirety. The robust antitumor efficacy has been demonstrated in preclinical animal models. However, there are still safety concerns due to the systemic instability and poor PK profile of the conjugates. There remains a need for improved therapeutic agents by developing stable and homogeneous conjugates through sitespecific conjugation technology.
[0082] In one embodiment, TLR-1 (l-(4-(aminomethyl)benzyl)-2-butyl-U/-imidazo[4,5- c]quinolin-4-amine), a commercially available TLR 7/8 agonist, was used as a payload for antibody conjugates with both cleavable linker and covalently modified to form new compounds for antibody conjugates with non-cleavable linkers.
Figure imgf000016_0001
[0083] In one embodiment, TLR-2 (A-(4-((4-amino-2-butyl-U/-imidazo[4,5-c]quinolin-l- yl)methyl)benzyl)-2-hydroxyacetamide), a novel TLR7/8 agonist can be used. This compound was used as a payload for antibody conjugates with cleavable linkers
Figure imgf000016_0002
[0084] In one embodiment, TLR-3 (l-(4-aminobutyl)-2-butyl-U/-imidazo[4,5-c]quinolin- 4-amine), a commercially available TLR 7/8 agonist, was covalently modified to form new compounds for antibody conjugates with non-cleavable linkers.
Figure imgf000016_0003
Linker-Payload Constructs
[0085] The immune stimulants disclosed herein can be reacted with various linkers via known chemistry. In some embodiments, linkers disclosed in Anami et al., Nature Communications 2018, 9, 2512 can be used, which is incorporated herein by reference in its entirety. Exemplary linker-payload constructs are represented by Formulae A1-A4 as described herein. Additional linker-payload constructs are shown as follows.
[0086] Formula A-I
Figure imgf000017_0001
wherein m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 (e.g. 1-8, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0091] TLR-1 is covalently modified with non-cleavable linkers.
[0092] Formula A-IV
Figure imgf000018_0001
wherein m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 (e.g. 1-8, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0099] TLR-3 is covalently modified with non-cleavable linkers.
[0100] Formula A- VIII
Figure imgf000019_0001
wherein n = 1-20 (e.g. 1-8, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
[0105] TLR-1 is covalently modified with non-cleavable linkers.
Antibody Conjugates
[0106] Various coupling methods known in the art can be used to conjugate the linkerpayload constructs disclosed herein to an antibody or antibody construct. Examples include: 1. Cysteine conjugation; 2. Site specific microbial transglutaminase (mTGase) catalyzed conjugation. Suitable antibodies can include immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD 152), T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced TNFR-related protein (GITR, also known as TNFRSF18), inducible T cell costimulatory (ICOS, also known as CD278), CD96, poliovirus receptor-related 2 (PVRL2, also known as CD112R), programmed cell death protein 1 (PD-1, also known as CD279), programmed cell death 1 ligand 1 (PD-L1, also known as B7-H3 and CD274), programmed cell death ligand 2 (PD-L2, also known as B7-DC and CD273), lymphocyte activation gene-3 (LAG-3, also known as CD223), B7-H4, killer immunoglobulin receptor (KIR), Tumor Necrosis Factor Receptor superfamily member 4 (TNFRSF4, also known as 0X40 and CD134) and its ligand OX40L (CD252), indoleamine 2,3 -dioxygenase 1 (IDO-1), indoleamine 2,3 -dioxygenase 2 (IDO-2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuator (BTLA, also known as CD272), T- cell membrane protein 3 (TIM3), the adenosine A2A receptor (A2Ar), and V-domain Ig suppressor of T cell activation (VISTA protein). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-L1.
[0107] In some embodiments, the antibody is selected from: ipilimumab (also known as Yervoy®) pembrolizumab (also known as Keytruda®), nivolumab (also known as Opdivo®), atezolizumab (also known as Tecentrig®), avelumab (also known as Bavencio®), and durvalumab (also known as Imfinzi™). In some embodiments, the antibody is selected from: ipilimumab (also known as Yervoy®), pembrolizumab (also known as Keytruda®), nivolumab (also known as Opdivo®), and atezolizumab (also known as Tecentrig®).
[0108] In some embodiments, the antibody is selected from anti-HER2 antibody (e.g., Trastuzumab or trastuzumab biosimilar DP001), and antibodies targeting CEACAM5, CAIX, Nectin-4, PD-L1, Claudin 18.2, etc.
[0109] In some embodiments, the antibody is anti-HER2 antibody.
[0110] In some embodiments, the antibody is anti-HER2 antibody, which contains a heavy chain and a light chain, wherein the heavy chain contains:
(a) a heavy chain CDRH1 having an amino acid sequence of GFNIKDTY (SEQ ID NO: 5);
(b) a heavy chain CDRH2 having an amino acid sequence of IYPTNGYT (SEQ ID NO: 6);
(c) a heavy chain CDRH3 having an amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 7); wherein the light chain contains:
(a) a light chain CDRL1 having an amino acid sequence of QDVNTA (SEQ ID NO: 8);
(b) a light chain CDRL2 having an amino acid sequence of SAS (SEQ ID NO: 9);
(c) a light chain CDRL3having an amino acid sequence of QQHYTTPPT (SEQ ID NO: 10); wherein the complementarity-determining regions (CDRs) are defined by using the IMGT numbering system
[0111] In some embodiments, the antibody is anti-HER2 antibody, which contains a heavy chain variable domain and a light chain variable domain; wherein the sequence of the heavy chain variable domain contains an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 11); wherein the sequence of the light chain variable domain contains an amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 12).
[0112] In some embodiments, the antibody is anti-HER2 antibody, which comprises a heavy chain having an amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1), and a light chain having an amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).
[0113] In some embodiments, the antibody is anti-HER2 antibody, which comprises a heavy chain having an amino acid sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLS SGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKP KGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 3), and a light chain having an amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAPTVS IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 4).
[0114] In some embodiments, the compound of the present invention, for example those of formulae A1-A4 or pharmaceutically acceptable salts or an isotopic derivative thereofs, are conjugated to Q295 of the antibody, via site specific microbial transglutaminase (mTGase) catalyzed conjugation, and the N297 of the antibody is glycosylated.
[0115] Suitable mTGase include, but is not limited to, bacterial transglutaminase (BTG). In some embodiments, the transglutaminase is wildtype, for example the TGase is from Strep Ladakanum (TG SL, SEQ ID NO: 13) or from Strep Mobaraensis (TG-SM, SEQ ID NO: 14). In some embodiments, the transglutaminase is an engineered transglutaminase, such as an engineered transglutaminase comprising an amino acid sequence having at least about 80% (for example, at least about 85%, 90%, 95%, or 99%) identity to SEQ ID NO: 13 or SEQ ID NO: 14.
SEQ ID NO: 13
DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQ REWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGR VAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNED ARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPE AFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNH YHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP SEQ ID NO: 14 DSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGRKQQMTEE QREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRPRSGETRAEFEGR VAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLKKELANGNDALRNEDA RSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFWSGQDRSSSADKRKYGDPDA FRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNFDYGWFGAQTEADADKTVWTHGNHY HAPNGSLGAM
HVYESKFRNWSEGYSDFDRGAYVITFIPKSWNTAPDKVKQGWP
[0116] Exemplary antibody conjugates are represented by Formulae 1-4 disclosed herein. Additional examples are as follows.
[0117] Formula B-I
Figure imgf000023_0001
wherein m = 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 or 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or 1, 2, 3, 4, 5, 6, 7, or 8), d = 1-4, or 1-2, e.g., 1,
2, 3 or 4; or 1 or 2.
[0120] Formula B-IV
Figure imgf000024_0001
wherein m = 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) and n = 1-20 or 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or 1, 2, 3, 4, 5, 6, 7, or 8), d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0124] Formula B- VIII
Figure imgf000025_0001
wherein n = 1-20 or 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or 1, 2, 3, 4, 5, 6, 7, or 8), d = 1-2, e.g., 1 or 2.
[0127] It should be noted that according to the above formulae B-I to B-X, the structure formula does not mean that the conjugation only occurs on one heavy chain, conjugation can happen on both of the heavy chains. This also applies to other formulae herein, if appropriate. In fact, the conjugation to the antibody can occur on each heavy chain of the antibody, and a small molecule (or one compound according to the present invention, i.e. one compound of formulae A-I to A-X) can conjugate onto either heavy chain of the antibody, preferably at the Q295 of the heavy chains of the antibody, Q295 is numbered by EU index.
[0128] It should be understood that according to the present invention, the actual number of the linker-drug molecule(s) conjugated to the antibody or antigen-binding fragment thereof is represented by the number d, which can be an individual value or an average value. The individual value stands for the actual number of the linker-drug molecule(s) conjugated to the antibody or antigen-binding fragment thereof in one antibody-conjugate; and the average value stands for the average of the actual numbers of the linker-drug molecule(s) conjugated to the antibody or antigen-binding fragment thereof in a mixture of antibody-conjugates.
Pharmaceutical Compositions and Use
[0129] While conjugates of the present disclosure can be administered as isolated compounds, these compounds can also be administered as part of a pharmaceutical composition. The subject disclosure thus further provides compositions comprising one or more conjugates disclosed herein in association with at least one pharmaceutically acceptable carrier. Conjugates and compositions containing them can be administered to a subject locally at or adjacent to a site of intended action (e.g., a tumor or lesion), or systemically (e.g., intravascularly). The conjugate and pharmaceutical composition can be adapted for various routes of administration, such as enteral, parenteral, intravenous, intramuscular, topical, subcutaneous, and so forth. Administration can be continuous or at distinct intervals, as can be determined by a person of ordinary skill in the art. Optionally, the antagonists and/or immune effector molecules of the conjugates, and suitable bioactive agents that are optionally administered with the conjugates separately or within the same formulation, can be formulated as pharmaceutically acceptable salts or solvates.
[0130] In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like (See, for example, Handbook of Pharmaceutical Excipients, Arthur H. Kibbe (ed., 2000, which is incorporated by reference herein in its entirety), Am. Pharmaceutical Association, Washington, D.C.
[0131] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions contain a therapeutically effective amount of a therapeutic agent preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In some embodiments, the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.
[0132] In some embodiments, it may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering the conjugate, care must be taken to use materials to which the conjugate does not absorb.
[0133] In some embodiments, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
[0134] In some embodiments, the composition can be delivered in a controlled release or sustained release system. In some embodiments, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). In some embodiments, polymeric materials can be used to achieve controlled or sustained release of the antibodies of the disclosure or fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71 : 105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and poly orthoesters. In some embodiments, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). [0135] Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the disclosure or fragments thereof. See, e.g., U.S. Pat. No. 4,526,938; PCT Publication No. WO 91/05548; PCT Publication No. WO 96/20698; Ning et al., 1996, Radiotherapy & Oncology 39: 179- 189; Song et al., 1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.
[0136] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. In some embodiments, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In some embodiments, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection.
[0137] The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0138] The compositions can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
[0139] Pharmaceutically acceptable salts of compounds may be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
[0140] Generally, the ingredients of the compositions disclosed herein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0141] In particular, the disclosure provides that the conjugates, or pharmaceutical compositions thereof, can be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In some embodiments, the conjugates, or pharmaceutical compositions thereof is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Preferably, the conjugates, or pharmaceutical compositions thereof is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized agents, or pharmaceutical compositions herein should be stored at between 2 °C and 8 °C in its original container and the therapeutic agents, or pharmaceutical compositions of the disclosure should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In some embodiments, the pharmaceutical composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. Preferably, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at between 2 °C and 8 °C in its original container.
[0142] In some embodiments, the disclosure provides that the composition of the disclosure is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the conjugate.
[0143] The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack.
[0144] The amount of the conjugate or composition comprising the conjugate which is effective in the treatment of one or more symptoms associated with ancer can be determined by standard clinical techniques. The precise dose to be employed in the formulation can also depend on the route of administration and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[0145] In various embodiments, the conjugates and compositions can be used to treat different types of cancer, which can include, but are not limited to, lung cancer (e.g., nonsmall cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer (e.g., melanoma), choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, and multiple myeloma.
Abbreviation [0146] The abbreviations used in the context of the present specification have the following meanings:
Figure imgf000031_0002
Examples
[0147] The reaction materials and agents used in the following examples were commercially available, and therefore their specific sources will not be specialized. Example 1: Synthesis of TLR-2
Figure imgf000031_0001
, ,
TLR-1 TLR-2
[0148] To a solution of compound 1 (0.69 mmol, 1.0 eq) in DCM (30 mL) was added a mixture of compound 3, 2-hydroxyacetic acid (0.69 mmol, 1.0 eq), DIEA (2.08 mmol, 3.0 eq) and HATU (0.76 mmol, 1.1 eq) at room temperature. Then the mixture was stirred at room temperature for 3 h. The reaction was monitored by LCMS. The reaction mixture was filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by prep-HPLC to give TLR-2 (32%) as white solid.
LCMS: [M+l] = 418
HNMR (400 MHz, (CD3OD): 8 7.89-7.87 (m, 1H), 7.53 (m, 1H), 7.30 (m, 1H), 7.20 (m, 2H), 6.96-6.94 (m, 2H), 5.84 (s, 2H), 4.30 (s, 2H), 3.89 (s, 2H), 2.92-2.88 (t, J= 7.2 Hz, 2H), 1.77-1.73 (m, 2H), 1.39-1.34 (m, 2H) and 0.86-0.83 (t, J= 7.0 Hz, 3H).
Example 2: Synthesis of TM1
Figure imgf000032_0001
Preparation of compound 3
[0149] To a solution of compound 1 (11.76 mmol, 1.0 eq) in dimethylformamide (100 mL) were added 2-(7 -aza- 1 H-benzotri azole- 1 -yl)- 1 , 1 ,3 , 3 -tetramethyluronium hexafluorophosphate (14.11 mmol, 1.2 eq) and MA-diisopropylethylamine (23.52 mmol, 2.0 eq). Then a solution of compound 2 (11.76 mmol, 1.0 eq) in dimethylformamide (300 mL) was added to the mixture. The mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS and TLC. The reaction mixture was then poured in tertbutyl methyl ether (750 mL), stirred for 15 min, then filtered. The resulting solid was washed with di chloromethane: tert-butyl methyl ether: methanol (5:5: 1) mixture to give compound 3 (5.8 g, crude) as yellow solid.
Preparation of compound 5
[0150] To a solution of compound 3 (6.87 mmol, 1.0 eq) and A,7V-diisopropylethylamine (13.74 mmol, 2.0 eq) in dimethylformamide (500 mL) was added compound 4 (13.74 mmol, 2.0 eq). Then the mixture was stirred at room temperature for 8 h. The reaction was monitored by LCMS. The reaction mixture was poured into tert-Butyl methyl ether (500 mL) and stirred. After 15 min, the mixture was filtered, the filter cake dispersed in dichloromethane (100 mL) for 20 min then filtered to give compound 5 (3.3 g, crude) as light yellow solid.
Preparation of compound 7
[0151] To a solution of compound 5 (3.47 mmol, 1.0 eq) and A,A-diisopropylethylamine (13.88 mmol, 4.0 eq) in dimethylformamide (50 mL) was added compound 6 (3.47 mmol, 1.0 eq). Then the mixture was stirred at room temperature for 5 h. The reaction was monitored by LCMS. The reaction mixture was used for next step without further purification.
Preparation of compound 8
[0152] To a solution of compound 7 in DMF was added diethylamine (20.82 mmol, 6.0 eq) under nitrogen atmosphere. Then the mixture was stirred at room temperature for 8 h. The reaction was monitored by LCMS. The reaction mixture was purified by reverse phase column (water: acetonitrile, 5%-55%) to give compound 8 (32%) as light yellow solid.
Preparation of compound 10
[0153] To a solution of compound 8 (1.13 mmol, 1.0 eq) and N, 7V-di isopropyl ethyl amine (0.29 g, 2.25 mmol, 2.0 eq) in dimethylformamide (20 mL) was added compound 9 (1.70 mmol, 1.5 eq). Then the mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure. The residue was triturated with ethyl acetate (5 mL) for 0.5 h, filtered and concentrated under reduced pressure to give compound 10 (990 mg, crude) as light yellow solid.
Preparation of TM1
[0154] The mixture of compound 10 (0.79 mmol, 1.0 eq) in trifluoroacetic acid/dichloromethane (30 mL, 1 :3) was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure and the residue was purified by prep-HPLC to give TM1 (408 mg, 39%) as white solid. The recycled raw material (0.16 mmol, 1.0 eq) in trifluoroacetic acid/dichloromethane (8 mL, 1 :3) was stirred at room temperature for 6 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure and the residue was purified by prep-HPLC to give TM1 (113 mg, 53%) as white solid.
LCMS: [M+l] = 1205, [M-l] = 1203
HNMR (400 MHz, CD3OD): 89.80 (m, 1H), 8.29-8.27 (m, 1H), 7.94 (m, 2H), 7.71 (m, 1H), 7.62 (m, 1H), 7.51 (m, 2H), 7.35 (m, 1H), 7.25 (m, 4H), 7.02 (m, 2H), 6.79 (s, 2H), 5.92 (s,
2H), 5.00 (s, 2H), 4.46-4.44 (m, 2H), 4.20 (m, 3H), 3.75 (m, 4H), 3.60 (m, 4H), 3.36 (m, 2H), 3.18 (m, 2H), 3.00-2.97 (m, 2H), 2.46 (m, 6H), 2.08 (m, 2H), 1.77 (m, 6H), 1.52 (m, 2H), 1.42 (m, 2H) and 0.96-0.91 (m, 9H).
Example 3: Synthesis of TM-2
Figure imgf000034_0001
Preparation of compound 12
[0155] To a solution of compound 11 (23.04 mmol, 1.0 eq) and cesium carbonate (57.5 mmol, 2.5 eq) in dimethylformamide (80 mL) was added 3 -bromoprop- 1-ene (46.07 mmol, 2.0 eq) dropwise. The mixture was stirred at 0-10 °C for 4 h under nitrogen atmosphere. The reaction was monitored by TLC and HPLC. The reaction mixture was filtered and the filtrate was diluted with ethyl acetate (50 mL), extracted with water (80 mL x 2) and brine (100 mL). The combined organic layer was concentrated under vacuum to give compound 12 (5.6 g, crude) as colorless oil.
Preparation of compound 13 [0156] To a mixture of compound 12 (3.89 mmol, 1.0 eq) in dichloromethane (9 mL) was added trifluoroacetic acid (3 mL). The mixture was stirred at room temperature for 4 h under nitrogen atmosphere. The reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure to give compound 13 (crude) as brown oil.
Preparation of compound 15
[0157] To a solution of compound 14 (3.69 mmol, 1.0 eq) and compound 13 (3.69 mmol, 1.0 eq) in tetrahydrofuran (15 mL) were added 2-(7-aza-lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (4.43 mmol, 1.2 eq) and N,N- diisopropylethylamine (18.45 mmol, 5.0 eq). Then the mixture was stirred at room temperature for 2 h under nitrogen atmosphere. The reaction was monitored by LCMS. The reaction mixture was extracted with ethyl acetate (20 mL x 2). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by column chromatography on a silica gel (petroleum ether: ethyl acetate, 80:20) to give compound 15 (67%) as white solid.
Preparation of compound 16
[0158] To a solution of compound 15 (8.86 mmol, 1.0 eq) and piperidine (44.3 mmol, 5.0 eq) in di chloromethane (75 mL) was added tetrakis(triphenylphosphine)palladium (0.886 mmol, 0.1 eq) at room temperature under nitrogen atmosphere. Then the mixture was stirred at room temperature for 2 h under nitrogen atmosphere. The reaction was monitored by LCMS. The reaction mixture was extracted with di chloromethane and water (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by column chromatography on a silica gel (di chloromethane: methanol, 99: 1) to give compound 16 (74%) as white solid.
Preparation of compound 19
[0159] To a mixture of compound 17 (6.42 mmol, 1.2 eq) in dimethylformamide (40 mL) were added 2-(7-aza- 1 H-benzotri azole- 1 -yl)- 1 , 1 ,3 , 3 -tetramethyluronium hexafluorophosphate (6.96 mmol, 1.3 eq) and A-diisopropylethylamine (16.06 mmol, 3.0 eq) at 0 °C. Then the mixture was stirred at 0 °C for 20 min. Then compound 18 (0.73 mL, 5.35 mmol, 1.0 eq) was added to the reaction mixture at 0 °C. Then the mixture was warmed up and stirred at room temperature for 16 h. The reaction was monitored by TLC. The reaction mixture was extracted with ethyl acetate and water (3 x 40 mL). The residue was purified by column chromatography on a silica gel (petroleum ether: ethyl acetate, 100:0-60:40) to give compound 19 (96%) as white solid.
Preparation of compound 20
[0160] To a mixture of compound 19 (2.18 g, 5.14 mmol, 1.0 eq) in ethyl acetate (30 mL) was added hydrochloric acid/ethyl acetate (20 mL, 1 : 1). Then the mixture was stirred at room temperature overnight. The reaction was monitored by TLC. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography on a silica gel (di chloromethane: methanol, 100:0-98:2-96:4) to give compound 20 (63%) as white solid.
Preparation of compound 21
[0161] To a mixture of compound 20 (3.25 mmol, 1.0 eq) and lead tetraacetate (4.56 mmol, 1.4 eq) in tetrahydrofuran/toluene (45 mL/15 mL) was added pyridine (3.91 mmol, 1.2 eq) at room temperature. Then the mixture was warmed up and stirred at refluxed for 5 h. The reaction was monitored by TLC. The reaction mixture was cooled to room temperature, filtered and concentrated under reduced pressure. The residue was extracted with ethyl acetate and water (60 mL x 3). The residue was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on a silica gel (petroleum ether: ethyl acetate, 100:0-50: 1-9: 1-20: 1-60:40) to give compound 21 (29%) as white solid.
Preparation of compound 22
[0162] To a mixture of compound TLR-2 (1.08 mmol, 1.0 eq) in acetone (25 mL) were added compound 21 (1.62 mmol, 1.5 eq) and potassium carbonate (3.23 mmol, 3.0 eq) at room temperature. Then the mixture was stirred at room temperature overnight. The reaction was monitored by TLC. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography on a silica gel (di chloromethane: methanol, 94:6) to give compound 22 (586 mg, 73%) as white solid.
Preparation of compound 23
[0163] To a mixture of compound 22 (0.792 mmol, 1.0 eq) in dimethylformamide (10 mL) was added diethylamine (2.56 mmol, 3.0 eq) at room temperature. Then the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. The reaction mixture was filtered and concentrated under reduced pressure. The residue was triturated with ethyl acetate to give compound 23 (78%) as white solid.
Preparation of compound 24
[0164] To a mixture of compound 23 (0.802 mmol, 1.0 eq) and compound 16 (0.802 mmol, 1.0 eq) in dimethylformamide (20 mL) were added 7V,7V-diisopropylethylamine (2.406 mmol, 3.0 eq) and 2-(7-aza-lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (0.962 mmol, 1.2 eq) at 0 °C. Then the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. The reaction mixture was quenched with water, and filtered, and the filter cake was triturated with di chloromethane: methanol = 10: 1 to give compound 24 (475 mg, 58%) as gray solid. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on a silica gel (di chloromethane: methanol, 100:0-97:3-95:5-93:7) to give compound 24 (140 mg, 17%) as gray solid.
Preparation of compound 25
[0165] To a mixture of compound 24 (0.55 mmol, 1.0 eq) in dimethylformamide (10 mL) was added diethylamine (1.66 mmol, 3.0 eq) at room temperature. Then the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. The reaction mixture was filtered and concentrated under reduced pressure. The residue was triturated with ethyl acetate to give compound 25 (100%) as gray solid.
Preparation of compound 26
[0166] To a mixture of compound 25 (0.56 mmol, 1.0 eq) in dimethylformamide (10 mL) was added compound 9 (0.67 mmol, 1.2 eq) at room temperature. Then triethylamine (1.12 mmol, 2.0 eq) was added to the mixture. The mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography on a silica gel (dichloromethane: methanol, 92:8) to give compound 26 (72%) as white solid.
Preparation of TM-2
[0167] The mixture of compound 26 (0.21 mmol, 1.0 eq) in TFA/di chloromethane (6 mL) was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-2 (90 mg, 41%) as white solid.
LCMS: [1/2M+1] = 529
HNMR (400 MHz, CD3OD): 8 8.45-8.44 (d, J= 4.8 Hz, 1H), 8.01 (m, 1H), 7.85 (m, 2H), 7.68-7.64 (t, J= 7.6 Hz, 1H), 7.40 (m, 1H), 7.28 (m, 2H), 7.01 (m, 2H), 6.78 (s, 2H), 5.92 (s, 2H), 5.23-5.20 (d, J = 14.4 Hz, 1H), 4.38-4.34 (m, 4H), 4.05 (m, 1H), 3.98 (s, 2H), 3.75 (m, 4H), 3.52 (m, 5H), 3.45 (m, 2H), 3.27 (m, 1H), 2.98 (m, 2H), 2.44 (m, 4H), 2.34 (m, 2H), 2.01 (m, 4H), 1.85-1.81 (m, 4H), 1.43 (m, 6H), 1.25 (m, 1H), 1.18-1.15 (m, 1H), 0.94 (m, 3H), 0.81 (m, 3H) and 0.75 (m, 3H).
Example 4: Synthesis of TM-3
Figure imgf000038_0001
[0168] To a solution of compound TLR-1 (1.16 mmol, 1.0 eq) in dimethylformamide (5 mL) was added a mixture of compound 9 (1.16 mmol, 1.0 eq) and triethylamine (3.47 mmol, 3.0 eq) at room temperature. Then the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction mixture was quenched with water and extracted with ethyl acetate (3 x 5 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-3 (46%) as yellow solid.
LCMS: [M+l] = 670
HNMR (400 MHz, (CD3OD): 8 7.81-7.79 (m, 1H), 7.64 (m, 1H), 7.40 (m, 1H), 7.25 (m, 2H), 7.11 (m, 1H), 7.02-7.00 (m, 2H), 6.74 (s, 2H), 5.85 (s, 2H), 4.36-4.34 (m, 2H), 3.74-3.71 (m, 4H), 3.51 (m, 2H), 3.43 (m, 2H), 3.30 (m, 2H), 3.17 (m, 2H), 2.95-2.93 (m, 2H), 2.46- 2.39 (m, 4H), 1.80-1.74 (m, 2H), 1.46-1.40 (m, 2H) and 0.94-0.90 (m, 3H).
Example 5: Synthesis of TM-5
Figure imgf000038_0002
Preparation of compound 28
[0169] To a solution of TLR-1 (1.07 mmol, 1.0 eq) in dimethylformamide (10 mL) were added triethylamine (3.2 mmol, 3.0 eq) and compound 27 (1.07 mmol, 1.0 eq). The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure. The residue was suspended in ethyl acetate (6 mL) and stirred for 10 min. The suspension was filtered and the filter cake was dried in vacuum to give compound 28 (470 mg, 71%) as off white solid.
Preparation of compound 29
[0170] To a solution of compound 28 (470 mg, 0.76 mmol, 1.0 eq) in di chloromethane (15 mL) was added trifluoroacetic acid (5 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure to give compound 29 (crude) as colorless oil.
Preparation of compound 31
[0171] To a solution of compound 29 (0.76 mmol, 1.0 eq) in dimethylformamide (12 mL) were added triethylamine (2.28 mmol, 3.0 eq) and compound 30 (0.76 mmol, 1.0 eq). The reaction mixture was stirred at room temperature for 4 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure to give compound 31 (crude) as off white solid.
Preparation of TM-5
[0172] To a solution of compound 31 (0.76 mmol, 1.0 eq) in dichloromethane (15 mL) was added trifluoroacetic acid (4 mL). The reaction mixture was stirred at room temperature for 6 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-5 (32%) as white solid.
LCMS: [M+l] = 590, [M+Na] = 612
HNMR (400 MHz, CD3OD): 8 8.43 (m, 1H), 7.96-7.94 (m, 1H), 7.76 (m, 1H), 7.60 (m, 1H), 7.30 (m, 1H), 7.27 (m, 2H), 7.05-7.03 (m, 2H), 5.92 (s, 2H), 4.36-4.34 (m, 2H), 3.72 (m, 2H), 3.52 (m, 2H), 3.50 (m, 2H), 3.46-3.43 (m, 2H), 3.30-3.28 (m, 2H), 3.14 (m, 2H), 3.00 (m, 2H), 2.56 (m, 2H), 2.47-2.44 (m, 2H), 1.84-1.80 (m, 2H), 1.44-1.30 (m, 2H) and 0.95-0.92 (m, 3H).
Example 6: Synthesis of TM-6
Figure imgf000039_0001
[0173] To a solution of TLR-3 (0.45 mmol, 1.0 eq) in dimethylformamide (5 mL) was added compound 9 (0.45 mmol, 1.0 eq) and triethylamine (0.9 mmol, 2.0 eq) at room temperature. Then the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-6 (22%) as white solid.
LC-MS: [M+l] = 622, [M-l] = 620
HNMR (400 MHz, CD3OD): 3 8.23-8.21 (m, 1H), 7.75-7.69 (m, 2H), 7.64-7.60 (m, 1H), 6.76 (s, 2H), 4.65-4.60 (m, 2H), 3.61 (m, 4H), 3.45-3.40 (m, 4H), 3.30-3.28 (m, 2H), 3.25 (m, 2H), 3.20 (m, 2H), 3.03-2.99 (m, 2H), 2.41-2.34 (m, 4H), 1.93-1.91 (m, 4H), 1.72 (m, 2H),
1.54-1.51 (m, 2H) and 1.04-1.00 (m, 3H).
Example 7: Synthesis of TM-7
Figure imgf000040_0001
Preparation of Compound 33
[0174] To a solution of TLR-3 (0.964 mmol, 1.0 eq), compound 32 (1.01 mmol, 1.05 eq) and 2-(7-aza-lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (1.25 mmol, 1.3 eq) in dimethylformamide (10 mL) was added A,7V-diisopropylethylamine (2.41 mmol, 2.5 eq). The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. Then the mixture was diluted with water (20 ml) and extracted with ethyl acetate (10 mL x 2). The organic layers were washed with brine (10 mL x 2). The ethyl acetate phase was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on a silica gel (methanol :dichloromethane, 0~3%) to give compound 33 (crude) as colorless oil.
Preparation of Compound 34
[0175] A solution of compound 33 (0.964 mmol, 1.0 eq) in dichloromethane/trifluoroacetic acid (4:1, 5mL) was stirred at room temperature for 20 min. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure to compound 34 (crude), which was used directly in the next step without further purification. Preparation of TM- 7
[0176] To a solution of compound 9 (0.616 mmol, 1.0 eq) and compound 34 (0.492 mmol, 0.8 eq) in dimethylformamide (5 mL) was added triethylamine (3.08 mmol, 5.0 eq). Then the mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction mixture was concentrated in vacuum. The residue was purified by prep- HPLC to give TM-7 (17%) as white solid.
LC-MS: [M+l] = 762
HNMR (400 MHz, DMSO-</d): 8 8.64 (s, 2H), 8.21-8.20 (m, 1H), 7.90 (m, 1H), 7.84 (m, 2H), 7.74-7.72 (m, 2H), 7.61-7.58 (m, 1H), 6.94 (s, 2H), 4.62-4.60 (m, 2H), 3.66 (m, 2H), 3.63 (m, 2H), 3.52 (s, 4H), 3.42-3.40 (m, 2H), 3.22 (m, 12H), 3.01-2.98 (m, 4H), 2.60 (m, 2H), 2.51 (s, 2H), 2.39 (m, 2H), 1.86-1.85 (m, 4H), 1.53 (m, 2H), 1.49 (m, 2H) and 1.01-0.98 (m, 3H).
Example 8: Synthesis of TM-8
Figure imgf000041_0001
TM-8
Preparation of compound 36
[0177] To a solution of compound 35 (1.92 mmol, 1.0 eq), compound 34 (2.11 mmol, 1.05 eq) and 7V,7V-diisopropylethylamine (6.00 mmol, 3.0 eq) in dimethylformamide (35 mL) was added 2-(7-aza-lH-benzotriazole-l-yl)-l, 1,3,3-tetramethyluronium hexafluorophosphate (2.60 mmol, 1.3 eq) at room temperature. The reaction mixture was stirred at room temperature for 1 h under nitrogen atmosphere. The reaction was monitored by TLC. Then the mixture was extracted with ethyl acetate (50 mL x 2). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give compound 36 (960 mg, crude) as yellow oil.
[0178] TLC: dichloromethane:methanol = 10: 1, UV 254 nm. Rf (compound 35) = 0.6, Rf (compound 34) = 0.5. Preparation of compound TM-8
[0179] To a solution of compound 36 (1.06 mmol, 1.0 eq) in dimethylformamide (10 mL) was added diethylamine (3 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. Then the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-8 (48%) as white solid. LCMS: [M+l] = 682; [1/2M+1] = 341.8
HNMR (400 MHz, CD3OD): 8 8.22 (d, J= 8.2 Hz, 1H), 7.79 (dd, J= 8.4, 1.2 Hz, 1H), 7.75- 7.69 (m, 1H), 7.62 (dd, J= 8.4, 7.2, 1.2 Hz, 1H), 4.64 (t, J= 7.6 Hz, 2H), 3.76 (t, J= 6.0 Hz, 2H), 3.59 (d, J= 4.0 Hz, 4H), 3.53 (t, J= 5.6 Hz, 2H), 3.46 - 3.31 (m, 8H), 3.26 (dd, J= 14.0, 6.8 Hz, 6H), 3.17 (t, J= 6.4 Hz, 2H), 3.02 (t, J = 7.6 Hz, 2H), 2.70 (dd, J= 9.6, 4.4 Hz, 4H), 2.60 (t, J= 6.4 Hz, 2H), 2.01-1.87 (m, 4H), 1.76-1.63 (m, 2H), 1.54 (dd, J= 15.2, 7.6 Hz, 2H) and 1.03 (t, J = 7.6 Hz, 3H).
Example 9: Synthesis of TM-9
Figure imgf000042_0001
Preparation of compound 37 [0180] To a solution of TLR-1 (3.90 mmol, 1.0 eq) in dimethylformamide (15 mL) were added compound 32 (4.09 mmol, 1.05 eq), 2-(7-aza-lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (5.07 mmol, 1.3 eq) and N,N- diisopropylethylamine (9.75 mmol, 2.5 eq). The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS and TLC. Then the mixture was concentrated under reduced pressure. The residue was suspended in ethyl acetate (10 mL) and stirred for 10 min. The precipitated solid was filtered and purified by column chromatography on a silica gel (di chloromethane: methanol, 93:7) to give compound 37 1.7 g, 73%).
Preparation of compound 38
[0181] To a solution of compound 37 (2.84 mmol, 1.0 eq) in dichloromethane (20 mL) was added trifluoroacetic acid (34 mL). The reaction mixture was stirred at room temperature for 0.5 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure to give compound 38 (crude).
Preparation of TM-9
[0182] To a solution of compound 38 (0.167 mmol, 1.0 eq) in dimethylformamide (5 mL) were added compound 39 (0.134 mmol, 0.8 eq) and triethylamine (0.835 mmol, 5.0 eq). The reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS and TLC. Then the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-9 (123 mg, 69%) as colorless gum.
LCMS: [M/2+1] = 538
HNMR (400 MHz, CD3OD) 8 7.96 (t, J = 4.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 6.80 (s, 2H), 5.93 (s, 2H), 4.36 (s, 2H), 3.78 - 3.69 (m, 4H), 3.64 - 3.54 (m, 38H), 3.50 - 3.41 (m, 4H), 3.26 (d, J = 5.6 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H), 2.43 (t, J = 6.8 Hz, 2H), 1.91 - 1.79 (m, 2H), 1.46 (dd, J = 15.2, 7.6 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H).
Example 10: Synthesis of TM-10
Figure imgf000043_0001
Preparation of compound 41
[0183] To a solution of compound 38 (1.1 mmol, 1.0 eq) in dimethylformamide (10 mL) were added triethylamine (5.5 mmol, 5.0 eq) and compound 40 (0.99 mmol, 0.9 eq). The reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure to give compound 41 (crude).
Preparation of TM-10
[0184] To a solution of compound 41 (1.08 mmol, 1.0 eq) in dichloromethane (10 mL) was added trifluoroacetic acid (3 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Then the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give TM-10 (48%) as colorless gum.
LCMS: [M/2+1] = 462
HNMR (400 MHz, CD3OD) 8 7.95 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 7.6 Hz, 2H), 5.93 (s, 2H), 4.35 (s, 2H), 3.77 - 3.70 (m, 4H), 3.68 (d, J = 5.2 Hz, 4H), 3.44 (t, J = 6.8 Hz, 32H), 3.15 (d, J = 4.8 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H), 2.74 (dd, J = 21.0, 14.0 Hz, 4H), 1.84 (t, J = 7.6 Hz, 2H), 1.45 (dd, J = 15.2, 7.6 Hz, 2H), 0.93 (t, J = 7.6 Hz, 3H).
[0185] The monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
[0186] In the following examples, the monoclonal antibodies (mAb), DP001 and mDPOOl were purchased from Biointron Biological Inc by contract. DP001 has the same amino acid sequence as trastuzumab; and mDPOOl is DP001 in which some amino acids replaced with mouse IgG2a.
[0187] DP001 and mDPOOl were made by recombinant DNA methods, their amino acid sequences are showed as below:
Figure imgf000044_0001
Figure imgf000045_0001
Example 11: General procedure for the preparation of antibody conjugates through cysteine coupling
Figure imgf000046_0001
TLR agonist
Figure imgf000046_0002
wherein m and n are integers, and m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7), n = 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-4, or 1-2, e.g., 1, 2, 3 or 4; or 1 or 2.
[0188] The mAh was exchanged into PBS (10 mM PB (disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 1 : 1), pH 6.0, 5 mM EDTA (disodium edetate), 137mM NaCl) via molecular weight cut-off centrifugal filtration (Millipore, 30 kDa). The resultant mAh solution was transferred to a 50 mL conical tube. The mAb concentration was determined to be 10 mg/mL by A280 (280nm ultraviolet absorption). To the ImL mAb solution was added TCEP (1.1 eq-1.2eq, 7.48 pL-8.16 pL, 10 mM stock) at room temperature and the resultant mixture was incubated at 37 °C for 1 hr, with gentle shaking. Upon being cooled to room temperature, with stirring, the corresponding TM compound (i.e. the payload-linker compound) (3.0 eq, 20.4 pL,10 mM dissolved in DMSO) was added dropwise and the resultant reaction mixture was allowed to be stirred at ambient temperature for 30 minutes, at which point L-NAC (10.0 eq, 6.8 pL,100 mM) was added. After an additional 10-15 minutes of stirring, the crude conjugate was then purified by Protein A chromatography (Mab selectsure) using 5 mM citric acid and 50 mM sodium citrate solution.
Example 12: General procedure for the preparation of antibody conjugates through Transglutaminase (mTgase) catalyzed site-specific coupling
Figure imgf000047_0001
wherein m and n are integers, and m = 1-7 (e.g. 1, 2, 3, 4, 5, 6, or 7), n = 1-20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), wherein d is a number, and d = 1-2, e.g., 1 or 2.
[0189] The mAh was exchanged into Tris-HCl (25 mM, pH 7.5) via molecular weight cutoff centrifugal filtration (Millipore, 30 kDa). The resultant mAh solution was transferred to a 50 mL conical tube. The mAb concentration was determined to be 30 mg/mL by A280. To the 333.3 pL mAb solution was added lOOuL reaction buffer (500 mM Tris-OAc, 20 mM EDTA, 0.2% Tween-20), 100 pL mTgase (15 mg/mL, SEQ ID NO: 13, wild type), 26 pL the corresponding TM compound (i.e. the payload-linker compound) (50 eq, 92mg/mL dissolved in DMSO) and 441 pL purified water, at room temperature and the resultant mixture was incubated at 30°C for no more than 120 hr, until modification rate >95%, with gentle shaking. At which point, the crude conjugate was then purified by Protein A chromatography (MabSelect Sure) using 5 mM citric acid and 50 mM sodium citrate solution.
Example 13: General procedure for the determination of the drug-antibody-ratios by hydrophobic interaction chromatography (HIC)
[0190] Sample (20 pg) was directly injected onto a TSKgel Butyl-NPR analytical column (Tosoh, 4.6 mm* 100 mm) at 30°C. Solvent A consisted of 1.5 M ammonium sulfate, 25 mM potassium phosphate, pH 7.0 and solvent B consisted of 25mM potassium phosphate (pH 7.0) and 25% isopropanol. The analytes were separated with a gradient from 0 % to 50 % solvent B from 0 to 20 minutes. The column was maintained at 30°C with a flow rate of 0.8 mL/min.
The elution profile was monitored at 214 nm.
Figure imgf000047_0002
Figure imgf000048_0002
Example 14: General procedure for the determination of the purity by SEC-HPLC
[0191] Sample (30 pg) was directly injected onto a XBridge®BEH200A analytical column (Waters, 7.8*300mm, 3.5pm) at ambient temperature. A mobile phase consisting of 100 mM potassium phosphate with 100 mM sodium chloride, pH 6.7, and 10% isopropanol, was used. The flow rate was 0.8 mL/min, run for 20 minutes. The elution profile was monitored at 280 nm.
Example 15: Analytical data for mAh (Trastuzumab biosimilar DP001)-TLR conjugates
[0192] Fig. 2 shows the HIC, SEC and MS spectra of DP001-TM1.
Figure imgf000048_0001
[0196] Fig. 6 shows the HIC, SEC and MS spectra of DP001-TM6.
Figure imgf000049_0001
It should be noted that the above-mentioned formulae in Example 15 do not show the actual numbers of linker-drug molecules conjugated to the antibody or antigen-binding fragment thereof, the DAR value can be seen in the figures. Confirmation of Conjugation on Heavy Chain Only.
[0202] Confirmation of Site-Specific Conjugation at Q295 (Sequential Q295 was appointed by EU numbering system). [0203] To confirm that drug molecule was conjugated to Q295 of IgGl’s heavy chain (HC) but not light chain (LC), Purified DAR2 from DP001-TM5, DP001-TM8 and DP001-TM10 and naked IgGl were deglycosylated, reduced, alkylated, digested into peptides using trypsin and/or chymotrypsin (Promega, Madison, Wis.) and separated by reversed phase chromatography (Cl 8) prior to mass spectrometry analysis. Digested peptides were monitored on HPLC by UV absorbance at 214 nm (/.max of TM5, TM8 and TM10). Only one peak at 214 nm was identified in DAR2 samples, whereas no peak was detected in control antibody. MALDI-TOF analysis identified that peak as a single charged peptide EEQYNSTYR from trypsin digestion or NAKTKPREEQY from chymotrypsin digestion containing Glutamine 295 with exactly one TM5, TM8 or TM10.
To exclude other Glutamines (other than Q295) as additional conjugation sites, full peptide mapping experiments were performed using unmodified IgGl and DP001-TM5, DP001-TM8, DP001-TM10. The digested samples were directly analyzed without purification to identify all glutamine-containing peptides on heavy chain. Out of all 16 glutamines, Q295 was the only conjugation site with TM5, TM8 and TM10, while all other glutamine containing peptides remain unchanged.
Example 16: TLR agonist activity
[0204] Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB)-induced secreted embryonic alkaline phosphatase (SEAP) activity of HEK-Blue hTLR7 or hTLR8 reporter cells 6-8 h after incubation with TLR 7/8 agonists and analyzed by fluorescence. Data shown are from three (hTLR8 or hTLR7) independent experiments.
[0205] Fig. 12: hTLR 7 activation: R848 (a known TLR7/8 Agonist); TM3, TM5, TM9, TM10
[0206] Fig. 13: hTLR 7 activation: TM6, TM7, TM8
[0207] Fig. 14: hTLR 8 activation: R848; TM5
Example 17: Activation of TLR7 and TLR8 signal transduction pathway of the antibody conjugates.
[0208] Calu3 cell lines and PBMCs (peripheral blood mononuclear cells) co-cultures were stimulated with antibody conjugates or antibody for 18 h and analyzed by ELISA for TNF-a production. Data shown are from n= 3 individual donors and are representative of three independent experiments
[0209] Fig. 15 shows in vitro human myeloid cell activation by DP001-TM1, DP001-TM3, DP001-TM5, DP001-TM6, DP001-TM7.
Example 18: In vivo antitumor activity [0210] Calu-3 tumor cells were implanted into NU/NU mice subcutaneously for the xenograft studies.
[0211] Tumor size was recorded twice a week. Once tumors reached 80- 120mm3, treatments were initiated. Mice were randomized by tumor size into treatment groups before initial treatment. Her2 antibody and its TLR 7/8 agonist conjugates were administered at 3-6 mg/kg at qw *4 schedule or 5-10 mg/kg at qw x 2 schedule.
[0212] As shown in Fig. 16 and Fig. 17, antibody-TLR7/8 agonist conjugates elicited antitumor immunity in Calu-3 tumor xenograft model. The Tumor growth inhibition rate of antibody-TLR7/8 agonist conjugates were show in Table 1.
[0213] HCC1954 tumor cells were implanted into NU/NU mice subcutaneously for the xenograft studies.
[0214] Tumor size was recorded twice a week. Once tumors reached 80- 120mm3, treatments were initiated. Mice were randomized by tumor size into treatment groups before initial treatment. Her2 antibody and its TLR 7/8 agonist conjugates were administered at 5-10 mg/kg at q2w*2 schedule.
[0215] As shown in Fig. 18, antibody-TLR7/8 agonist conjugates elicited antitumor immunity in HCC1954 tumor xenograft model.
[0216] The Tumor growth inhibition rate of antibody-TLR7/8 agonist conjugates were shown in Table 2.
Example 19: Stability of the conjugates in buffer, human, or mouse plasma
[0217] mAb conjugates were incubated in buffer, human and mouse plasma at 37 °C for 120 hours. The test samples were analyzed by LC-MS to measure released payload.
[0218] Fig. 19 shows stability of the non-cleavable antibody conjugate of DP001-TM5 in buffer, human, or mouse plasma.
Table 1. The Tumor growth inhibition rate of antibody-TLR7/8 agonist conjugates
Figure imgf000052_0001
Table 2. The Tumor growth inhibition rate of antibody-TLR7/8 agonist conjugates
Figure imgf000053_0001
MODIFICATIONS
[0219] Modifications and variations of the described methods and compositions of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure are intended and understood by those skilled in the relevant field in which this disclosure resides to be within the scope of the disclosure as represented by the following claims.
INCORPORATION BY REFERENCE
[0220] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
REFERENCES:
1. Fridman, W. H., Pages, F., Sautes-Fridman, C. & Galon, J. The immune contexture in human tumours: impact on clinical outcome. Nat. Rev. Cancer 12, 298-306 (2012).
2. Galon, J. & Bruni, D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat. Rev. Drug Discov. 18, 197-218 (2019).
3. Joyce, J. A. & Fearon, D. T. T cell exclusion, immune privilege, and the tumor microenvironment. Science 348, 74-80 (2015).
4. Urban-Wojciuk, Z. et al. The role of TLRs in anti-cancer immunity and tumor rejection. Front. Immunol. 10, 2388 (2019).
5. Carmi, Y. et al. Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity. Nature 521, 99-104 (2015).
6. Sagiv-Barfi, I. et al. Eradication of spontaneous malignancy by local immunotherapy. Sci. Transl. Med. 10, eaan4488 (2018).
7. Shelley E. Ackerman, et al. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature Cancer 2, 18-33 (2021).
8. Michael Nathaniel Alonso et al US 10,675,358 B2, 9/2020
9. Doris Stoermer et al, US 8,951, 528 B2, 2/2015
10. Kyle J. Lindstrom et al. US 8,871,782 B2
11. Moon Sung Ju et al. W02020/168017 Al 12. Alex Cortez et al. US2017/0121421 Al
13. Steward D. et al. W02019/040491 Al
14. Valerie Odegard et al. US2020/0113912 Al, 4/2020
15. David Craig MeGowan et al. US9,499,549 B2, 11/2016 16. Sean Hu et al. US2017/0106096 Al, 4/2017
17. Robert L.Coffman et al. US2019/0151462 Al, 05/2019
Sequence Listing Information :
DTD Version : Vl_3
File Name : CSPC Dophen . xml
Software Name : WIPO Sequence
Software Version : 2.1. 2
Production Date : 2022-09-15
General Information :
Current application / IP Office : US
Current application / Applicant file reference : FPCH22160258P
Earliest priority application / IP Office : US
Earliest priority application / Application number : US63/245, 331
Earliest priority application / Filing date : 2021-09-17
Applicant name : CSPC Dophen Corporation
Applicant name / Language : en
Invention title : Antibody Immune- stimulant Conj ugates and Use Thereof ( en )
Sequence Total Quantity : 14
Sequences :
Sequence Number ( ID) : 1
Length : 450
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . .450
> mol_type, protein
> organism, unidentified
Residues :
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY 60
ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS 120
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180
GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 240
PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300
STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE 360
MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450
Sequence Number ( ID) : 2
Length : 214
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 214
> mol_type, protein
> organism, unidentified
Residues :
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASF LYSGVPS 60
RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFI FPP 120
SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180
LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214
Sequence Number ( ID) : 3 Length : 450 Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . .450
> mol_type, protein
> organism, unidentified
Residues :
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY 60
ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS 120
AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT WNSGSLSSGV HTFPAVLQSD 180
LYTLSSSVTV TSSTWPSQSI TCNVAHPASS TKVDKKI EPR GPTIKPCPPC KCPAPNLLGG 240
PSVFIFPPKI KDVLMISLSP IVTCVWDVS EDDPDVQISW FVNNVEVHTA QTQTHREDYN 300
STLRWSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE 360
MTKKQVTLTC MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW 420
VERNSYSCSV VHEGLHNHHT TKSFSRTPGK 450
Sequence Number ( ID) : 4
Length : 214
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 214
> mol_type, protein
> organism, unidentified
Residues :
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASF LYSGVPS 60
RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRAD AAPTVSI FPP 120
SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL NSWTDQDSKD STYSMSSTLT 180
LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC 214
Sequence Number ( ID) : 5
Length : 8
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 8
> mol_type, protein
> organism, unidentified
Residues :
GFNIKDTY 8
Sequence Number ( ID) : 6
Length : 8
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 8
> mol_type, protein
> organism, unidentified
Residues :
IYPTNGYT 8
Sequence Number ( ID) : 7 Length : 13 Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 13
> mol_type, protein
> organism, unidentified Residues :
SRWGGDGFYA MDY 13
Sequence Number (ID) : 8
Length : 6
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 6
> mol_type, protein
> organism, unidentified Residues :
QDVNTA
Sequence Number (ID) : 9 Residues : 000
Sequence Number (ID) : 10
Length : 9
Molecule Type : AA
Features Location/Qualifiers :
- source, 1. .9
> mol_type, protein
> organism, unidentified
Residues :
QQHYTTPPT 9
Sequence Number (ID) : 11
Length : 120
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 120
> mol_type, protein
> organism, unidentified
Residues :
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY 60
ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS 120
Sequence Number (ID) : 12
Length : 107
Molecule Type : AA
Features Location/Qualifiers :
- source, 1 . . 107
> mol_type, protein > organism, unidentified
Residues :
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60
RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIK 107
Sequence Number (ID): 13 Length: 331
Molecule Type: AA
Features Location/Qualifiers :
- source, 1. .331
> mol_type, protein
> organism unidentified
Residues :
DSDERVTPPA EPLDRMPDPY RPSYGRAETI VNNYIRKWQQ VYSHRDGRKQ QMTEEQREWL 60 SYGCVGVTWV NSGQYPTNRL AFAFFDEDKY KNELKNGRPR SGETRAEFEG RVAKDSFDEA 120 KGFQRARDVA SVMNKALENA HDEGAYLDNL KKELANGNDA LRNEDARSPF YSALRNTPSF 180 KDRNGGNHDP SKMKAVIYSK HFWSGQDRSG SSDKRKYGDP EAFRPDRGTG LVDMSRDRNI 240
PRSPTSPGES FVNFDYGWFG AQTEADADKT VWTHGNHYHA PNGSLGAMHV YESKFRNWSD 300 GYSDFDRGAY WTFVPKSWN TAPDKVTQGW P 331
Sequence Number (ID): 14 Length: 331
Molecule Type: AA
Features Location/Qualifiers:
- source, 1. .331
> mol_type, protein
> organism unidentified
Residues :
DSDDRVTPPA EPLDRMPDPY RPSYGRAETV VNNYIRKWQQ VYSHRDGRKQ QMTEEQREWL 60 SYGCVGVTWV NSGQYPTNRL AFASFDEDRF KNELKNGRPR SGETRAEFEG RVAKESFDEE 120 KGFQRAREVA SVMNRALENA HDESAYLDNL KKELANGNDA LRNEDARSPF YSALRNTPSF 180 KERNGGNHDP SRMKAVIYSK HFWSGQDRSS SADKRKYGDP DAFRPAPGTG LVDMSRDRNI 240
PRSPTSPGEG FVNFDYGWFG AQTEADADKT VWTHGNHYHA PNGSLGAMHV YESKFRNWSE 300 GYSDFDRGAY VITFIPKSWN TAPDKVKQGW P 331
END

Claims

1. A compound having Formula A3 or Formula A4, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000060_0001
Formula A4 wherein m is an integer from 1 to 7 and n is an integer from 1 to 20.
2. The compound of claim 1, wherein X is -NH2.
3. The compound of claim 1, wherein the compound is a compound having Formula A-IV:
Figure imgf000060_0002
Formula A-IV wherein m is an integer from 1 to 7 and n is an integer from 1 to 20, especially m is an integer from 1 to 7 and n is an integer from 1 to 8.
4. The compound of claim 1, wherein the compound is a compound having Formula A- VIII:
Figure imgf000060_0003
Formula A- VIII wherein m is an integer from 1 to 7 and n is an integer from 1 to 20, especially m is an integer from 1 to 7 and n is an integer from 1 to 8.
5. The compound of claim 1, wherein the compound is a compound having Formula A-X:
Figure imgf000061_0001
Formula A-X wherein m is an integer from 1 to 7 and n is an integer from 1 to 20, especially m is an integer from 1 to 7 and n is an integer from 1 to 8.
6. An antibody conjugate, comprising the compound or a pharmaceutically acceptable salt or an isotopic derivative thereof according to any one of claims 1-5 conjugated to an antibody or an antigen-binding fragment thereof.
7. The antibody conjugate according to claim 6, wherein each antibody or fragment thereof attaches to 1-4, especially 1-2, compounds conjugated thereon.
8. The antibody conjugate according to any of claims 6-7, wherein the compound is conjugated via site specific microbial transglutaminase (mTGase) catalyzed conjugation.
9. The antibody conjugate according to any of claims 6-8, wherein the antibody is selected from anti-HER2 antibody, anti-CEACAM5 antibody, anti-CAIX antibody, anti-Nectin-4 antibody, anti-PD-Ll antibody, and/or anti-Claudin 18.2 antibody.
10. The antibody conjugate according to any of claims 6-9, comprising the compound of Formula A3 or Formula A4, or a pharmaceutically acceptable salt or an isotopic derivative thereof and having a structure of Formula 3 or Formula 4 or Formula 5 or Formula 6, or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000061_0002
61
Formula 3 wherein m = 1-7, n = 1-20, d = 1-4;
Figure imgf000062_0001
Formula 5 wherein n = 1-20, d = 1-4;
Figure imgf000062_0002
Formula 6 wherein n = 1-20, d = 1-2.
11. The antibody conjugate of claim 10, comprising the compound of Formula A-IV or a pharmaceutically acceptable salt or an isotopic derivative thereof and having the structure of Formula 4-1 or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000062_0003
Formula 4-1 wherein m=l-7, n=l-20, d=l-2.
12. The antibody conjugate of claim 10, comprising the compound of Formula A- VIII or a pharmaceutically acceptable salt or an isotopic derivative thereof and having the structure of Formula 4-4 or a pharmaceutically acceptable salt or an isotopic derivative thereof:
Figure imgf000063_0001
Formula 4-4 wherein m = 1-7 and n = 1-20, d = 1-2.
13. The antibody conjugate of claim 10, comprising the compound or salt of Formula A-X and having the structure of Formula 6-4:
Figure imgf000063_0002
Formula 6-4 wherein n = 1-20, d = 1-2.
14. The antibody conjugate according to any of claims 6-13, wherein the antibody is anti- HER2 antibody or antigen-binding fragment thereof, the heavy chain of which comprises:
(a) a CDRH1 comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 5);
(b) a CDRH2 comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 6);
(c) a CDRH3 comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 7); and the light chain of which contains:
(a) a CDRL1 comprising the amino acid sequence of QDVNTA (SEQ ID NO: 8);
(b) a CDRL2 comprising the amino acid sequence of SAS (SEQ ID NO: 9);
(c) a CDRL3 comprising the amino acid sequence of QQHYTTPPT (SEQ ID NO: 10); wherein the complementarity-determining regions (CDRs) are defined by using the IMGT numbering system.
15. The antibody conjugate according to any of claims 6-14, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12.
16. The antibody conjugate according to any of claims 6-15, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain comprising the amino acid sequence of SEQ ID NO: 2.
17. The antibody conjugate according to any of claims 6-15, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 4.
18. The antibody conjugate according to any of claims 6-17, wherein the compound or a pharmaceutically acceptable salt or an isotopic derivative thereof according to any one of claims 1-5 is conjugated to Q295 of the antibody, via site specific microbial transglutaminase (mTGase) catalyzed conjugation, and the N297 of the antibody is glycosylated.
19. An antibody conjugate, which is selected from the group consisting of:
Figure imgf000064_0003
, , , d=2 or about 2.0;
Figure imgf000064_0001
, wherein n=7, d=2 or about
2.0; wherein the antibody represented
Figure imgf000064_0002
comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain comprising the amino acid sequence of SEQ ID NO: 2; preferably, the conjugation occurs at the Q295s of the antibody, preferably via site specific microbial transglutaminase (mTGase) catalyzed conjugation; and the N297s of the antibody are glycosylated.
20. A pharmaceutical composition, comprising the antibody conjugate of any one of claims 6-19, and a pharmaceutically acceptable excipient.
21. The antibody conjugate of any one of claims 6-19, for use in the treatment of cancer.
22. A method for treating cancer, comprising administering a therapeutically effective amount of the antibody conjugate of any one of claims 6-19 or the pharmaceutical composition of claim 20 to a subject in need thereof.
23. Use of the antibody conjugate of any one of claims 6-19 or the pharmaceutical composition of claim 20 in the manufacture of a medicament for treating cancer.
24. The antibody conjugate of claim 21, the method of claim 22, or the use of claim 23, wherein said cancer is selected from solid cancers, lymphomas, and leukemias, including, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer (e.g., melanoma), choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, and multiple myeloma.
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