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WO2023044016A2 - Conjugués d'anticorps immunostimulants et leur utilisation - Google Patents

Conjugués d'anticorps immunostimulants et leur utilisation 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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WO2023044016A3 (fr
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

La présente invention concerne, dans certains modes de réalisation, des conjugués d'anticorps immunostimulants pour l'immunothérapie anticancéreuse. L'invention concerne également des procédés de fabrication et d'utilisation desdits conjugués d'anticorps immunostimulants.
PCT/US2022/043820 2021-09-17 2022-09-16 Conjugués d'anticorps immunostimulants et leur utilisation Ceased WO2023044016A2 (fr)

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