WO2024050031A2 - Novel camptothecin derivatves as antibody-drug conjugates (adc) payloads - Google Patents

Novel camptothecin derivatves as antibody-drug conjugates (adc) payloads Download PDF

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WO2024050031A2
WO2024050031A2 PCT/US2023/031737 US2023031737W WO2024050031A2 WO 2024050031 A2 WO2024050031 A2 WO 2024050031A2 US 2023031737 W US2023031737 W US 2023031737W WO 2024050031 A2 WO2024050031 A2 WO 2024050031A2
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antibody
cancer
adc
gly
antibodies
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PCT/US2023/031737
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French (fr)
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WO2024050031A3 (en
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Dinnguo LIU
Haifeng Bao
Yuan WEI
Haihong Zhong
Shen LIU
Lijin CAO
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Bionecure Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention describes novel camptothecin derivatives and methods for preparing these derivative payloads bearing a linker with a functional group that can be used for conjugation to cell binding agents to generate cytotoxic drug conjugates.
  • the present invention further relates to therapeutic use of these conjugates for treatment of cancer as the conjugates are targeted and selectively delivered to a specific tumor cell population.
  • the present invention further relates to antibody drug conjugates made with camptothecin derivative payloads bearing a linker moiety conjugated to tumor associated antigen (TAA) binding agents, preparation methods, pharmaceutical compositions and uses thereof for the treatment of cancer.
  • TAA tumor associated antigen
  • Cancer immunotherapy is enjoying a renaissance, and in the past few years the rapidly advancing field has produced several new methods of treating cancer.
  • Numerous cancer immunotherapy strategies have been the focus of extensive research and clinical evaluation including, but not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates: treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints); treatment using bispecific T cell engaging antibodies (BITE®) such as blinatumomab; treatment involving administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21 , GM-CSF IFN- a, IFN-p and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating
  • ADCs Antibody-drug conjugates combine the binding specificity of an antibody with the potency of drugs such as, for example, cytotoxic agents, anticancer and immunosuppressive drugs.
  • drugs such as, for example, cytotoxic agents, anticancer and immunosuppressive drugs.
  • the use of ADCs allows the target-specific delivery of drugs which, if administered as unconjugated drugs, may result in unacceptable levels of toxicity to normal cells.
  • the mechanism of an ADC is to recognize and bind to specific antigen through the antibodies, trigger a series of reactions, and then enter the cytoplasm through the endocytosis, where the highly cytotoxic drug is dissociated from the antibody after the degradation by lysosomal enzymes to kill cancer cells.
  • targeting drug delivery can make the drug act on cancer cells directly and reduce the damage to normal cells.
  • cytotoxic compounds used in antibody-drug conjugates inhibit various essential cellular targets, such as microtubules (maytansinoids, auristatins, taxanes: U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333.410; 6,441 ,163; 6,340,701 : 6,372,738; 6,436,931; 6,596,757: 7.276,497; 7,301,019; 7,303,749; 7,368,565; 7,473,796; 7,585,857; 7,598,290: 7.495,114;
  • microtubules maytansinoids, auristatins, taxanes: U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333.410; 6,441 ,163; 6,340,701 : 6,372,738; 6,436,931; 6,596,757: 7.276,497; 7,301,019; 7,303,749; 7,36
  • Camptothecin (Figure 1) was originally isolated from the bark of Camptotheca acuminata, a tree native to the rocky slopes of north China isolated by Wall et al. [Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J Am Chem Soc 1966; 88: 3888-3890], CPT has been demonstrated to be effective against a broad spectrum of tumors.
  • CPT has attracted much attention because of a very good spectrum of its antitumor activity against experimental animal tumor models, such as L1210 leukemia in mice and Walker 256 sarcoma in rats.
  • experimental animal tumor models such as L1210 leukemia in mice and Walker 256 sarcoma in rats.
  • its clinical development failed due to the reversible bone marrow depression and hemorrhagic cystitis, which are the major dose-limiting toxicity
  • CPT-11 is a water-soluble pro-drug and undergoes carboxylesterase-mediated hydrolysis to form SN-38, a potent Topo I inhibitor (Fig. 1) [Bencharit S, Morton CL, Howard-Williams EL, Danks MK, Potter PM, Redinbo MR. Structural insights into CPT-11 activation by mammalian carboxyesterases.
  • camptothecin derivatives for use as ADC payloads for use in the treatment or to prevent recurrence of cancers and/or immunological disorders.
  • the present invention describes novel camptothecin derivatives and methods for preparing payloads derived from them with linkers containing functional groups that can be used for conjugation to cell binding agents to generate cytotoxic drug conjugates.
  • the present invention relates to an antibody drug conjugate (ADC) comprising an antibody (e.g., a cell binding antibody) chemically linked to a camptothecin analog residue represented by the following formula (I):
  • ADC antibody drug conjugate
  • Ab is an antibody (e.g., a cell binding antibody) or antigen binding fragment thereof;
  • camptothecin derivative drug moieties (D) have the structure:
  • Ri is a hydrogen atom or a C1-C6 alkyl
  • L is a bivalent linker comprising a peptide moiety of 2-4 amino acid represented by the following formula (II):
  • L 1 is a sulfhydryl reactive linker attached to the antibody
  • L 2 is peptide moiety
  • L 3 is a self-immolative moiety connected to the drug moiety that is: [021] and wherein R 2 is a hydrogen atom or a C1-C6 alkyl, n 1 is 1, 2, 3, 4 or 5, and n 2 is 2, 3, 4 or 5.
  • Another aspect of the invention is a pharmaceutical composition including a Formula I ADC compound, a mixture of Formula I ADC compounds, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient
  • Another aspect provides a pharmaceutical combination comprising a Formula I ADC compound and a second compound having anticancer properties or other therapeutic effects.
  • Another aspect is a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating the cells with an amount of an antibody-drug conjugate of Formula I, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.
  • Another aspect is a method of treating cancer comprising administering to apatient a therapeutically effective amount of a pharmaceutical composition including a Formula I ADC.
  • the invention relates to antibody drug conjugates wherein x is about 1 to about 8. In various embodiments, x is about 4 to about 7. In various embodiments, x is about 4. in various embodiments, x is about 6. In various embodiments, x is about 7.
  • the invention relates to derivatized camptothecin analogs wherein L 1 comprises pyrroline-dione.
  • the heterocyclyl ring is selected from saturated or unsaturated 4-6 membered nitrogen containing heterocyclic rings.
  • saturated heterocyclic radicals include saturated 3 to 6- membered heteromonocylic group containing 1 to 4 nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidine, piperazinyl]; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.
  • heterocyclic radicals also termed “heteroaryl” radicals, indude unsaturated 5 to 6 membered heteromonocycly! group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4- pyridyl.
  • the cyclic alkyl ring also know as a cycloalkyl ring, is a saturated cyclic alkyl group derived by the removal of one hydrogen atom from a single carbon atom of a parent cycloalkane.
  • Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane, and the like.
  • the present invention provides an anti-Her2 antibody (Herceptin) that is conjugated with camptothecin analogs, thus targeting disease cells or tissues.
  • the anti-Her2 antibody binds to an antigen in the disease cells or tissues.
  • a drug conjugated to the antibody exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen-expressing cells to treat or prevent recurrence of Her2 positive cancers.
  • the high affinity of the antibody drug conjugate ensures that the camptothecin analogs targets the tumor cells.
  • the present technology provides a method to treat cancers by exerting cellular inhibitory or killing effect of camptothecin analogs on the Her2 positive cells.
  • the ADC’s comprise an L that is a cleavable linker.
  • the ADC is an anti-Her2 antibody (Herceptin) conjugated with camptothecin analogs.
  • the ADC is an anti-Her2 antibody conjugated with a camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that is peptidase cathepsin sensitive.
  • the ADC is an anti-Her2 antibody conjugated with camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that is not acid labile.
  • the ADC is an anti-Her2 antibody conjugated with camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that does not contain a disulfide bond.
  • the ADC is an anti-Her2 antibody conjugated with camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that provides stability during circulation while being able to release the drug once inside the cells.
  • linkers are contemplated to provide stability to the conjugated molecule prior to endocytosis, such as during circulation, to prevent premature degradation of the linker and release of the toxic drug, thus minimize the toxic effect of the drug.
  • the number of bonds formed between the drug-linker and cysteine residue on the anti-Her2 antibody is from 3 to 8. In various embodiments, the number of such bonds is at least 2, or alternatively at least 4, or 5. In various embodiments, the number of such formed bonds is no more than 8, or alternatively no more than 7, 6, 5, or 4. In various embodiments, each anti-Her2 antibody, on average, is conjugated with about 4-7 drug molecules through cysteines.
  • the drug load on an anti-Her2 antibody may vary depending on many factors, such as the potency of the drug, the size, stability of the anti-Her2 antibody, conjugatable groups available on the anti-Her2 antibody, etc. in various embodiments, 1 to 8 camptothecin analogs molecules are conjugated with 1 anti-Her2 antibody molecule. In various embodiments, an average of about 4 to 7 camptothecin analogs drug molecules are conjugated with an anti- Her2 antibody molecule.
  • Another aspect of the invention relates to methods of inhibiting abnormal cell growth or treating a proliferative disorder, an autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis or kidney disease in a mammal comprising administering to said mammal a therapeutically effective amount of the conjugate of formulas I, optionally, a chemotherapeutic agent.
  • Another aspect of the invention relates to pharmaceutical compositions of the cell binding agent conjugates of formula I and a pharmaceutically acceptable carrier, additive or diluent thereof.
  • the ADC constructs of the present invention comprise an Ab that is a targeting moiety, such as an antibody or antibody fragment capable of binding to a tumor associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, extracellular matrix protein or protease(s), or any post-translational modification residue(s).
  • TAA tumor associated antigen
  • the ADC constructs of the present invention comprise an Ab that is a targeting moiety that exhibits binding affinity to a diseased cell or tissue.
  • the antibody or antibody fragment is capable of binding to a TAA selected from the group consisting of: tumor-associated calcium signal transducer 2 (also known as Trop-2), Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CDS, CD7, CD13, CD19, CD20, CD21 , CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71 , CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1, uPA, MAGES, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250
  • TAA tumor-associated calcium
  • the ADC comprises a TAA binding Ab selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab’, a Fab2, a Fab'2, a IgG, a IgM , a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, and an diabody.
  • the antibody is a chimeric antibody.
  • the antibody is a humanized monoclonal antibody. In various embodiments, the antibody is a fully human monoclonal antibody.
  • the present invention provides a pharmaceutical composition comprising the isolated ADC constructs in admixture with a pharmaceutically acceptable carrier. [040] In another aspect, the invention provides uses of the ADC constructs for the preparation of a medicament for the treatment of cancer.
  • the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof.
  • the subject is a human subject.
  • the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, or rhabdomyosarcoma or any cancer.
  • the subject previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”).
  • a recurrent cancer a cancer that has a resistant or refractory cancer.
  • the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, iPS induced CAR-T or iPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG).
  • a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, iPS induced CAR-T or iPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG).
  • BCG Bacille Calmette-Guerine
  • the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1.
  • immunotherapy including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1.
  • Figure 1 depicts the structures of CRT, CPT-11, SN-38, exatecan and DXd.
  • Figure 2 depicts conjugation procedure for camptothecin analog payload with reduced mAb.
  • Figure 3 depicts line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P-352 and BI-P353 in Her2 positive breast cancer cell line BT-474.
  • Figure 4 depicts line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P352 and BI-P353 in Her2 positive gastric cancer cell line NCI-N87.
  • Figure 5A and figure 5B depict line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P352 and BI-P353 in Her2 positive breast cancer cell line SK-BR-3, respectively.
  • Figure 6 depicts line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P352 and BI-P353 in Her2 negative pancreatic cancer cell line BxPC-3.
  • Figure 7 depicts the plasm stability of Herceptin-BI-P353 ADC
  • Figure 8 depicts line graphs depicting the in vivo efficacy of Herceptin-BI-P353
  • Figure 8A depicts mean tumor volume (mm 3 ) and Figure 8B depicts body weight (g).
  • the present invention provides novel camptothecin linker-payloads, and novel antibody drug conjugates comprising a camptothecin linker-payloads of the present invention linked to an antibody for targeted delivery to disease tissues
  • the present invention provides antibody-drug conjugates (ADCs) and ADC derivatives and methods relating to the use of such conjugates to treat cancer.
  • ADCs antibody-drug conjugates
  • the antibody, or other targeting moiety in the ADC binds to e.g., a tumor associated antigen (TAA) on the cancer cell.
  • TAA tumor associated antigen
  • the antibody is conjugated to a novel camptothecin linker-payload which exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen expressing cells to treat or prevent recurrence of the antigen expressing cancers or immunological disorders.
  • the ADCs of the present invention have superior drug/antibody ratios (DARs), demonstrate improved solubility, enhanced CMC characteristics, and increased therapeutic efficacy particulary against high antigen expressing tumors while sparing the normal tissues expressing low or no level of antigen.
  • the ADCs provide for the targeting of broader patient populations and patients having a refractory cancer or who previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”).
  • alkyl refers to a fully saturated branched or unbranched hydrocarbon moiety having up to 20 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, n-decyl and the like.
  • heterocyclyl referes to a saturated or unsaturated non-aromatic ring or ring system, and contains at least one heteroatom selected from O, S, and N.
  • the heterocyclyl can be attached at a heteroatom, a carbon atom, or both.
  • aryl refers to an aromatic hydrocarbon group having 6- 20 carbon atoms in the ring portion. Typically, aryl is monocyclic, bicyclic or tricyclic aryl having 6-20 carbon atoms. Furthermore, the term “aryl” as used herein, refers to an aromatic moiety which can be a single aromatic ring, or multiple aromatic rings that are fused together.
  • Non- limiting examples include phenyl, naphthyl or tetrahydronaphthyl, each of which may optionally be substituted with 1-4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S- nitro, cyano, carboxy, alkyl-O-C(O)--, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, phenyl, and heterocyclyl.
  • substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl
  • cyclic alkyl or “cycloalkyl” refers to a saturated or unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms. Unless otherwise provided, cycloalkyl refers to cyclic hydrocarbon moiety having between 3 and 9 ring carbon atoms or between 3 and 7 ring carbon atoms, each of which can be optionally substituted with one, or two, or three, or more substituents independently selected from the group consisting of alkyl, halo, oxo, hydroxy, alkoxy, alkyl-C(O)-, acylamino, carbamoyl, alkyl-NH--, (alkyl)2N-, thiol, alkyl-S-, nitro, cyano, carboxy, alkyl-O--C(O)--, sulfonyl, sulfonamide, sulfamoyl, and heterocyclyl
  • the term "optionally substituted” refers to a group that is unsubstituted or is substituted with one or more, typically 1 , 2, 3 or 4, suitable non-hydrogen substituents.
  • polypeptide polypeptide
  • peptide polypeptide
  • protein protein
  • peptides polypeptides
  • proteins are chains of amino acids whose alpha carbons are linked through peptide bonds.
  • the terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group.
  • amino terminus refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide.
  • carboxy terminus refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide.
  • Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether bond as opposed to an amide bond.
  • Polypeptides of the invention include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties.
  • single or multiple amino acid substitutions e.g., conservative amino acid substitutions
  • may be made in the naturally occurring sequence e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a "conservative amino acid substitution” refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid.
  • non-conservative amino acid substitution refers to the substitution of a member of one of these classes for a member from another class.
  • the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5): cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4).
  • the substitution of amino acids whose hydrophilicity values are within + 2 is included, in various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.
  • polypeptide fragment and “truncated polypeptide” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to a corresponding full-length protein.
  • fragments can be, e.g., at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length.
  • fragments can also be, e.g., at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids in length.
  • a fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence ⁇ e.g., an artificial linker sequence).
  • polypeptide variant and “polypeptide mutant” as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
  • the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 2/5, at least 300, at least 350, at least 400, at least 450 or at least 500 amino adds in length.
  • Variants of the present invention indude fusion proteins.
  • a "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • % sequence identity is used interchangeably herein with the term “% identity” and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm and means that a given sequence is at least 80% identical to another length of another sequence.
  • the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
  • % sequence homology is used interchangeably herein with the term ’’% homology” and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program.
  • 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence.
  • the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In various embodiments, the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
  • BLAST programs e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN
  • Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases.
  • the BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. See Id.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1, less than about 0.01 , or less than about 0.001.
  • polypeptide region has a sequence with at least 70%, typically at least 80%, more typically at least 85%, or at least 90% or at least 95% sequence similarity to a reference sequence.
  • a polypeptide is substantially similar to a second polypeptide, for example, where the two peptides differ by one or more conservative substitution(s).
  • recombinant polypeptide is intended to include all polypeptides, including fusion molecules and ADCs that are prepared, expressed, created, derived from, or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell.
  • heterologous refers to a composition or state that is not native or naturally found, for example, that may be achieved by replacing an existing natural composition or state with one that is derived from another source.
  • expression of a protein in an organism other than the organism in which that protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.
  • TAA tumor associated antigen
  • the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length.
  • anti-TAA antagonist antibody refers to an antibody that is able to bind to TAA and inhibit TAA biological activity and/or downstream pathway(s) mediated by TAA signaling.
  • An anti-TAA antagonist antibody encompasses antibodies that block, antagonize, suppress or reduce (including significantly) TAA biological activity, including downstream pathways mediated by TAA signaling, such as receptor binding and/or elicitation of a cellular response to TAA.
  • an anti-TAA antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby the TAA itself, an TAA biological activity (including but not limited to its ability to mediate any aspect of headache), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
  • an anti-TAA antagonist antibody binds TAA and prevents TAA binding to a TAA receptor
  • an anti-TAA antibody binds TAA and prevents activation of a TAA receptor. Examples of anti-TAA antagonist antibodies are provided herein.
  • [Target] antibody should be interpreted as similar to “anti-[Target] antibody” and means an antibody capable of binding to the [Target],
  • the term “Target” or [Target] shall be interpreted as a TAA or any molecule present at the surface of cells, preferably tumoral cells, more preferably mammals and human cells, and which can be used for drug delivery.
  • the Target is specifically express or overexpress on the surface of tumoral cells in comparison with normal cells.
  • antibody is used herein to refer to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes and having specificity to a tumor antigen or specificity to a molecule overexpressed in a pathological state.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as subtypes of these genes and myriad of immunoglobulin variable region genes.
  • Light chains (LC) are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3 (and in some instances, CH4).
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the extent of the framework region and CDRs has been defined.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1, lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1 , CDR2, CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • Antibodies with different specificities j.e. different combining sites for different antigens
  • the Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions.
  • the Kabat database is now maintained online and CDR sequences can be determined, for example, see IMGT/V-QUEST programme version: 3.2.18 March 29, 2011 , available on the internet and Brochet, X. et al., Nucl. Acids Res. 36, W503-508, 2008).
  • the Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17, 1986; Chothia et al., Nature, 342: 877-83, 1989.
  • the AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272, 1989
  • AbMTM A Computer Program for Modeling Variable Regions of Antibodies, ” Oxford, UK; Oxford Molecular, Ltd.
  • the AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, 1999.
  • the contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. BioL, 5:732-45, 1996.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody.
  • the Fc region may be a native sequence Fc region or a variant Fc region.
  • the Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain.
  • the Fc portion of an antibody mediates several important effector functions e.g.
  • cytokine induction ADCC
  • phagocytosis phagocytosis
  • complement dependent cytotoxicity CDC
  • half-life/clearance rate of antibody and antigen-antibody complexes e.g., the neonatal FcR (FcRn) binds to the Fc region of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG).
  • FcRn neonatal FcR
  • replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (see, e.g., Winter et al., U.S. Patent No 5,648,260 and 5,624,821).
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab 2 , F(ab)’ 2 fragments, single chain Fv proteins (“scFv”) and disulfide stabilized Fv proteins (“dsFv”), that bind to the target antigen.
  • a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • antibody encompasses e.g., monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab') 2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments or antigen binding fragments of any of the above.
  • monoclonal antibodies including full-length monoclonal antibodies
  • polyclonal antibodies multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab'
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site.
  • a “Fab fragment” comprises one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a “Fab' fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.
  • F(ab')2 fragment that has two antigen- combining sites and is still capable of cross-linking antigen.
  • a "F(ab')2 fragment” contains two iight chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • the "Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.
  • Single-chain antibodies are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region.
  • Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649, U.S. Patent No. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.
  • an antigen-binding fragment and “antigen-binding protein” as used herein means any protein that binds a specified target antigen.
  • Antigen-binding fragment includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments.
  • An exemplary antigen-binding fragment of an antibody is the heavy chain and/or light chain CDR(s), or the heavy and/or light chain variable region.
  • immunoglobulin chain (heavy or light chain) antigen binding protein is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen.
  • fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope.
  • the fragments are neutralizing fragments.
  • such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and/or light chain or portion thereof.
  • These biologically active fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies.
  • Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody, Fab', F(ab') 2 , Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit.
  • a functional portion of the antigen binding proteins disclosed herein could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life.
  • Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL regions joined by a linker that is too short to allow for pairing between two regions on the same chain, thus allowing each region to pair with a complementary region on another polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-48, 1993; and Poljak et al., Structure, 2:1121-23, 1994). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites.
  • Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). In various embodiments bispecific antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807, 1981 ; by "polydoma” techniques (see, e.g., U.S. Patent No.
  • bispecific antibodies of the present invention can have binding specificities for at least two different epitopes at least one of which is a tumor associate antigen.
  • the antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal” is not to be construed as requiring production of the antibody by any specific method.
  • chimeric antibody refers to an antibody which has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds targeted antigen.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo ⁇ ., for example in the CDRs and in particular CDR3.
  • the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • humanized antibody refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • the framework regions are chosen from human germline exon XH, JH, VK and JK sequences.
  • acceptor sequences for humanization of FR of a VH domain can be chosen from genuine V H exons VH 1-18 (Matsuda et al., Nature Genetics 3:88-94, 1993) or VH1-2 (Shin et al., EMBO J. 10:3641-3645, 1991) and for the hinge region (J H ), exon JH-6 (Mattila et al., Eur. J. Immunol. 25:2578-2582, 1995).
  • germline VK exon B3 Cox et al., Eur. J. Immunol. 24:827-836, 1994
  • JK exon JK-1 Hieter et al., J. Biol. Chem. 257:1516-1522, 1982
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant, combinatorial human antibody library; antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. All such recombinant means are well known to those of ordinary skill in the art.
  • epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule.
  • Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be "linear” or “conformational.” In a linear epitope, all the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.
  • a desired epitope on an antigen it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present invention.
  • the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope.
  • An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, e.g., the antibodies compete for binding to the antigen.
  • the competition for binding to the epitope can be determined by any methods or techniques known by the person skilled in the art such as, without limitation, radioactivity, Biacore, ELISA, Flow cytometry, etc.
  • which competes for binding to the epitope it is meant a competition of at least 20%, preferentially at least 50% and more preferentially at least 70%.
  • An antigen binding protein including an antibody, "specifically binds" to an antigen if it binds to the antigen with a high binding affinity as determined by a dissociation constant (K D , or corresponding Kb, as defined below) value of at least 1 x 10‘ 6 M, or at least 1 x 10’ 7 M, or at least 1 x 10‘ 8 M, or at least 1 x 10" 9 M, er at least 1 x 10 '" J M, or at least 1 x 10’ 11 M.
  • K D dissociation constant
  • An antigen binding protein that specifically binds to the human antigen of interest may be able to bind to the same antigen of interest from other species as well, with the same or different affinities.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction.
  • pharmaceutical composition refers to a composition suitable for pharmaceutical use in an animal.
  • a pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier "Pharmacologically effective amount” refers to that amount of an agent effective to produce the intended pharmacological result.
  • “Pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton.
  • a “pharmaceutically acceptable salt” is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.
  • treat refers to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms.
  • to "alleviate” a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (e.g., metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total).
  • treatment is a reduction of pathological consequence of a proliferative disease.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • an effective amount or “therapeutically effective amount” as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms.
  • an effective amount comprises an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferabiy stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • An effective amount can be administered in one or more administrations.
  • ECsc half maximal effective concentration
  • concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after some specified exposure time. It is commonly used as a measure of drug's potency.
  • the ECso of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed.
  • the ECso of a quantal dose response curve represents the concentration of a compound where 50% of the population exhibits a response, after specified exposure duration. Concentration measures typically follow a sigmoidal curve, increasing rapidly over a relatively small change in concentration. This can be determined mathematically by derivation of the best-fit line.
  • Adjuvant setting refers to a clinical setting in which an individual has had a history of a proliferative disease, particularly cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (such as surgical resection), radiotherapy, and chemotherapy. However, because of their history of the proliferative disease (such as cancer), these individuals are considered at risk of development of the disease.
  • Treatment or administration in the "adjuvant setting” refers to a subsequent mode of treatment.
  • the degree of risk i.e., when an individual in the adjuvant setting is considered as "high risk” or "low risk) depends upon several factors, most usually the extent of disease when first treated.
  • the terms "co-administration”, “co-administered” and “in combination with”, referring to the fusion molecules of the invention and one or more other therapeutic agents, is intended to mean, and does refer to and include the following: simultaneous administration of such combination of fusion molecules of the invention and therapeutic agent] s) to an individual in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said individual; substantially simultaneous administration of such combination ef fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said individual, whereupon said components are released at substantially the same time to said individual; sequential administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said individual with a significant time interval between each administration, whereupon said components are released at substantially
  • therapeutic protein refers to proteins, polypeptides, antibodies, peptides or fragments or variants thereof, having one or more therapeutic and/or biological activities.
  • Therapeutic proteins encompassed by the invention include but are not limited to, proteins, polypeptides, peptides, antibodies, and biologies (the terms peptides, proteins, and polypeptides are used interchangeably herein). It is specifically contemplated that the term “therapeutic protein” encompasses the fusion molecules of the present invention.
  • patient may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine).
  • domesticated mammals e.g., canine or feline
  • laboratory mammals e.g., mouse, rat, rabbit, hamster, guinea pig
  • agricultural mammals e.g., equine, bovine, porcine, ovine
  • the patient can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context.
  • the patient may be an immunocompromised patient or a patient with a weakened immune system including, but not limited to patients having primary immune deficiency, AIDS; cancer and transplant patients who are taking certain immunosuppressive drugs; and those with inherited diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency).
  • the patient has an immunogenic cancer, including, but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high rate of mutations (Lawrence et al., Nature, 499(7457): 214-218, 2013).
  • an immunogenic cancer including, but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high rate of mutations (Lawrence et al., Nature, 499(7457): 214-218, 2013).
  • administering refers to the actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a patient, that control and/or permit the administration of the agent(s)/compound(s) at issue to the patient.
  • Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic regimen, and/or prescribing particular agent(s)/compounds for a patient.
  • Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like. Where administration is described herein, "causing to be administered” is also contemplated.
  • Resistant or refractory cancer refers to tumor cells or cancer that do not respond to previous anti-cancer therapy including, e.g., chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy.
  • Tumor cells can be resistant or refractory at the beginning of treatment, or they may become resistant or refractory during treatment.
  • Refractory tumor cells include tumors that do not respond at the onset of treatment or respond initially for a short period but fail to respond to treatment.
  • Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but fail to respond to subsequent rounds of therapies.
  • refractory tumor cells also encompass tumors that appear to be inhibited by treatment with anticancer therapy but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.
  • the anticancer therapy can employ chemotherapeutic agents alone, radiation alone, targeted therapy alone, surgery alone, or combinations thereof.
  • the refractory tumor cells are interchangeable with resistant tumor.
  • tumor microenvironment refers to the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow- derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM).
  • Components in the tumor microenvironment can modulate the growth of tumor cells, e.g., their ability to progress and metastasize.
  • the tumor microenvironment can aiso be influenced by the tumor releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance.
  • proliferative disease includes tumor disease (including benign or cancerous) and/or any metastases.
  • a proliferative disease may include hyperproliferative conditions such as hyperplasias, fibrosis (especially pulmonary, but aiso other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.
  • the proliferative disease is cancer.
  • the proliferative disease is a nan-cancerous disease.
  • the proliferative disease is a benign or malignant tumor.
  • immunogenicity refers to the ability of an antibody or antigen binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient and includes, for example, the human anti-mouse antibody (HAMA) response.
  • HAMA human anti-mouse antibody
  • a HAMA response is initiated when T-cells from a subject make an immune response to the administered antibody. The T-cells then recruit B-cells to generate specific "anti-antibody” antibodies.
  • an immune cell means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen).
  • an immune cell is, e.g., a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, Langerhan’s cells, or Kuffer cells.
  • Polynucleotide refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds
  • nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-0- methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • PNAs peptide-nucleic acids
  • nucleic acid typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5‘-end of the RNA transcript are referred to as “upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3' end of the coding RNA transcript are referred to as “downstream sequences.”
  • “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides.
  • the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
  • a first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.
  • hybridizing specifically to or “specific hybridization” or “selectively hybridize to” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences.
  • Stringent hybridization and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence-dependent and are different under different environmental parameters.
  • highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the Tm for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than about 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCI at 72°C for about 15 minutes.
  • stringent wash conditions is a 0.2 x SSC wash at 65°C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A high stringency wash can be preceded by a low stringency wash to remove background probe signal.
  • An exemplary low stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 4-6 x SSC at 40" C for 15 minutes
  • a signal to noise ratio of 2 x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Primer refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e. , in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as ONA polymerase.
  • a primer is typically single-stranded but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications.
  • a primer is complementary' to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • Probe when used in reference to a polynucleotide, refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide.
  • a probe specifically hybridizes to a target complementary polynucleotide but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions.
  • Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In instances where a probe provides a point of initiation for synthesis of a complementary polynucleotide, a probe can also be a primer.
  • Linker refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5’ end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences.
  • a “cleavable linker” refers to a linker that can be degraded or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like.
  • Cleavable linkers may also be cleaved by environmental cues, such as, for example, changes in temperature, pH, salt concentration, etc.
  • Non-cleavable linkers are linkers that release an attached payload via lysosomal degradation of the antibody following internalization.
  • label refers to incorporation of another molecule in the antibody.
  • the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods).
  • the label or marker can be therapeutic, e.g., a drug conjugate or toxin.
  • Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, !4 C, 15 N, 35 S, 90 Y, "Tc, 111 In, 125 l, 131 l), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium
  • a "vector” is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell.
  • a “plasmid” refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated.
  • a viral vector e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g , non-episomal mammalian vectors
  • An "expression vector” is a type of vector that can direct the expression of a chosen polynucleotide.
  • a "regulatory sequence” is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked.
  • the regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g, polypeptides that bind to the regulatory sequence and/or the nucleic acid).
  • Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • a nucleotide sequence is “operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g, the level, timing, or location of expression) of the nucleotide sequence.
  • a "host cell” is a cell that can be used to express a polynucleotide of the invention.
  • a host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g, a yeast or other fungus), a plant cell (e.g, a tobacco or tomato plant ceil), an animal cell (e.g, a human cell, a monkey ceil, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
  • a prokaryote for example, E. coli
  • a eukaryote for example, a single-celled eukaryote (e.g, a yeast or other fungus)
  • a plant cell e.g, a tobacco or tomato plant ceil
  • an animal cell e.g, a human
  • a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
  • the phrase "recombinant host cell” can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed.
  • a host cell also can be a ceil that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g, mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • isolated molecule (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art.
  • Molecule purity or homogeneity may be assayed by a number of means well known in the art.
  • the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art.
  • higher resolution may be provided by using HPLC or other means well known in the art for purification.
  • a protein or polypeptide is "substantially pure,” “substantially homogeneous,” or “substantially purified” when at least about 60% to 75% of a sample exhibits a single species of polypeptide.
  • the polypeptide or protein may be monomeric or multimeric.
  • a substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
  • Reference to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of "X”.
  • the present invention is directed to an ADC of the following formula (I):
  • [D-L-] x -Ab or a pharmaceutically acceptable salt thereof wherein Ab is an antibody, or an antigen binding antibody fragment thereof; L is a linker; and D is a camptothecin analog drug moiety.
  • the Ab is an antibody (e g., a cell binding antibody) or antigen binding fragment thereof;
  • camptothecin analog drug moieties of the invention have the structure:
  • Ri is a hydrogen atom or a C1-C6 alkyl
  • L is a bivalent linker comprising a peptide moiety of 2-4 amino acid represented by the following formula (II):
  • L 1 is a linker attached to the antibody comprising a reactive functional group selected from maleimide, thiol, amino, alkyl bromide, alkyl iodide, carboxyl, and NHS ester.
  • the reactive functional group include NHR 2 , OH, SH, - CH 2 CH 2 SH, - CO 2 H, and NHS ester;
  • L 2 is a 2-4 AA peptide moiety
  • L 3 is a self-immolative moiety connected to the drug moiety that is:
  • R 2 is a hydrogen atom or a C1-C6 alkyl
  • n 1 is 1 , 2, 3, 4 or 5, and n 2 is 2
  • the drug moiety reagents include the structures:
  • R2 is hydrogen atom or C1-C6 alkyl, m is 0, 1, 2, 3 or 4.
  • linker attaches the antibody to a drug moiety through covalent bond(s).
  • the linker is a bifunctional or multifunctional moiety which can be used to link one or more drug moiety (D) and an antibody unit (Ab) to form antibody-drug conjugates (ADC) of Formula I.
  • the linker (L) may be stable outside a cell, • e., extracellular, or it may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions.
  • Antibody-drug conjugates (ADC) can be conveniently prepared using a linker having reactive functionality for attaching to the drug moiety and to the antibody.
  • a cysteine thiol, or an amine, e.g., N- terminus or amino acid side chain such as lysine, of the antibody (Ab) can form a bond with a functional group of a linker reagent, drug moiety (D) or drug-linker reagent (D-L).
  • the linkers are preferably stable outside the target cell.
  • the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the drug moiety remains linked to the antibody.
  • An effective linker will: (I) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (ill) remain stable and intact, i.e., not cleaved, until the conjugate has been delivered to its targeted site: and (iv) maintain a cytotoxic, cell- killing effect or a cytostatic effect of the camptothecin analog drug moiety.
  • Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation/analysis technique LC/MS.
  • Covalent attachment of the antibody and the drug moiety requires the linker to have two reactive functional groups.
  • Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G.T. (1996) Bioconjugate Techniques: Academic Press: New York, p 234-242).
  • the linker may be substituted with groups which modulate solubility or reactivity.
  • a sulfonate substituent may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or the drug moiety or facilitate the coupling reaction of Ab-L with D, or D-L with Ab, depending on the synthetic route employed to prepare the ADC.
  • Nucleophilic groups on antibodies include but are not limited to: (i) N- terminal amine groups, (ii) side chain amine groups, e.g., lysine, (ill) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Amine, thiol, and hydroxyi groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol).
  • a reducing agent such as DTT (dithiothreitol).
  • Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles.
  • Additional nucleophilic groups can be introduced into antibodies through the reaction oflysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non- native cysteine amino acid residues).
  • US 2007/0092940 teaches engineering antibodies by introduction of reactive cysteine amino acids.
  • a linker has a reactive nucleophilic group which is reactive with an electrophilic group present on an antibody.
  • Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
  • the heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the electrophilic group on an antibody provides a convenient site for attachment to a Linker.
  • Nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • Linkers can be peptidic, comprising one or more amino acid units.
  • Peptide linker reagents may be prepared by solid phase or liquid phase synthesis methods (E. Schroder and K. Lubke, The Peptides, volume 1 , pp 76-136 (1965) Academic Press) that are well known in the field of peptide chemistry, including t-BOC chemistry (Geiser et al "Automation of solid- phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R. Uss, Inc., 1988, pp. 199-218) and Fmoc/HBTU chemistry (Fields, G. and Noble, R.
  • Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine- phenylalanine (af or ala-phe).
  • Exemplary tripeptides include: glycine-valine-citrulline (gly- val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • Exemplary tetrapeptides include: glycine-glycine- valine-citruiline (gly-gly-val-cit) and glycine-glycine-phenylalanine-citrulline (gly-gly- phe-gly).
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • Embodiments of drug-linker reagents include:
  • R1 is H or C1-C6 alkyl, n ⁇ is 0, 1 , 2, 3, or 4;
  • R 2 and R 3 are independently an amino acid side chain selected from hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, - CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, - 2)3NHCOCH3, - 4NHCOCH3, ⁇ (CH2)4NHCHO, 2NH2, 2-pyridylmethyl-, 3- pyridylmethyi-, 4-pyridylmethyl-, phenyl and cyclohexyl.
  • the ADC or ADC derivative is internalized by targeted tumor cells or by activated immune cells, where the ADC or ADC derivative exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen expressing ceils to treat or prevent recurrence of the antigen expressing cancers or immunological disorders.
  • the ADC or ADC derivative is not internalized, and the anti-Target Ab is effective to deplete or inhibiting target antigen-expressing cells by binding to the cell membrane.
  • the ADC or ADC derivatives thereof can be targeted to a biological molecule in a cell (e.g., an inflammatory agent) and accumulate at or adjacent cells secreting or binding the biological molecule, where the therapeutic drug moiety exerts an effect (e.g., a cytotoxic, cytostatic, or immunosuppressive effect).
  • a biological molecule in a cell e.g., an inflammatory agent
  • the therapeutic drug moiety exerts an effect (e.g., a cytotoxic, cytostatic, or immunosuppressive effect).
  • the ADCs of the present invention have superior drug/antibody ratios (DARs), demonstrate improved solubility, enhanced CIVIC characteristics, and increased therapeutic efficacy against high antigen expressing tumor cells while having less effect on low or no antigen expressing cells i.e. normal cells.
  • DARs drug/antibody ratios
  • the ADCs provide for the targeting of broader patient populations and patients with a refractory cancer or who previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”).
  • Tumor antigens expressed on the cell membrane are potential targets in immunotherapy, with the ideal tumor antigen absent on normal cells and overexpressed on the tumor cell surface.
  • the ADCs used in the methods of the present invention may comprise an antibody, or antigen binding antibody fragment, specific to any of the tumor associated antigens described in the art, including any biosimilar, biogeneric, follow-on biologic, or follow-on protein version of any TAA described in the art.
  • the TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which the skilled artisan wishes to induce an immune response.
  • the TAA, TAA variant, or TAA mutant contemplated for use in the combination methods of the present invention is selected from, or derived from, the list provided in Table 1.
  • the TAA has an amino acid sequence that shares an observed homology of, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 9/%, 98%, or at least about 99% with any one of the sequences disclosed in Table 1.
  • a method for generating a monoclonal antibody that binds specifically to a targeted antigen polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the targeted antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeioma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the targeted antigen polypeptide.
  • antibody-producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to targeted antigen polypeptide.
  • the monoclonal antibody may be purified from the cell culture. A variety of different techniques are then available for testing an antigen/antibody interaction to identify particularly desirable antibodies.
  • Antibodies can be engineered in numerous ways. They can be made as single- chain antibodies (including small modular immunopharmaceuticals or SMIPsTM), Fab and F(ab')z fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.
  • Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al.
  • a humanized antibody has one or more amino acid residues introduced from a source that is nonhuman, in addition to the nonhuman CDRs.
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525, 1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al.. Science, 239:1534-1536, 1988), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.
  • U.S. Patent No. 5,693,761 to Queen et al discloses a refinement on Winter et al. for humanizing antibodies, and is based on the premise that ascribes avidity loss to problems in the structural motifs in the humanized framework which, because of steric or other chemical incompatibility, interfere with the folding of the CDRs into the binding-capable conformation found in the mouse antibody.
  • Queen teaches using human framework sequences closely homologous in linear peptide sequence to framework sequences of the mouse antibody to be humanized. Accordingly, the methods of Queen focus on comparing framework sequences between species. Typically, all available human variable region sequences are compared to a particular mouse sequence and the percentage identity between correspondent framework residues is calculated.
  • the human variable region with the highest percentage is selected to provide the framework sequences for the humanizing project. Queen also teaches that it is important to retain in the humanized framework, certain amino acid residues from the mouse framework critical for supporting the CDRs in a binding-capable conformation. Potential criticality is assessed from molecular models. Candidate residues for retention are typically those adjacent in linear sequence to a CDR or physically within 6.A of any CDR residue.
  • framework shuffling Another method of humanizing antibodies, referred to as “framework shuffling", relies on generating a combinatorial library with nonhuman CDR variable regions fused in frame into a pool of individual human germline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). The libraries are then screened to identify clones that encode humanized antibodies which retain good binding.
  • variable regions both light and heavy
  • sequence of the variable region of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closest to that of the rodent is then accepted as the human framework region (framework region) for the humanized antibody (Sims et al., J. Immunol., 151 :2296, 1993; Chothia et al., J. Mol. Biol., 196:901 , 1987).
  • Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions.
  • the same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. (U.S.A.), 89:4285, 1992: Presta et al., J. Immunol., 151 :2623, 1993).
  • the choice of nonhuman residues to substitute into the human variable region can be influenced by a variety of factors. These factors include, for example, the rarity of the amino acid in a particular position, the probability of interaction with either the CDRs or the antigen, and the probability of participating in the interface between the light and heavy chain variable domain interface. (See, for example, U.S. Patent Nos. 5,693,761 , 6,632,927, and 6,639,055).
  • One method to analyze these factors is through the use of three-dimensional models of the non-human and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • a method for producing a TAA antibody or antigen-binding fragment thereof comprises the steps of synthesizing a library of human antibodies on phage, screening the library with TAA or an antibody-binding portion thereof, isolating phage that bind TAA, and obtaining the antibody from the phage.
  • one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with TAA or an antigenic portion thereof to create an immune response, extracting antibody-producing ceils from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage.
  • Recombinant anti-TAA antibodies of the invention may be obtained in this way.
  • recombinant human anti-TAA antibodies of the invention can also be isolated by screening a recombinant combinatorial antibody library.
  • the library is a scFv phage display library, generated using human VL and VH CDNAS prepared from mRNA isolated from B cells.
  • Kits for generating phage display libraries are commercially available (e g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612).
  • Methods for generating phage display libraries are commercially available (e g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612).
  • There also are other methods and reagents that can be used in generating and screening antibody display libraries see, e.g., U.S. Pat. No. 5,223,409; PCT Publication Nos.
  • WO 92/18619 WO 91/17271 , WO 92/20791 , WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology, 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85, 1992; Huse et al., Science, 246:1275-1281, 1989; McCafferty et al., Nature, 348:552-554, 1990; Griffiths et al., EMBO J., 12:725-734, 1993; Hawkins et al., J. Mol.
  • Human antibodies are also produced by immunizing a non-human, transgenic animal comprising within its genome some or all of human immunoglobulin heavy chain and light chain loci with a human IgE antigen, e.g., a XenoMouseTM animal (Abgenix, Inc./Amgen, Inc.- Fremont, Calif.).
  • XenoMouseTM mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 , 1994 and U.S. Pat. Nos.
  • XenoMouseTM mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies.
  • the XenoMouseTM mice contain approximately 80% of the human antibody V gene repertoire through introduction of megabase sized, germline configuration fragments of the human heavy chain loci and kappa light chain loci in yeast artificial chromosome (YAC).
  • YAC yeast artificial chromosome
  • XenoMouseTM mice further contain approximately all of the human lambda light chain locus. See Mendez et al , Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J Exp. Med., 188:483-495, 1998; and WO 98/24893.
  • the ADCs of the present invention utilize an antibody or antigen-binding fragment thereof is a polyclonal antibody, a monoclonal antibody or antigen- binding fragment thereof, a recombinant antibody, a diabody, a chimerized or chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen- binding fragment thereof, a single chain antibody, an Fv, an Fd, an Fab, an Fab', or an F(ab') 2 , and synthetic or semi-synthetic antibodies.
  • the ADCs of the present invention utilize an antibody or antigen-binding fragment that binds to a TAA with a dissociation constant (KD) of, e.g., at least about 1x10' 3 M, at least about 1x10' 4 M, at least about 1x10' 5 M, at least about 1x10’ 8 M, at least about 1x1 O' 7 M, at least about 1x1 O' 8 M, at least about 1x10' 9 M, at least about 1x10' 10 M, at least about 1x1 O' 11 M, or at least about 1x1 O' 12 M.
  • KD dissociation constant
  • the fusion molecules of the present invention utilize an antibody or antigen-binding fragment that binds to a TAA with a dissociation constant (KD) in the range of, e.g., at least about 1x10' 3 M to at least about 1x1 O' 4 M.
  • KD dissociation constant
  • the ADCs of the present invention utilize an antibody or antigen-binding fragment that cross-com petes for binding to the same epitope on the TAA as a reference antibody which comprises the heavy chain variable region and light chain variable region set forth in the references and sequence listings provided herein.
  • antibodies contemplated for use in the ADCs of the present invention include but are not limited to LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101 , anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 or KC4 (both anti- mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5), MN- 15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p).
  • LL1 anti-CD74
  • LL2 or RFB4 anti-CD
  • Immu 31 an anti-alpha- fetoprotein
  • R1 anti-IGF-1R
  • A19 anti-CD19
  • TAG-72 e.g., CC49
  • Tn J591 or HuJ591
  • AB-PG1-XG1-026 anti-PSMA dimer
  • D2/B anti- PSM.A
  • G250 an anti-carbonic anhydrase IX MAb
  • L243 anti-HLA-DR
  • alemtuzumab anti- CD52
  • bevacizumab anti-EGFR
  • gemtuzumab anti-CD33
  • ibritumomab tiuxetan anti-CD20
  • panitumumab anti-EGFR
  • tositumomab anti-CD20
  • PAM4 aka clivatuzumab, anti-mucin
  • hPAM4 U.S. Pat. No. 7,282,567
  • hA20 U.S. Pat. No. 7,251,164
  • hA19 U.S. Pat. No. 7,109,304
  • hlMMU-31 U.S. Pat. No. 7,300,655
  • hLL1 U.S. Pat. No. 7,312,318,
  • hLL2 U.S. Pat. No. 7,074,403
  • hMu-9 U.S. Pat. No. 7,387,773
  • hL243 U.S.
  • the ADCs of the present invention include at least one antibody or fragment thereof that binds to Her2.
  • the present invention features bispecific molecules comprising an anti-Her2 antibody, or antigen-binding fragment thereof, of the invention.
  • An antibody of the invention, or antigen-binding fragment thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • the antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein.
  • an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.
  • the invention includes bispecific molecules capable of binding both to FcyR or FcaR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing Her2.
  • the bispecific molecules target Her2 expressing cells to effector cell and trigger Fc receptor-mediated effector cell activities, e.g., phagocytosis of a Her2 expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion
  • Fc receptor-mediated effector cell activities e.g., phagocytosis of a Her2 expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion
  • another functional molecule which is linked to the anti-Her2 antibody
  • signaling molecules such as Trop-2, Her-3, EGFR, IGF-R, c-Met, EphA2, EphB2, and MUC16
  • agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecoles such as PD-1, PD-L1, OX-40, CS137, GITR, LAG3, TIM-3, and VISTA
  • Bispecific antibodies or fragments can be of several configurations.
  • bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions).
  • antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807, 1981 ; by "polydoma” techniques (see, e.g., U.S. Patent No. 4,474,893); or by recombinant DNA techniques.
  • bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes at least one of which is a tumor associate antigen.
  • the antibodies and fragments can also be heteroantibodies.
  • Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.
  • any agent that exerts a therapeutic effect on cancer cells or activated immune ceils can be used as the warhead conjugated to an anti- target antigen antibody.
  • useful classes of cytotoxic or immunosuppressive agents include, for example, antitubulin agents (e.g., auristatins and maytansinoids), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and-carboplatin), anthracyclines, antibiotics, antifolates, anti metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins,
  • antitubulin agents e.g., a
  • cytotoxic or immunosuppressive agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5-fiuordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlore
  • AMC
  • the therapeutic drug moiety is a cytotoxic agent.
  • Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, a-amanitin, and mitoxantrone.
  • dolastatins e.
  • the cytotoxic agent is a conventional chemotherapeutic such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide.
  • chemotherapeutic such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide.
  • potent agents such as CC-1065 analogues, calicheamicin, maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can be used in the ADCs of the present invention.
  • Linkers in ADCs may have significant impacts on the biological activities. For example, in vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation method in developing safe and efficacious ADCs for cancer therapy.
  • Some embodiments of the invention relate to camptothecin payloads linked to antibodies via a cleavable linker.
  • antibody was reduced by adding antibody to TCEP (Tris (2-carboxy ethyl) phosphine) dissolved in a pH-adjusted PBS EDTA buffer.
  • TCEP Tris (2-carboxy ethyl) phosphine
  • the antibody/TCEP solution was incubated at 37°C for 2-3 hrs.
  • the reduced antibodies were buffer exchanged into conjugation reaction buffer.
  • the camptothecin payload dissolved in DMSO was added to a solution of reduced monoclonal antibody at payload/antibody ratio of 7 ⁇ 30:1 in order to achieve different drug to antibody ratios (DARs).
  • DARs drug to antibody ratios
  • the payload/antibody solution was incubated for 1-2 hr at 20°C while the reduced antibody was conjugated to the payload via the maleimide group. After conjugation was completed, the reaction mixture was desalted and concentrated to yield the ADCs.
  • the biochemical properties of the resulting ADCs were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC- HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to confirm drug loading (DAR).
  • SEC- HPLC size-exclusion chromatography high pressure liquid chromatography
  • HIC-HPLC hydrophobic interaction chromatography HPLC
  • the final ADC products are comprised of four or six or seven payload-linker molecules.
  • the cysteine conjugation method used in the conjugation process produces more homogenouse ADCs compared to lysine conjugation method.
  • high drug loading e.g., drug ratio >5
  • drug moieties conjugated to an antibody during a conjugation reaction are less than the theoretical maximum.
  • the drug loading also referred as the Drug-Antibody ratio (DAR) is the average number of drugs per antibody.
  • DAR Drug-Antibody ratio
  • drug loading may range from 1 to 8 drugs (D) per antibody, i.e., where 2, 4, 5, 6, and 8 drug moieties are covalently attached to the antibody.
  • compositions of ADCs include collections of cell binding agents, e.g., antibodies, conjugated with a range of drugs, from 1 to 8 or 1 to 12.
  • the average number of drugs per antibody in preparations of ADCs from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis.
  • the ADCs of the present invention has much higher solubility compared to those ADCs containing SMCC- DM1 or vc-MMAE payload, therefore allow more drugs to be conjugated to the antibody i.e., DAR > 7.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an ADC as described herein, with one or more pharmaceutically acceptable excipient(s).
  • the pharmaceutical compositions and methods of uses described herein also encompass embodiments of combinations (co-administration) with other active agents, as detailed below.
  • the ADCs provided herein can be formulated by a variety of methods apparent to those of skill in the art of pharmaceutical formulation. Such methods may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all GMP regulations of the U.S. Food and Drug Administration.
  • ADCs of the invention are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s), or carriers.
  • pharmaceutically acceptable excipients and carriers are well known and understood by those of ordinary skill and have been extensively described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990)
  • the pharmaceutically acceptable carriers may be included for purposes of modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine): antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins
  • amino acids such as glycine, glutamine, asparagine, arginine or lysine
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof.
  • compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution.
  • optional formulation agents Remington's Pharmaceutical Sciences, supra
  • the therapeutic composition may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a patient and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like.
  • the pharmaceutical composition is formulated for parenteral administration via a route selected from, e.g., subcutaneous injection, intraperitoneal injection, intramuscular Injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.
  • a route selected from, e.g., subcutaneous injection, intraperitoneal injection, intramuscular Injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.
  • the therapeutic pharmaceutical compositions may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired ADC in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a polypeptide is formulated as a sterile, isotonic solution, properly preserved.
  • pharmaceutical formulations suitable for injectable administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Other parentally administrate formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • Any method for formulating and administering peptides, proteins, antibodies, and immunoconjugates accepted in the art may suitably be employed for administering the ADCs of the present invention.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • ADC ADC to be employed in the methods of the present invention
  • dosage values may include single or multiple doses, and that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the dosage regimen with the compositions of this disclosure may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the subject, the severity of the condition, the route of administration, and the particular antibody employed.
  • the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values.
  • the present invention encompasses intra-subject dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
  • the total monthly dose of the ADCs of the invention can be in the range of 0.002-500 mg per patient, 0.002-400 mg per patient, 0.002-300 mg per patient, 0.002-200 mg per patient, 0.002-100 mg per patient, 0.002-50 mg per patient, 0.006-500 mg per patient, 0.006-400 mg per patient, 0.006-300 mg per patient, 0.006-200 mg per patient, 0.006-100 mg per patient, 0.006-50 mg per patient, 0.02-500 mg per patient, 0.02- 400 mg per patient, 0.02-300 mg per patient, 0.02-200 mg per patient, 0.02-100 mg per patient, 0.02-50 mg per patient, 0.06-500 mg per patient, 0.06-400 mg per patient, 0.06-300 mg per patient, 0.06-200 mg per patient, 0.06-100 mg per patient, 0.06-50 mg per patient, 0.2-500 mg per patient, 0.2-400 mg per patient, 0.2-300 mg per patient, 0 2-200 mg per patient, 0.2-
  • An exemplary, non-limiting weekly dosing range for a therapeutically effective amount of the ADCs of the invention can be about 0.0001 to about 0.9 mg/kg, about 0.0001 to about 0.8 mg/kg, about 0.0001 to about 0.7 mg/kg, about 0.0001 to about 0.6 mg/kg, about 0.0001 to about 0.5 mg/kg, about 0.0001 to about 0.4 mg/kg, about 0.0001 to about 0.3 mg/kg, about 0.0001 to about 0.2 mg/kg, about 0.0001 to about 0.1 mg/kg, about 0.0003 to about 0.9 mg/kg, about 0.0003 to about 0.8 mg/kg, about 0.0003 to about 0.7 mg/kg, about 0.0003 to about 0.6 mg/kg, about 0.0003 to about 0.5 mg/kg, about 0.0003 to about 0.4 mg/kg, about 0.0003 to about 0.3
  • compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the composition should provide a sufficient quantity of at least one of the ADCs disclosed herein to effectively treat the patient.
  • the dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • the dosing frequency of the administration of the ADC pharmaceutical composition depends on the nature of the therapy and the particular disease being treated.
  • the patient can be treated at regular intervals, such as weekly or monthly, until a desired therapeutic result is achieved, or treated with a loading dose followed by maintenance dose at regular intervals.
  • Exemplary dosing frequencies include, but are not limited to: once weekly without break: once weekly, every other week; once every 2 weeks; once every 3 weeks; weakly without break for 2 weeks, twice weekly without break for 2 weeks, twice weekly without break for 3 weeks, twice weekly without break for 4 weeks, twice weekly without break for 5 weeks, twice weekly without break for 6 weeks, twice weekly without break for 7 weeks, twice weekly without break for 8 weeks, monthly; once every other month; once every three months; once every four months; once every five months; or once every six months, or yearly.
  • Toxicity and therapeutic index of the pharmaceutical compositions of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effective dose is the therapeutic index and it can be expressed as the ratio LD S0 /ED S0 .
  • Compositions that exhibit iarge therapeutic indices are generally preferred.
  • the present invention relates to a method of treating a proliferative disease (such as cancer) in an individual, comprising administering to the individual a therapeutically effective amount of an ADC.
  • a proliferative disease such as cancer
  • the ADCs and methods described herein can be used to effectively treat cancers, including recurrent, resistant, or refractory cancers, at surprisingly low doses.
  • the methods of the present invention are useful in treating certain cellular proliferative diseases.
  • diseases include, but are not limited to, the following: a) proliferative diseases of the breast, which include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma, lobular carcinoma in situ and metastatic breast cancer; b) proliferative diseases of lymphocytic cells, which include, but are not limited to, various T cell and B cell lymphomas, non-Hodgkins lymphoma, cutaneous T cell lymphoma, Hodgkins disease, and lymphoma of the central nervous system; (c) multiple myeloma, chronic neutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelomonocytic leukemia, atypical chronic
  • sarcomas which include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma
  • proliferative diseases of the kidneys which include, but are not limited to, renal cell carcinoma, clear cell carcinoma of the kidney; and renal cell adenocarcinoma
  • precursor B-lymphoblastic leukemia/lymphoma precursor B-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia), B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B- cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell le
  • lymphoma mediastinal large B-cell lymphoma, primary effusion lymphoma and Burkitt's lymphoma/Burkitt cell leukemia; (s) precursor T- lymphoblastic lymphoma/leukemia (precursor T-cell acute lymphoblastic leukemia), T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell lymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides/Sezary syndrome, anaplastic large-cell lymphoma, T/null cell, primary cutaneous type, peripheral T-cell lymphoma, not otherwise characterized, angioi
  • AML with 11q23 (MLL) abnormalities AML minimally differentiated, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroid leukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelosis with myelofibrosis.
  • the proliferative disease is a cancer selected from the group consisting of: B cell lymphoma; a lung cancer (small cell lung cancer and non-small cell lung cancer); a bronchus cancer; a colorectal cancer; a prostate cancer; a breast cancer; a pancreas cancer; a stomach cancer; an ovarian cancer; a urinary bladder cancer; a brain or central nervous system cancer; a peripheral nervous system cancer; an esophageal cancer; a cervical cancer; a melanoma; a uterine or endometrial cancer; a cancer of the oral cavity or pharynx; a liver cancer; a kidney cancer; a biliary tract cancer; a small bowel or appendix cancer; a salivary gland cancer; a thyroid gland cancer; a adrenal gland cancer; an osteosarcoma; a chondrosarcoma; a liposarcoma; a testes cancer; and a malignant fibr
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an ADC, wherein the ADC is administered to the individual at a weekly dosage selected from the group consisting of .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.
  • the ADC is administered to the individual at a dosage (e g., at a weekly dosage) included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.03 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.1 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg.
  • a dosage included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about
  • the ADC is administered to the individual at a dosage (e.g., at a weekly dosage) of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.
  • the cancer expresses the TAA of the ADC of the present invention.
  • the cancer is a non -TAA expressing cancer in the tumor microenvironment of a TAA expressing cancer.
  • the methods may inhibit or prevent the growth or proliferation of TAA expressing or non-TAA expressing tumor cells in an individual, such as for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • the modulation may reduce the size of the solid tumor by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • the inhibition of the tumor cell proliferation can be measured by cell-based assays, such as bromodeoxyuridine (BRDU) incorporation (Hoshino et al., Int. J. Cancer 38, 369, 1986; Campana et a!., J. Immunol. Meth. 107:79, 1988; [ 3 H]-thymidine incorporation (Chen, J., Oncogene 13: 1395-403, 1996; Jeoung, J., J. Biol. Chem. 270:18367-73, 1995; the dye Alamar Blue (available from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998).
  • BRDU bromodeoxyuridine
  • the anchorage independent growth of cancer cells is assessed by colony formation assay in soft agar, such as by counting the number of cancer cell colonies formed on top of the soft agar (see Examples and Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989).
  • a xenograft comprises human cells from a pre-existing tumor or from a tumor cell line.
  • Tumor xenograft assays are known in the art and described herein (see, e.g., Ogawa et al., Oncogene 19:6043-6052, 2000).
  • tumorigenicity is monitored using the hollow fiber assay, which is described in U.S. Patent No. 5,698,413, which is incorporated herein by reference in its entirety.
  • the percentage of the inhibition is calculated by comparing the tumor cell proliferation, anchorage independent growth, or tumor cell growth under modulator treatment with that under negative control condition (typically without modulator treatment). For example, where the number of tumor cells or tumor cell colonies (colony formation assay), or PRDU or pH]-thymidine incorporation is A (under the treatment of modulators) and C (under negative control condition), the percentage of inhibition would be (C-A)/Cx100%.
  • tumor cell lines derived from human tumors and available for use in the in vitro and in vivo studies include, but are not limited to, leukemia cell lines (e.g., CCRF- CEM, HL-60(TB), K-562, MOLT-4, RPM1-8226, SR, P388 and P388/ADR, H292, MV-4-11); non-small cell lung cancer cell lines (e.g., A549/ATCC, EKVX, HOP-62, HOP- 92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522 and LXFL 529); small cell lung cancer ceil lines (e.g., DMS 114 and SHP-77); colon cancer cell lines (e.g., COLO 205, HOC-2998, HCT-116, HCT-15.
  • leukemia cell lines e.g., CCRF- CEM, HL-60(TB), K-562, MOLT
  • HT29, KM12, SW-620, DLD-1 and KM20L2; central nervous system (CNS) cancer cell lines e.g., SF-268, SF-295, SF-539, SNB-19, SNB-75, U251 , SNB-78 and XF 498); melanoma cell lines (e.g., LOX I MVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, RPMI-7951 and M19-MEL): ovarian cancer cell lines (e.g., IGROV1 , OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8 and SK-OV-3); renal cancer cell lines (e.g., 786- 0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, RXF-631 and SN12K1); prostate
  • the present invention relates to a method of activating or stimulating an non-TAA expressing immune cell located in the tumor microenvironment of a TAA expressing tumor, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an ADC; wherein the ADC is administered to the individual at a dosage (e.g., at a weekly dosage) included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.03 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.1 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg.
  • a dosage e.
  • the ADC is administered to the individual at a weekly dosage selected from the group consisting of about .0001 mg/kg, of about .0003 mg/kg, of about .001 mg/kg, of about .003 mg/kg, of about .01 mg/kg. of about .03 mg/kg, of about 0.1 mg/kg, of about 0.2 mg/kg, of about 0.3 mg/kg, of about 0.4 mg/kg, of about 0.5 mg/kg, of about 0.6 mg/kg, of about 0.7 mg/kg, of about 0.8 mg/kg, and of about 0.9 mg/kg.
  • the ADC is administered to the individual at a dosage (e.g., at a weekly dosage) of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.
  • a dosage e.g., at a weekly dosage of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.
  • the methods described herein may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders, such approaches including, but not limited to chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.
  • approaches including, but not limited to chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.
  • methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy.
  • the present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through use of the fusion molecules described herein.
  • a wide array of conventional compounds has been shown to have anti-neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant T-cells in leukemic or bone marrow malignancies.
  • chemotherapy has been effective in treating various types of malignancies, many anti -neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.
  • the ADC disclosed herein When the ADC disclosed herein is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such fusion molecule may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti-neoplastic agent in resistant T-cells.
  • a second anti-cancer agent such as a chemotherapeutic agent, will be administered to the patient.
  • chemotherapeutic agent includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fluradabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and doxetaxel) and/
  • the dosages of such chemotherapeutic agents include, but is not limited to, about any of 10 mg/m 2 , 20 mg/m 2 , 30 mg/m 2 , 40 mg/m 2 , 50 mg/m 2 , 60 mg/m 2 , 75 mg/m 2 , 80 mg/m 2 , 90 mg/m 2 , 100 mg/m 2 , 120 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg/m 2 , 210 mg/m 2 , 220 mg/m 2 , 230 mg/m 2 , 240 mg/m 2 , 250 mg/m 2 , 260 mg/m 2 , and 300 mg/m 2 .
  • the present invention relates to combination therapies designed to treat a proliferative disease (such as cancer) in an individual, comprising administering to the individual: a) a therapeutically effective amount of an ADC, and b) immunotherapy, wherein the combination therapy optionally provides increased effector cell killing of tumor cells, i.e. , a synergy exists between the ADC and the immunotherapy when co- administered.
  • a proliferative disease such as cancer
  • the proliferative disease is a cancer selected from the group consisting of: B cell lymphoma; a lung cancer (small cell lung cancer and non-small cell lung cancer); a bronchus cancer; a colorectal cancer; a prostate cancer; a breast cancer; a pancreas cancer; a stomach cancer; an ovarian cancer; a urinary bladder cancer; a brain or central nervous system cancer; a peripheral nervous system cancer; an esophageal cancer; a cervical cancer; a melanoma; a uterine or endometrial cancer: a cancer of the oral cavity or pharynx; a liver cancer; a kidney cancer; a biliary tract cancer: a small bowel or appendix cancer; a salivary gland cancer; a thyroid gland cancer; a adrenal gland cancer; an osteosarcoma; a chondrosarcoma; a liposarcoma; a testes cancer; and a malignant fibr
  • the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody- drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1 , CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec 7, Siglec 8, Siglec 9, Siglec 15 and VISTA; treatment using bispecific T ceil engaging antibodies (BITE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-a, IFN-p and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric
  • immunotherapy including, but are
  • a combination therapy method of treating a proliferative disease in an individual comprising administering to the individual a) an effective amount of an ADC; and b) immunotherapy; wherein the combination therapy provides increased effector cell killing.
  • the immunotherapy is treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules.
  • the immunotherapy is treatment using chimeric antigen receptor (CAR)-T cells.
  • the immunotherapy is treatment using CAR-NK cells.
  • the immunotherapy is treatment using bispecific T cell engaging antibodies (BITE®).
  • the cancer expresses the TAA of the ADC of the present invention.
  • the cancer is a non-TAA expressing cancer in the tumor microenvironment of a TAA expressing cancer.
  • the immunotherapy will target a TAA that is different than the TAA targeted by the ADC.
  • kits for the treatment of cancer and/or in an adjunct therapy typically comprise a container containing an ADC of the present invention.
  • the ADC can be present in a pharmacologically acceptable excipient.
  • the kits may optionally include an immunotherapy cancer agent.
  • the kits can optionally include instructional materials disclosing means of use of the ADC and/or immunotherapy to treat a cancer.
  • the instructional materials may also, optionally, teach preferred dosages, counter-indications, and the like.
  • kits can also include additional components to facilitate the particular application for which the kit is designed.
  • additional components can also include additional components to facilitate the particular application for which the kit is designed.
  • additional components for example, and additionally comprise means for disinfecting a wound, for reducing pain, for attachment of a dressing, and the like.
  • instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials
  • HATU (2.85g,7.5mmol) was added to a solution of 6-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1-yl) hexanoic acid (1.06g, 5mmol), (S)-tert- butyl 2-amino-3-methylbutanoate hydrochloride (1.1g , 5.25mmol) in DMF (15 mL). DIPEA (1.1g, 8.5 mmol) was added to the mixture, which was stirred overnight at room temperature. The mixture was diluted with DCM, which was washed with aqueous sodium bicarbonate and brine.
  • HATU (1.48g, 3.9mmol) was added to a solution of compound 1 (0.93g, 3mmol), (S)-tert-butyl 2-aminopropanoate hydrochloride (0.54mg 3mmol) in DMF (20 mL). DIPEA (0.58g, 4.5mmol) was added to the mixture and it was stirred overnight at room temperature. The mixture was diluted with DCM, which was washed with aqueous sodium bicarbonate and brine.
  • HATU (38.0mg,0.1mmol) was added to a solution of compound 34 (30mg,0.05mmol), compound 36 (32.1mg , 0.064mmol) in DMF (3 mL).
  • DIPEA (19.1mg, 0.15 mmol) was added to the mixture and stirred overnight at room temperature.
  • the mixture was diluted with DCM, washed with aqueous sodium bicarbonate and brine.
  • MS: m/z 1093.3 (M+1).
  • HATU 43.0mg, 0.113mmoi
  • exatecan mesylate 50mg , 0.094mmol
  • 2-methyithioacetic add 20 mg, 0.192mmol
  • camptothecin derivatives were conjugated to camptothecin derivatives to form ADCs and evaluated for their ability to inhibit the growth of multiple cancer cell lines expressing different levels of Her2. Camptothecin derivative stabilizes the topoisomerase I complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.
  • Some embodiments of the invention relate to camptothecin derivatives linked to antibodies via a cleavable linker.
  • Herceptin was reduced by adding antibody to TCEP (Tris (2-carboxyethy! phosphine) dissolved in a pH-adjusted PBS EDTA buffer.
  • the antibody/TCEP solution was incubated at 37°C for 2 hrs.
  • the excessive TCEP was removed by ultrafiltration using an amicon ultra filter.
  • the reduced antibodies were buffer exchanged into conjugation reaction buffer.
  • the solution of camptothecin payloads in DMA was added to a solution of reduced anti-Her2 monoclonal antibody at payload/antibody ratio of 7 - 30:1 to achieve different drug to antibody ratios (DARs).
  • the payload/antibody solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the payload via the thiol maleimide ligation After conjugation was completed, the reaction mixture was desalted and concentrated to yield anti-Her2 ADCs.
  • the biochemical properties of the resulting ADCs were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to confirm drug loading (DAR).
  • SEC-HPLC size-exclusion chromatography high pressure liquid chromatography
  • HIC-HPLC hydrophobic interaction chromatography HPLC
  • DAR drug loading
  • Anti-Her2 ADCs were prepared by conjugation of Herceptin with different camptothecin derivatives with similar DARs, and were tested against BT-474 breast cancer cells and N87 gastric cancer cells representing high Her2 expression, and BxPC-3 pancreatic cancer cells with low/none level of Her2.
  • In vitro cytotoxicity assay was performed. Free camptothecin derivatives were also tested in the same assay. Briefly, all cell lines were cultured in a suitable culture medium at 37°C in a humidified incubator atmosphere of 5% CO2. Cells were plated in 96-well flat bottom plates. Cell seeding number ranged from 500 cells/ /100 ul/ well to 6,000 cells/100 pl/well.
  • ADCs or free camptothecin derivatives were prepared from stock solution and diluted into appropriated working concentration 24 hours after cell seeding. A serial ten-fold dilution for seven points was performed with culture medium. The final concentrations were ranging from 10,000 nM to 0.001 nM. The cells were incubated with ADCs for 5 days. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot#PL701) was added to the wells for 1-4 hours at 37°C and the absorbance at 450 nm was measured using a Microplate Reader (SpectraMax M5, Molecular Devices) and SoftMax Pro5.4.1 software. Dose-response curves were generated and IC50 was calculated using GraphPad Prism 7 three-parameter curve fitting.
  • Figure 3 - Figure 6 show the representative killing curves of free camptothecin derivatives and ADCs containing payload BI-P352, BI-P353 in BT-474 (Figure 3), NCI-N87 (Figure 4), SK-BR-3 ( Figure 5) and BxPC-3 ( Figure 6) cells.
  • ADCs containing various camptothecin derivative payloads demonstrated specific and potent in vitro killing activities in Her2-expressingcells.
  • Table 2 and Table 3 summarize the IC50 values from in vitro cytotoxicity assays. Results demonstrate that Herceptin-352 and Herceptin-353 induced potent cytotoxicity against BT-474, SK-BR-3 and NCI-N87 tumor cells that have high Her2 expression, with IC50 in the sub nM range. In the Her2 negative BxPC-3 cells, IC50 of both ADCs was around 100 nM. On the other hand, the free payload BI-340 and BI-341 was able to kill all three cell lines with similar IC50s. These in vitro cytotoxicity results suggest a wide therapeutic window for the BI-340 and BI-341 containing ADCs.
  • Herceptin-BI-P353 (0.1 mg/ml) was incubated in biank human plasma at 37°C for up to 96 hours. At each time point, samples were drawn and the remaining amount of total antibody (naked + conjugated) and conjugated antibody (ADC) were measured using a quantitative sandwich enzyme linked immunoassay (ELISA).
  • ELISA sandwich enzyme linked immunoassay
  • human Her2 protein was coated onto the microplate to capture Herceptin antibodies. After removing the unbound antibodies, the bound total Herceptin antibody was detected with horseradish peroxidase (HRP) conjugated goat anti- human IgG Fc specific polycolonal antibody.
  • HRP horseradish peroxidase
  • Herceptin-BI-P353 ADC For the determination of Herceptin-BI-P353 ADC only, a mouse anti-DXd antibody was added as the secondary antibody. The concentration of Herceptin- BI-P353 ADC was then detected with HRP conjugated goat anti-mouse lgG(H+L) antibody. Figure xx shows the percentage of remaining total antibody and Herceptin-BI-P353 at different time points. The trend for Herceptin-BI-P353 was similar to that of total antibody. Both of them remained at 88% after 96 hours. These results indicate that Herceptin-BI-P353 is stable in human plasma.
  • Figure 8A shows the results of the in vivo NCI-N87 xenograft study. As shown, the administration of Herceptin-353 at both dose levels resulted in partial tumor regression, similar to the effects induced by Herceptin-DXd. Both ADCs had no obvious effect on mouse body weigh change compared to vehicle control as show in Figure 8B.

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Abstract

The present invention describes novel camptothecin derivatives and methods for preparing camptothecin derivative payloads bearing a linker with a functional group that can be used for conjugation to cell binding agents to generate cytotoxic drug conjugates. The present invention further relates to antibody drug conjugates made with camptothecin derivative payloads bearing a linker moiety conjugated to tumor associated antigen (TAA) antibody, preparation methods, pharmaceutical compositions and uses thereof for the treatment of cancer. Methods relating to the use of the novel ADCs to treat antigen positive cells in cancers and immunological disorders are also provided herein.

Description

NOVEL CAMPTOTHECIN DERIVA TVES AS ANTIBODY-DRUG CONJUGATES (ADC) PAYLOADS
RELATED APPLICATIONS
[001] This application claims benefit of U.S. Provisional Application No. 63/402,603 filed on August 31 , 2022, incorporated in its entirety by reference herein.
FIELD OF THE INVENTION
[002] The present invention describes novel camptothecin derivatives and methods for preparing these derivative payloads bearing a linker with a functional group that can be used for conjugation to cell binding agents to generate cytotoxic drug conjugates. The present invention further relates to therapeutic use of these conjugates for treatment of cancer as the conjugates are targeted and selectively delivered to a specific tumor cell population. The present invention further relates to antibody drug conjugates made with camptothecin derivative payloads bearing a linker moiety conjugated to tumor associated antigen (TAA) binding agents, preparation methods, pharmaceutical compositions and uses thereof for the treatment of cancer.
BACKGROUND OF THE INVENTION
[003] Today, cancer remains a major cause of death worldwide despite the numerous advanced diagnostic and therapeutic methods that have been developed. The major barrier to successful treatment and prevention of cancer lies in the fact that many cancers still fail to respond to the current chemotherapeutic and immunotherapy intervention, and many individuals suffer a recurrence or death, even after aggressive therapy.
[004] Cancer immunotherapy is enjoying a renaissance, and in the past few years the rapidly advancing field has produced several new methods of treating cancer. Numerous cancer immunotherapy strategies have been the focus of extensive research and clinical evaluation
Figure imgf000002_0001
including, but not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates: treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints); treatment using bispecific T cell engaging antibodies (BITE®) such as blinatumomab; treatment involving administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21 , GM-CSF IFN- a, IFN-p and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating lymphocytes (TILs); and treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and/or TOR transgenic).
[005] Antibody-drug conjugates (ADCs) combine the binding specificity of an antibody with the potency of drugs such as, for example, cytotoxic agents, anticancer and immunosuppressive drugs. The use of ADCs allows the target-specific delivery of drugs which, if administered as unconjugated drugs, may result in unacceptable levels of toxicity to normal cells. The mechanism of an ADC is to recognize and bind to specific antigen through the antibodies, trigger a series of reactions, and then enter the cytoplasm through the endocytosis, where the highly cytotoxic drug is dissociated from the antibody after the degradation by lysosomal enzymes to kill cancer cells. Compared with the traditional chemotherapy which causes damage to both cancer cells and normal tissues indiscriminately, targeting drug delivery can make the drug act on cancer cells directly and reduce the damage to normal cells.
[006] The cytotoxic compounds used in antibody-drug conjugates inhibit various essential cellular targets, such as microtubules (maytansinoids, auristatins, taxanes: U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333.410; 6,441 ,163; 6,340,701 : 6,372,738; 6,436,931; 6,596,757: 7.276,497; 7,301,019; 7,303,749; 7,368,565; 7,473,796; 7,585,857; 7,598,290: 7.495,114;
7,601 ,354, U.S. Patent Application Nos. 20100092495, 20100129314, 20090274713, 20090076263, 20080171865) and DNA (calicheamicin, doxorubicin, CC-1065 analogues: U.S. Pat. Nos. 5,475,092: 5,585,499; 5,846,545; 6,534,660; 6,756,397; 6,630,579; 7,388,026;
7,655,660; 7,655,661)
Figure imgf000003_0001
[007] Camptothecin (CPT) (Figure 1) was originally isolated from the bark of Camptotheca acuminata, a tree native to the rocky slopes of north China isolated by Wall et al. [Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J Am Chem Soc 1966; 88: 3888-3890], CPT has been demonstrated to be effective against a broad spectrum of tumors. Its molecular target has been firmly established to be DNA topoisomerase I (Topo I) that changes the topological state of duplex DNA by single-strand breakage and religation. [Chen AY, Liu LF. DNA topoisomerases: essential enzymes and lethal targets. Annu Rev Pharmacol Toxicol 1994; 34: 191-218; Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873-14878. Hsiang YH, Liu LF. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 1988; 48: 1722-1726], Biochemical studies in vitro have revealed that CPT binds at the interface between Topo I and DNA and inhibits specifically the religation step in the cleavage/religation reaction [Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985; 260: 14873-14878. Svejstrup JQ, Christiansen K, Gromova II, Anderson AH, Westergaard O. New technique for uncoupling the cleavage and relegation reactions of eukaryotic topoisomerase I: the mode of action of camptothecin at a specific recognition site. J Mol Biol 1991 ; 222: 669-678],
[008] CPT has attracted much attention because of a very good spectrum of its antitumor activity against experimental animal tumor models, such as L1210 leukemia in mice and Walker 256 sarcoma in rats. However, its clinical development failed due to the reversible bone marrow depression and hemorrhagic cystitis, which are the major dose-limiting toxicity [Muggia FM, Creaven PJ, Hansen HH, Cohen MH, Selawry OS. Phase I clinical trial of weekly and yearly treatment with camptothecin (NSC-100880); correlation with preclinical studies. Cancer Chemother Res 1972; 56: 515-521. Schappi U, Fleischmann RW, Cooney DA. Toxicity of camptothecin (NSC-100880). Cancer Cemother Rep Part 1974; 35: 25-36], Later, efforts directed at finding new camptothecin derivatives with higher anticancer activity and less toxicity led to the discovery of a potent and safer camptothecin derivative, named irinotecan or CPT-11 .
Figure imgf000004_0001
As one of the prominent anti-neoplastic drugs widely used in clinical practice today, CPT-11 is a water-soluble pro-drug and undergoes carboxylesterase-mediated hydrolysis to form SN-38, a potent Topo I inhibitor (Fig. 1) [Bencharit S, Morton CL, Howard-Williams EL, Danks MK, Potter PM, Redinbo MR. Structural insights into CPT-11 activation by mammalian carboxyesterases. Nat Struct Biol 2002: 9: 337-342. Andoh T, Ishii K, Suzuki Y, Ikegami Y, Kusunoki Y, Takemoto Y, et al. Characterization of mammalian mutant with a camptothecinresistant DNA topoisomerase I. Proc Natl Acad Sci USA 1987; 84: 5565-5569],
[009] Recently, with the FDA approval of Trodelvy® and Enhertu®, two camptothecin derivatives, SN-38 and DXd, have been validated as ADC payloads.
[010] There still exists a great need for camptothecin derivatives for use as ADC payloads for use in the treatment or to prevent recurrence of cancers and/or immunological disorders.
SUMMARY OF THE INVENTION
[011] The present invention describes novel camptothecin derivatives and methods for preparing payloads derived from them with linkers containing functional groups that can be used for conjugation to cell binding agents to generate cytotoxic drug conjugates.
[012] In one aspect, the present invention relates to an antibody drug conjugate (ADC) comprising an antibody (e.g., a cell binding antibody) chemically linked to a camptothecin analog residue represented by the following formula (I):
[D-L-]x-Ab (I)
[013] wherein x is about 1 to about 8;
[014] Ab is an antibody (e.g., a cell binding antibody) or antigen binding fragment thereof;
[015] wherein one or more camptothecin derivative drug moieties (D) have the structure:
Figure imgf000005_0001
Figure imgf000006_0001
[016] wherein Ri is a hydrogen atom or a C1-C6 alkyl
[017] X is -C(=O)-(CH2)n1-S-, -C(=O)-(CH2)n1-O-NR2-, -C(=O)-(CH2)n1-N(OR2)-, - C(=O)-(CH2)n1-NR2-, -S(=O)2-CH2-(CH2)n1-S-, -S(-O)2-CH2-(CH2)n1-O-1 -S(=O)2-CH2-(CH2)n1-O- NR2-:-S(-O)2-CH2-(CH2)n'-N(OR2)-, S(=O)2-CH2-(CH2)n1-N(R2)-, -S(=O)2-NH-CH2-(CH2)n1-S-i - S(“O)2“NH-CH2-(CH2)n1-O“, -C(-O)-O-(CH2)n2-S-, -C(=O)-O-(CH2)n2-O-, -C(-O)-O-(CH2)n2-O- NR2-, -C(=O)-O-(CH2)n2-N(OR2)-, -C^OWCH^-NCRa)-, -C(=O)-NH-(CH2)n2-S-, -C(==O)-NH- (CH2)n2-O-, -C(=O)-NH-(CH2)n2-O-NR2-, -C(=O)-NH-(CH2)n2-N(OR2)-, or -C(=O)-NH-(CH2)n2- N(R2)-;
[018] wherein L is a bivalent linker comprising a peptide moiety of 2-4 amino acid represented by the following formula (II):
[019] U-L2-L3 (II)
[020] wherein L1 is a sulfhydryl reactive linker attached to the antibody, L2 is peptide moiety, L3 is a self-immolative moiety connected to the drug moiety that is:
Figure imgf000006_0002
[021] and wherein R2 is a hydrogen atom or a C1-C6 alkyl, n1 is 1, 2, 3, 4 or 5, and n2 is 2, 3, 4 or 5.
[022] Another aspect of the invention is a pharmaceutical composition including a Formula I ADC compound, a mixture of Formula I ADC compounds, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient
[023] Another aspect provides a pharmaceutical combination comprising a Formula I ADC compound and a second compound having anticancer properties or other therapeutic effects.
[024] Another aspect is a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating the cells with an amount of an antibody-drug conjugate of Formula I, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.
[025] Another aspect is a method of treating cancer comprising administering to apatient a therapeutically effective amount of a pharmaceutical composition including a Formula I ADC.
[026] In various embodiments, the invention relates to antibody drug conjugates wherein x is about 1 to about 8. In various embodiments, x is about 4 to about 7. In various embodiments, x is about 4. in various embodiments, x is about 6. In various embodiments, x is about 7.
[027] In various embodiments, the invention relates to derivatized camptothecin analogs wherein L1 comprises pyrroline-dione. In various embodiments of the invention, the heterocyclyl ring is selected from saturated or unsaturated 4-6 membered nitrogen containing heterocyclic rings. Examples of saturated heterocyclic radicals include saturated 3 to 6- membered heteromonocylic group containing 1 to 4 nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidine, piperazinyl]; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. morpholinyl]; saturated 3 to 6- membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl]. Examples of unsaturated heterocyclic radicals, also termed "heteroaryl"
Figure imgf000007_0001
radicals, indude unsaturated 5 to 6 membered heteromonocycly! group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4- pyridyl. pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g , 4H-1 ,2,4-triazolyl, 1 H-1 ,2,3-triazolyl, 2H- 1 ,2,3-triazolyl]; unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g., tetrazolo [1,5-b]pyridazinyl]; unsaturated 5- to 6- membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1 ,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1 ,2,5- oxadiazolyl]; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g. benzoxazolyl, benzoxadiazolyl]; and unsaturated 5 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g., 1 ,2,4-thiadiazolyl. In various embodiments of the invention, the cyclic alkyl ring, also know as a cycloalkyl ring, is a saturated cyclic alkyl group derived by the removal of one hydrogen atom from a single carbon atom of a parent cycloalkane. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane, and the like.
[028] The present invention provides an anti-Her2 antibody (Herceptin) that is conjugated with camptothecin analogs, thus targeting disease cells or tissues. The anti-Her2 antibody binds to an antigen in the disease cells or tissues. A drug conjugated to the antibody exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen-expressing cells to treat or prevent recurrence of Her2 positive cancers. The high affinity of the antibody drug conjugate ensures that the camptothecin analogs targets the tumor cells. The present technology provides a method to treat cancers by exerting cellular inhibitory or killing effect of camptothecin analogs on the Her2 positive cells.
[029] In various embodiments, the ADC’s comprise an L that is a cleavable linker. In various embodiments, the ADC is an anti-Her2 antibody (Herceptin) conjugated with camptothecin analogs. In various embodiments, the ADC is an anti-Her2 antibody conjugated with a camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that is peptidase cathepsin sensitive. In various embodiments, the ADC is an anti-Her2 antibody conjugated with camptothecin analogs, wherein the camptothecin analogs
Figure imgf000008_0001
is linked to an anti-Her2 antibody via a linker that is not acid labile. In various embodiments, the ADC is an anti-Her2 antibody conjugated with camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that does not contain a disulfide bond. In various embodiments, the ADC is an anti-Her2 antibody conjugated with camptothecin analogs, wherein the camptothecin analogs is linked to an anti-Her2 antibody via a linker that provides stability during circulation while being able to release the drug once inside the cells. Such linkers are contemplated to provide stability to the conjugated molecule prior to endocytosis, such as during circulation, to prevent premature degradation of the linker and release of the toxic drug, thus minimize the toxic effect of the drug.
[030] In various embodiments there is provided one or more of a set of ADCs of Formulas I wherein L and L1 are cysteine reactive linkers.
[031] in various embodiments there is provided one or more of a set of compounds of Formula I and II wherein L and L2 are cleavabie linkers.
[032] In various embodiments, the number of bonds formed between the drug-linker and cysteine residue on the anti-Her2 antibody is from 3 to 8. In various embodiments, the number of such bonds is at least 2, or alternatively at least 4, or 5. In various embodiments, the number of such formed bonds is no more than 8, or alternatively no more than 7, 6, 5, or 4. In various embodiments, each anti-Her2 antibody, on average, is conjugated with about 4-7 drug molecules through cysteines.
[033] The drug load on an anti-Her2 antibody may vary depending on many factors, such as the potency of the drug, the size, stability of the anti-Her2 antibody, conjugatable groups available on the anti-Her2 antibody, etc. in various embodiments, 1 to 8 camptothecin analogs molecules are conjugated with 1 anti-Her2 antibody molecule. In various embodiments, an average of about 4 to 7 camptothecin analogs drug molecules are conjugated with an anti- Her2 antibody molecule.
[034] Another aspect of the invention relates to methods of inhibiting abnormal cell growth or treating a proliferative disorder, an autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis or
Figure imgf000009_0001
kidney disease in a mammal comprising administering to said mammal a therapeutically effective amount of the conjugate of formulas I, optionally, a chemotherapeutic agent.
[035] Another aspect of the invention relates to pharmaceutical compositions of the cell binding agent conjugates of formula I and a pharmaceutically acceptable carrier, additive or diluent thereof.
[036] In various embodiments, the ADC constructs of the present invention comprise an Ab that is a targeting moiety, such as an antibody or antibody fragment capable of binding to a tumor associated antigen (TAA), a tissue-specific antigen, a cell surface molecule, extracellular matrix protein or protease(s), or any post-translational modification residue(s). In various embodiments, the ADC constructs of the present invention comprise an Ab that is a targeting moiety that exhibits binding affinity to a diseased cell or tissue.
[037] In various embodiments, the antibody or antibody fragment is capable of binding to a TAA selected from the group consisting of: tumor-associated calcium signal transducer 2 (also known as Trop-2), Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CDS, CD7, CD13, CD19, CD20, CD21 , CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71 , CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1, uPA, MAGES, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta 6, 5T4, CA9, IGF-1 R, Axl, B7H3, B7H4, CDH6, HAVCR1, STEAP-1, STEAP-2, UPK2, CLDN18.2, CLDN6, CLDN9, c-Met, MICA/B, LIV-1 , ROR1 , ADAM9, STn, Globo H, SSEA-4, MG7-Ag, Fucosyl GM-1 , DLK-1, CEACAM5.
[038] In various embodiments, the ADC comprises a TAA binding Ab selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab’, a Fab2, a Fab'2, a IgG, a IgM , a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, and an diabody. In various embodiments, the antibody is a chimeric antibody. In various embodiments, the antibody is a humanized monoclonal antibody. In various embodiments, the antibody is a fully human monoclonal antibody.
Figure imgf000010_0001
[039] In another aspect, the present invention provides a pharmaceutical composition comprising the isolated ADC constructs in admixture with a pharmaceutically acceptable carrier. [040] In another aspect, the invention provides uses of the ADC constructs for the preparation of a medicament for the treatment of cancer.
[041] In another aspect, the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, or rhabdomyosarcoma or any cancer.
[042] In various embodiments, the subject previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”). In various embodiments, the subject has a resistant or refractory cancer.
[043] In another aspect, the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, iPS induced CAR-T or iPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG). In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1. PD-L1 , CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec 7, Siglec 8, Siglec 9, Siglec 15 and VISTA: treatment using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL- 12, IL-21 , GM-CSF, IFN-a, IFN-p and IFN-y; treatment using therapeutic vaccines such as
Figure imgf000011_0001
sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and/or TCR transgenic); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using vaccine such as BCG; wherein the combination therapy optionally provides increased effector cell killing of tumor cells, i.e. , a synergy exists between the ADC constructs and the immunotherapy when co-administered.
[044] In another aspect, there are provided novel compounds described herein as well as methods of making thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[045] Figure 1 depicts the structures of CRT, CPT-11, SN-38, exatecan and DXd.
[046] Figure 2 depicts conjugation procedure for camptothecin analog payload with reduced mAb.
[047] Figure 3 depicts line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P-352 and BI-P353 in Her2 positive breast cancer cell line BT-474.
[048] Figure 4 depicts line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P352 and BI-P353 in Her2 positive gastric cancer cell line NCI-N87.
[049] Figure 5A and figure 5B depict line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P352 and BI-P353 in Her2 positive breast cancer cell line SK-BR-3, respectively.
[050] Figure 6 depicts line graphs depicting the results of in vitro cytotoxicity assay of ADCs containing BI-P352 and BI-P353 in Her2 negative pancreatic cancer cell line BxPC-3. [051] Figure 7 depicts the plasm stability of Herceptin-BI-P353 ADC
[052] Figure 8 depicts line graphs depicting the in vivo efficacy of Herceptin-BI-P353
ADC in NCI-N87 xenograft models. Figure 8A depicts mean tumor volume (mm3) and Figure 8B depicts body weight (g).
Figure imgf000012_0001
MODE(S) OF CARRYING OUT THE INVENTION
[053] The present invention provides novel camptothecin linker-payloads, and novel antibody drug conjugates comprising a camptothecin linker-payloads of the present invention linked to an antibody for targeted delivery to disease tissues, in various embodiments, the present invention provides antibody-drug conjugates (ADCs) and ADC derivatives and methods relating to the use of such conjugates to treat cancer. The antibody, or other targeting moiety in the ADC, binds to e.g., a tumor associated antigen (TAA) on the cancer cell. In various embodiments, the antibody is conjugated to a novel camptothecin linker-payload which exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen expressing cells to treat or prevent recurrence of the antigen expressing cancers or immunological disorders. Importantly, the ADCs of the present invention have superior drug/antibody ratios (DARs), demonstrate improved solubility, enhanced CMC characteristics, and increased therapeutic efficacy particulary against high antigen expressing tumors while sparing the normal tissues expressing low or no level of antigen. Moreover, the ADCs provide for the targeting of broader patient populations and patients having a refractory cancer or who previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer").
Definitions
[054] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art. The methods and techniques of the present invention are generally performed according to
Figure imgf000013_0001
conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.
[055] As used herein, the term “alkyl” refers to a fully saturated branched or unbranched hydrocarbon moiety having up to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, n-decyl and the like.
[056] As used herein, the term “heterocyclyl”, “heterocycloalkyl” or “heterocyclo” referes to a saturated or unsaturated non-aromatic ring or ring system, and contains at least one heteroatom selected from O, S, and N. The heterocyclyl can be attached at a heteroatom, a carbon atom, or both.
[057] As used herein, the term “aryl” refers to an aromatic hydrocarbon group having 6- 20 carbon atoms in the ring portion. Typically, aryl is monocyclic, bicyclic or tricyclic aryl having 6-20 carbon atoms. Furthermore, the term "aryl" as used herein, refers to an aromatic moiety which can be a single aromatic ring, or multiple aromatic rings that are fused together. Non- limiting examples include phenyl, naphthyl or tetrahydronaphthyl, each of which may optionally be substituted with 1-4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S- nitro, cyano, carboxy, alkyl-O-C(O)--, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, phenyl, and heterocyclyl.
Figure imgf000014_0001
[058] As used herein, “cyclic alkyl” or “cycloalkyl” refers to a saturated or unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms. Unless otherwise provided, cycloalkyl refers to cyclic hydrocarbon moiety having between 3 and 9 ring carbon atoms or between 3 and 7 ring carbon atoms, each of which can be optionally substituted with one, or two, or three, or more substituents independently selected from the group consisting of alkyl, halo, oxo, hydroxy, alkoxy, alkyl-C(O)-, acylamino, carbamoyl, alkyl-NH--, (alkyl)2N-, thiol, alkyl-S-, nitro, cyano, carboxy, alkyl-O--C(O)--, sulfonyl, sulfonamide, sulfamoyl, and heterocyclyl.
[059] As used herein, the term "optionally substituted" unless otherwise specified refers to a group that is unsubstituted or is substituted with one or more, typically 1 , 2, 3 or 4, suitable non-hydrogen substituents.
[060] The point of attachment of a given moiety to the parent structure can be readily determined by one of skill in art. Thus, although the point of attachment may not be explicitly shown, it would be evident to the skilled artisan based on common general knowledge in the chemical arts.
[061] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, "peptides", "polypeptides", and "proteins" are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term "amino terminus” (abbreviated N-terminus) refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether bond as opposed to an amide bond.
Figure imgf000015_0001
[062] Polypeptides of the invention include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A "conservative amino acid substitution" refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. A "non-conservative amino acid substitution” refers to the substitution of a member of one of these classes for a member from another class. In making such changes, according to various embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8): glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[063] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in various embodiments, the substitution of amino acids whose hydropathic indices are within + 2 is included. In various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.
[064] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein,
Figure imgf000016_0001
as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
[065] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5): cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in various embodiments, the substitution of amino acids whose hydrophilicity values are within + 2 is included, in various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.
[066] The term "polypeptide fragment" and “truncated polypeptide” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to a corresponding full-length protein. In various embodiments, fragments can be, e.g., at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length. In various embodiments, fragments can also be, e.g., at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids in length. A fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence {e.g., an artificial linker sequence).
[067] The terms "polypeptide variant" and “polypeptide mutant” as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at
Figure imgf000017_0001
least 175, at least 200, at least 225, at least 250, at least 2/5, at least 300, at least 350, at least 400, at least 450 or at least 500 amino adds in length. Variants of the present invention indude fusion proteins.
[068] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
[069] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm and means that a given sequence is at least 80% identical to another length of another sequence. In various embodiments, the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
[070] The term "% sequence homology" is used interchangeably herein with the term ’’% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence. In various embodiments, the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In various embodiments, the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
Figure imgf000018_0001
[071] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet at the NCBI website. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (with special reference to the published default setting, i.e., parameters w=4, t=17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. See Id.
[072] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1, less than about 0.01 , or less than about 0.001.
[073] The terms "substantial similarity" or "substantially similar," in the context of polypeptide sequences, indicate that a polypeptide region has a sequence with at least 70%, typically at least 80%, more typically at least 85%, or at least 90% or at least 95% sequence similarity to a reference sequence. For example, a polypeptide is substantially similar to a second polypeptide, for example, where the two peptides differ by one or more conservative substitution(s).
[074] The term "recombinant polypeptide", as used herein, is intended to include all polypeptides, including fusion molecules and ADCs that are prepared, expressed, created,
Figure imgf000019_0001
derived from, or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell.
[075] The term "heterologous" as used herein refers to a composition or state that is not native or naturally found, for example, that may be achieved by replacing an existing natural composition or state with one that is derived from another source. Similarly, the expression of a protein in an organism other than the organism in which that protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.
[076] The term “tumor associated antigen” (TAA) refers to, e.g., cell surface antigens that are selectively expressed by cancer cells or over-expressed in cancer cells relative to most normal cells. The terms "TAA variant" and “TAA mutant" as used herein refers to a TAA that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another TAA sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length.
[077] The term "anti-TAA antagonist antibody" (interchangeably termed "anti-TAA antibody") refers to an antibody that is able to bind to TAA and inhibit TAA biological activity and/or downstream pathway(s) mediated by TAA signaling. An anti-TAA antagonist antibody encompasses antibodies that block, antagonize, suppress or reduce (including significantly) TAA biological activity, including downstream pathways mediated by TAA signaling, such as receptor binding and/or elicitation of a cellular response to TAA. For purpose of the present invention, it will be explicitly understood that the term "anti-TAA antagonist antibody" encompasses all the previously identified terms, titles, and functional states and characteristics whereby the TAA itself, an TAA biological activity (including but not limited to its ability to mediate any aspect of headache), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree. In some embodiment, an anti-TAA antagonist antibody binds TAA and prevents TAA binding to a TAA
Figure imgf000020_0001
receptor, in other embodiments, an anti-TAA antibody binds TAA and prevents activation of a TAA receptor. Examples of anti-TAA antagonist antibodies are provided herein.
[078] The term "[Target] antibody" should be interpreted as similar to “anti-[Target] antibody" and means an antibody capable of binding to the [Target], The term "Target" or [Target] shall be interpreted as a TAA or any molecule present at the surface of cells, preferably tumoral cells, more preferably mammals and human cells, and which can be used for drug delivery. Preferably, the Target is specifically express or overexpress on the surface of tumoral cells in comparison with normal cells.
[079] The term "antibody" is used herein to refer to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes and having specificity to a tumor antigen or specificity to a molecule overexpressed in a pathological state. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as subtypes of these genes and myriad of immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one “heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
[080] in a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3 (and in some instances, CH4). Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of
Figure imgf000021_0001
three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs has been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1, lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass
[081] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1 , CDR2, CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities (j.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).
[082] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. The Kabat database is now maintained online and CDR sequences can be determined, for example, see IMGT/V-QUEST programme version: 3.2.18 March 29, 2011 , available on the internet and Brochet, X. et al., Nucl. Acids Res. 36, W503-508, 2008). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17, 1986; Chothia et al., Nature, 342: 877-83, 1989. The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272, 1989;
"AbM™, A Computer Program for Modeling Variable Regions of Antibodies, ” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those
Figure imgf000022_0001
described by Samudrala et al, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, 1999. The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. BioL, 5:732-45, 1996.
[083] The term "Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes (e.g., the neonatal FcR (FcRn) binds to the Fc region of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG). Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (see, e.g., Winter et al., U.S. Patent No 5,648,260 and 5,624,821).
[084] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab2, F(ab)’2 fragments, single chain Fv proteins (“scFv”) and disulfide stabilized Fv proteins (“dsFv”), that bind to the target antigen. A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody encompasses e.g., monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody
Figure imgf000023_0001
fragments that exhibit the desired biological activity, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments or antigen binding fragments of any of the above.
[085] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. A "Fab fragment" comprises one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" comprises one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.
[086] Pepsin treatment of an antibody yields an F(ab')2 fragment that has two antigen- combining sites and is still capable of cross-linking antigen. A "F(ab')2 fragment" contains two iight chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
[087] The "Fv region" comprises the variable regions from both the heavy and light chains but lacks the constant regions.
[088] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649, U.S. Patent No. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.
[089] The terms "an antigen-binding fragment" and “antigen-binding protein” as used herein means any protein that binds a specified target antigen. "Antigen-binding fragment" includes but is not limited to antibodies and binding parts thereof, such as immunologically functional fragments. An exemplary antigen-binding fragment of an antibody is the heavy chain and/or light chain CDR(s), or the heavy and/or light chain variable region.
Figure imgf000024_0001
[090] The term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen binding protein, as used herein, is a species of antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragments are neutralizing fragments. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, a diabody, Fab', F(ab')2, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged serum half-life.
[091] Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL regions joined by a linker that is too short to allow for pairing between two regions on the same chain, thus allowing each region to pair with a complementary region on another polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-48, 1993; and Poljak et al., Structure, 2:1121-23, 1994). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
Figure imgf000025_0001
[092] Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). In various embodiments bispecific antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807, 1981 ; by "polydoma" techniques (see, e.g., U.S. Patent No. 4,474,893); or by recombinant DNA techniques. In various embodiments bispecific antibodies of the present invention can have binding specificities for at least two different epitopes at least one of which is a tumor associate antigen. In various embodiments the antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.
[093] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any specific method.
[094] The term “chimeric antibody” as used herein refers to an antibody which has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds targeted antigen.
[095] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo}., for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include
Figure imgf000026_0001
antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
[096] The term “humanized antibody” as used herein refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. In various embodiments, the framework regions are chosen from human germline exon XH, JH, VK and JK sequences. For example, acceptor sequences for humanization of FR of a VH domain can be chosen from genuine VH exons VH 1-18 (Matsuda et al., Nature Genetics 3:88-94, 1993) or VH1-2 (Shin et al., EMBO J. 10:3641-3645, 1991) and for the hinge region (JH), exon JH-6 (Mattila et al., Eur. J. Immunol. 25:2578-2582, 1995). In other examples, germline VK exon B3 (Cox et al., Eur. J. Immunol. 24:827-836, 1994) and JK exon JK-1 (Hieter et al., J. Biol. Chem. 257:1516-1522, 1982) can be chosen as acceptor sequences for VL domain humanization.
[097] The term "recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant, combinatorial human antibody library; antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In various embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody
Figure imgf000027_0001
germline repertoire in vivo. All such recombinant means are well known to those of ordinary skill in the art.
[098] The term "epitope" as used herein includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or “conformational." In a linear epitope, all the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present invention. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, e.g., the antibodies compete for binding to the antigen. The competition for binding to the epitope can be determined by any methods or techniques known by the person skilled in the art such as, without limitation, radioactivity, Biacore, ELISA, Flow cytometry, etc. As “which competes for binding to the epitope" it is meant a competition of at least 20%, preferentially at least 50% and more preferentially at least 70%.
[099] An antigen binding protein, including an antibody, "specifically binds" to an antigen if it binds to the antigen with a high binding affinity as determined by a dissociation constant (KD, or corresponding Kb, as defined below) value of at least 1 x 10‘6 M, or at least 1 x 10’7 M, or at least 1 x 10‘8 M, or at least 1 x 10"9 M, er at least 1 x 10 '"J M, or at least 1 x 10’11 M. An antigen binding protein that specifically binds to the human antigen of interest may be able to bind to the same antigen of interest from other species as well, with the same or different affinities. The term "KD" as used herein refers to the equilibrium dissociation constant of a specific antibody-antigen interaction.
Figure imgf000028_0001
[0100] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier "Pharmacologically effective amount" refers to that amount of an agent effective to produce the intended pharmacological result. "Pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.
[0101] The terms "treat”, "treating" and "treatment" refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to "alleviate" a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. As used herein, “treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (e.g., metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total). Also encompassed by "treatment" is a reduction of pathological consequence of a proliferative disease. The methods of the invention contemplate any one or more of these aspects of treatment.
[0102] The term "effective amount" or “therapeutically effective amount” as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor
Figure imgf000029_0001
size; (iii) inhibit, retard, slow to some extent and preferabiy stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. An effective amount can be administered in one or more administrations.
[0103] The term “half maximal effective concentration” (“ECsc”) corresponds to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after some specified exposure time. It is commonly used as a measure of drug's potency. The ECso of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed. The ECso of a quantal dose response curve represents the concentration of a compound where 50% of the population exhibits a response, after specified exposure duration. Concentration measures typically follow a sigmoidal curve, increasing rapidly over a relatively small change in concentration. This can be determined mathematically by derivation of the best-fit line.
[0104] "Adjuvant setting" refers to a clinical setting in which an individual has had a history of a proliferative disease, particularly cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (such as surgical resection), radiotherapy, and chemotherapy. However, because of their history of the proliferative disease (such as cancer), these individuals are considered at risk of development of the disease. Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment. The degree of risk (i.e., when an individual in the adjuvant setting is considered as "high risk" or "low risk") depends upon several factors, most usually the extent of disease when first treated. [0105] As used herein, the terms "co-administration", "co-administered" and "in combination with", referring to the fusion molecules of the invention and one or more other therapeutic agents, is intended to mean, and does refer to and include the following: simultaneous administration of such combination of fusion molecules of the invention and therapeutic agent] s) to an individual in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said individual; substantially simultaneous administration of such combination
Figure imgf000030_0001
ef fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said individual, whereupon said components are released at substantially the same time to said individual; sequential administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said individual with a significant time interval between each administration, whereupon said components are released at substantially different times to said individual; and sequential administration of such combination of fusion molecules of the invention and therapeutic agent(s) to an individual in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said individual, where each part may be administered by either the same or a different route.
[0106] The term “therapeutic protein" refers to proteins, polypeptides, antibodies, peptides or fragments or variants thereof, having one or more therapeutic and/or biological activities. Therapeutic proteins encompassed by the invention include but are not limited to, proteins, polypeptides, peptides, antibodies, and biologies (the terms peptides, proteins, and polypeptides are used interchangeably herein). It is specifically contemplated that the term "therapeutic protein" encompasses the fusion molecules of the present invention.
[0107] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine). In various embodiments, the patient can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system including, but not limited to patients having primary immune deficiency, AIDS; cancer and transplant patients who are taking certain immunosuppressive drugs; and those with inherited
Figure imgf000031_0001
diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including, but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high rate of mutations (Lawrence et al., Nature, 499(7457): 214-218, 2013).
[OIOS] The phrase “administering’’ or "cause to be administered" refers to the actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a patient, that control and/or permit the administration of the agent(s)/compound(s) at issue to the patient. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic regimen, and/or prescribing particular agent(s)/compounds for a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like. Where administration is described herein, "causing to be administered" is also contemplated.
[0109] “Resistant or refractory cancer” refers to tumor cells or cancer that do not respond to previous anti-cancer therapy including, e.g., chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the beginning of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the onset of treatment or respond initially for a short period but fail to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but fail to respond to subsequent rounds of therapies. For purposes of this invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with anticancer therapy but recur up to five years, sometimes up to ten years or longer after treatment is discontinued. The anticancer therapy can employ chemotherapeutic agents alone, radiation alone, targeted therapy alone, surgery alone, or combinations thereof. For ease of description and not limitation, it will be understood that the refractory tumor cells are interchangeable with resistant tumor. [OHO] The term “tumor microenvironment” refers to the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow- derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). Components in the tumor microenvironment can modulate the growth of tumor cells,
Figure imgf000032_0001
e.g., their ability to progress and metastasize. The tumor microenvironment can aiso be influenced by the tumor releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance.
[0111] The term "proliferative disease” includes tumor disease (including benign or cancerous) and/or any metastases. A proliferative disease may include hyperproliferative conditions such as hyperplasias, fibrosis (especially pulmonary, but aiso other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. In some embodiments, the proliferative disease is cancer. In some embodiments, the proliferative disease is a nan-cancerous disease. In some embodiments, the proliferative disease is a benign or malignant tumor.
[0112] The term "immunogenicity" as used herein refers to the ability of an antibody or antigen binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient and includes, for example, the human anti-mouse antibody (HAMA) response. A HAMA response is initiated when T-cells from a subject make an immune response to the administered antibody. The T-cells then recruit B-cells to generate specific "anti-antibody” antibodies.
[0113] The term "immune cell" as used herein means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen). In various embodiments, an immune cell is, e.g., a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, Langerhan’s cells, or Kuffer cells.
[0114] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-0-
Figure imgf000033_0001
methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."
[0115] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3‘ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5‘-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3' end of the coding RNA transcript are referred to as “downstream sequences."
[0116] "Complementary" refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.
[0117] "Hybridizing specifically to” or "specific hybridization" or "selectively hybridize to", refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. "Stringent hybridization" and "stringent
Figure imgf000034_0001
hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence-dependent and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N.Y.; Sambrook et al., 2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3.sup.rd ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.
[0118] Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than about 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCI at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2 x SSC wash at 65°C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A high stringency wash can be preceded by a low stringency wash to remove background probe signal. An exemplary medium stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 1 x SSC at 45°C for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 4-6 x SSC at 40" C for 15 minutes In general, a signal to noise ratio of 2 x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
[0119] "Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e. , in the presence of nucleotides, a
Figure imgf000035_0001
complementary polynucleotide template, and an agent for polymerization such as ONA polymerase. A primer is typically single-stranded but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary' to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
[0120] "Probe,” when used in reference to a polynucleotide, refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In instances where a probe provides a point of initiation for synthesis of a complementary polynucleotide, a probe can also be a primer.
[0121] "Linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5’ end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers may also be cleaved by environmental cues, such as, for example, changes in temperature, pH, salt concentration, etc. Non-cleavable linkers are linkers that release an attached payload via lysosomal degradation of the antibody following internalization.
[0122] The terms "label" or "labeled" as used herein refers to incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties
Figure imgf000036_0001
that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, !4C, 15N, 35S, 90Y, "Tc, 111 In, 125l, 131l), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
[0123] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a “plasmid," which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g , non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a chosen polynucleotide.
[0124] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The
Figure imgf000037_0001
regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g, polypeptides that bind to the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06. A nucleotide sequence is “operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g, the level, timing, or location of expression) of the nucleotide sequence.
[0125] A "host cell" is a cell that can be used to express a polynucleotide of the invention. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g, a yeast or other fungus), a plant cell (e.g, a tobacco or tomato plant ceil), an animal cell (e.g, a human cell, a monkey ceil, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell also can be a ceil that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g, mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
[0126] The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally
Figure imgf000038_0001
originates, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
[0127] A protein or polypeptide is "substantially pure,” "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
[0128] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means “and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
[0129] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X" includes description of "X".
[0130] As used herein and in the appended claims, the singular forms “a," “or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that
Figure imgf000039_0001
aspects and variations of the invention described herein include "consisting" and/or "consisting essentially of" aspects and variations.
Antibody Drug Conjugates
[0131] In one aspect, the present invention is directed to an ADC of the following formula (I):
[D-L-]x-Ab
Figure imgf000040_0001
or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody, or an antigen binding antibody fragment thereof; L is a linker; and D is a camptothecin analog drug moiety.
[0132] wherein x is about 1 to about 8;
[0133] wherein the Ab is an antibody (e g., a cell binding antibody) or antigen binding fragment thereof;
[0134] In various embodiments, the camptothecin analog drug moieties of the invention have the structure:
Figure imgf000040_0002
[0135] wherein Ri is a hydrogen atom or a C1-C6 alkyl;
Figure imgf000040_0003
[0136] wherein X is -C(=O)-(CH2)n1-S-, -C(=O)-(CH2)n1-O-NR2-, -C(=O)-(CH2)n1- N(OR2)-, -C(=O)-(CH2)n1-NR2-, -S(=O)2-CH2-(CH2)n1-S-, -S(=O)2-CH2-(CH2)n1-O-, -S(=O)2-CH2- (CH2)n1-O-NR2-,-S(=O)2-CH2-(CH2)n1-N(OR2)-, S(=O)2-CH2-(CH2)n1-N(R2)-, -S(=O)2-NH-CH2- (CH2)n1-S-, -S^O^-NH-CHHCH^-O-, -C(=O)-O-(CH2)n2-S-s -C(=O)-O-(CH2)n2-O-, -C(=O)-O- (CH2)n2-O-NR2-, -C(=O)-O-(CH2)n2-N(OR2)-, -C(=O)-O-(CH2)n2-N(R2)-, -C(=O)-NH-(CH2)n2-S-, - C(=O)-NH-(CH2)n2-O-, -C(=O)-NH-(CH2)n2-O-NR2-, -C(=O)-NH~(CH2)n2-N(OR2)-, or -C(=O)-NH- (CH2)n2-N(R2)~;
[0137] wherein L is a bivalent linker comprising a peptide moiety of 2-4 amino acid represented by the following formula (II):
U~L2-L3 (II)
[0138] wherein L1 is a linker attached to the antibody comprising a reactive functional group selected from maleimide, thiol, amino, alkyl bromide, alkyl iodide, carboxyl, and NHS ester. In various embodiments, the reactive functional group include NHR2, OH, SH, - CH2CH2SH, - CO2H, and NHS ester;
[0139] wherein L2 is a 2-4 AA peptide moiety, L3 is a self-immolative moiety connected to the drug moiety that is:
Figure imgf000041_0001
[0140] wherein R2 is a hydrogen atom or a C1-C6 alkyl, n1 is 1 , 2, 3, 4 or 5, and n2 is 2,
3, 4 or 5.
[0141] Accordingly, the drug moiety reagents include the structures:
Figure imgf000041_0002
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000044_0001
Figure imgf000045_0001
[0142] wherein R2 is hydrogen atom or C1-C6 alkyl, m is 0, 1, 2, 3 or 4.
[0143] The linker, L, attaches the antibody to a drug moiety through covalent bond(s).
The linker is a bifunctional or multifunctional moiety which can be used to link one or more drug moiety (D) and an antibody unit (Ab) to form antibody-drug conjugates (ADC) of Formula I. The linker (L) may be stable outside a cell, • e., extracellular, or it may be cleavable by enzymatic activity, hydrolysis, or other metabolic conditions. Antibody-drug conjugates (ADC) can be conveniently prepared using a linker having reactive functionality for attaching to the drug moiety and to the antibody. A cysteine thiol, or an amine, e.g., N- terminus or amino acid side chain such as lysine, of the antibody (Ab) can form a bond with a functional group of a linker reagent, drug moiety (D) or drug-linker reagent (D-L).
[0144] The linkers are preferably stable outside the target cell. Before being internalized into a cell, the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the drug moiety remains linked to the antibody. An effective linker will: (I) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (ill) remain stable and intact, i.e., not cleaved, until the conjugate has been delivered to its targeted site: and (iv) maintain a cytotoxic, cell- killing effect or a cytostatic effect of the camptothecin analog drug moiety. Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation/analysis technique LC/MS. [0145] Covalent attachment of the antibody and the drug moiety requires the linker to have two reactive functional groups. Bivalent linker reagents which are useful to attach two or
Figure imgf000045_0002
more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G.T. (1996) Bioconjugate Techniques: Academic Press: New York, p 234-242).
[0146] In another embodiment, the linker may be substituted with groups which modulate solubility or reactivity. For example, a sulfonate substituent may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or the drug moiety or facilitate the coupling reaction of Ab-L with D, or D-L with Ab, depending on the synthetic route employed to prepare the ADC.
[0147] Nucleophilic groups on antibodies include but are not limited to: (i) N- terminal amine groups, (ii) side chain amine groups, e.g., lysine, (ill) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyi groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction oflysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non- native cysteine amino acid residues). US 2007/0092940 teaches engineering antibodies by introduction of reactive cysteine amino acids.
[0148] In some embodiments, a linker has a reactive nucleophilic group which is reactive with an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form
Figure imgf000046_0001
a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker.
[0149] Nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
[0150] Linkers can be peptidic, comprising one or more amino acid units. Peptide linker reagents may be prepared by solid phase or liquid phase synthesis methods (E. Schroder and K. Lubke, The Peptides, volume 1 , pp 76-136 (1965) Academic Press) that are well known in the field of peptide chemistry, including t-BOC chemistry (Geiser et al "Automation of solid- phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R. Uss, Inc., 1988, pp. 199-218) and Fmoc/HBTU chemistry (Fields, G. and Noble, R. (1990) “Solid phase peptide synthesis utilizing 9-fiuoroenylmethoxycarbonyl amino acids”, Int. J. Peptide Protein Res. 35:161-214), on an automated synthesizer such as the Rainin Symphony Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ), or Model 433 (Applied Biosystems, Foster City, CA).
[0151] Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine- phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly- val-cit) and glycine-glycine-glycine (gly-gly-gly). Exemplary tetrapeptides include: glycine-glycine- valine-citruiline (gly-gly-val-cit) and glycine-glycine-phenylalanine-citrulline (gly-gly- phe-gly). Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for
Figure imgf000047_0001
enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
[0152] Embodiments of drug-linker reagents include:
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000050_0002
Figure imgf000051_0001
Figure imgf000051_0002
Figure imgf000052_0001
[0153] wherein R1 is H or C1-C6 alkyl, n< is 0, 1 , 2, 3, or 4;
[0154] and wherein R2 and R3 are independently an amino acid side chain selected from hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, - CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, - 2)3NHCOCH3, - 4NHCOCH3, ~(CH2)4NHCHO,
Figure imgf000052_0002
2NH2, 2-pyridylmethyl-, 3- pyridylmethyi-, 4-pyridylmethyl-, phenyl and cyclohexyl.
[0155] In accordance with some of the methods described herein, the ADC or ADC derivative is internalized by targeted tumor cells or by activated immune cells, where the ADC or ADC derivative exerts a cytotoxic, cytostatic, or immunosuppressive effect on the antigen
Figure imgf000052_0003
expressing ceils to treat or prevent recurrence of the antigen expressing cancers or immunological disorders. In certain embodiments, the ADC or ADC derivative is not internalized, and the anti-Target Ab is effective to deplete or inhibiting target antigen-expressing cells by binding to the cell membrane. In certain embodiments, the ADC or ADC derivatives thereof can be targeted to a biological molecule in a cell (e.g., an inflammatory agent) and accumulate at or adjacent cells secreting or binding the biological molecule, where the therapeutic drug moiety exerts an effect (e.g., a cytotoxic, cytostatic, or immunosuppressive effect).
[0156] Importantly, the ADCs of the present invention have superior drug/antibody ratios (DARs), demonstrate improved solubility, enhanced CIVIC characteristics, and increased therapeutic efficacy against high antigen expressing tumor cells while having less effect on low or no antigen expressing cells i.e. normal cells. Moreover, the ADCs provide for the targeting of broader patient populations and patients with a refractory cancer or who previously responded to treatment with an anti-cancer therapy, but, upon cessation of therapy, suffered relapse (hereinafter “a recurrent cancer”).
Target Antigens and Exemplary Antibodies
[0157] Tumor antigens expressed on the cell membrane are potential targets in immunotherapy, with the ideal tumor antigen absent on normal cells and overexpressed on the tumor cell surface. The ADCs used in the methods of the present invention may comprise an antibody, or antigen binding antibody fragment, specific to any of the tumor associated antigens described in the art, including any biosimilar, biogeneric, follow-on biologic, or follow-on protein version of any TAA described in the art. The TAA can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which the skilled artisan wishes to induce an immune response.
[0158] In various embodiments, the TAA, TAA variant, or TAA mutant contemplated for use in the combination methods of the present invention is selected from, or derived from, the list provided in Table 1.
Table 1
Figure imgf000053_0001
| | | | | | | | | | | | | i | | i | | | | | | |
Figure imgf000054_0001
| | | | | | | | | | | i | | | | I | | | | | i | | | |
Figure imgf000055_0001
|
Figure imgf000056_0002
[0159] ki various embodiments, the TAA has an amino acid sequence that shares an observed homology of, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 9/%, 98%, or at least about 99% with any one of the sequences disclosed in Table 1.
[0160] Methods of generating antibodies that bind to the TAAs described herein are known to those skilled in the art. For example, a method for generating a monoclonal antibody that binds specifically to a targeted antigen polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the targeted antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g.,
Figure imgf000056_0001
cells from the spleen) from the mouse and fusing the antibody-producing cells with myeioma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the targeted antigen polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to targeted antigen polypeptide. The monoclonal antibody may be purified from the cell culture. A variety of different techniques are then available for testing an antigen/antibody interaction to identify particularly desirable antibodies.
[0161] Other suitable methods of producing or isolating antibodies of the requisite specificity can used, including, for example, methods which select recombinant antibody from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies. See e.g., Jakobovits et al., Proc. Natl. Acad. Sci. (U.S.A.), 90: 2551-2555, 1993; Jakobovits et al., Nature, 362: 255-258, 1993; Lonberg et al., U.S. Pat. No. 5,545,806; and Surani et al., U.S. Pat. No. 5,545,807.
[0162] Antibodies can be engineered in numerous ways. They can be made as single- chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')z fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.
[0163] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al., Science, 240:1041-1043, 1988; Liu et al., Proc. Natl. Acad.
Figure imgf000057_0001
Sci. (U.S.A.), 84:3439-3443, 1987; Liu et al., J. Immunol., 139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. (U.S.A.), 84:214-218, 1987; Nishimura et ai., Cane. Res., 47:999-1005, 1987; Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst., 80:1553- 1559, 1988).
[0164] Methods for humanizing antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced from a source that is nonhuman, in addition to the nonhuman CDRs. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525, 1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al.. Science, 239:1534-1536, 1988), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable region has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.
[0165] U.S. Patent No. 5,693,761 to Queen et al, discloses a refinement on Winter et al. for humanizing antibodies, and is based on the premise that ascribes avidity loss to problems in the structural motifs in the humanized framework which, because of steric or other chemical incompatibility, interfere with the folding of the CDRs into the binding-capable conformation found in the mouse antibody. To address this problem, Queen teaches using human framework sequences closely homologous in linear peptide sequence to framework sequences of the mouse antibody to be humanized. Accordingly, the methods of Queen focus on comparing framework sequences between species. Typically, all available human variable region sequences are compared to a particular mouse sequence and the percentage identity between correspondent framework residues is calculated. The human variable region with the highest percentage is selected to provide the framework sequences for the humanizing project. Queen also teaches that it is important to retain in the humanized framework, certain amino acid residues from the mouse framework critical for supporting the CDRs in a binding-capable conformation. Potential criticality is assessed from molecular models. Candidate residues for
Figure imgf000058_0001
retention are typically those adjacent in linear sequence to a CDR or physically within 6.A of any CDR residue.
[0166] In other approaches, the importance of particular framework amino acid residues is determined experimentally once a low-avidity humanized construct is obtained, by reversion of single residues to the mouse sequence and assaying antigen binding as described by Riechmann et al, 1988. Another example approach for identifying important amino acids in framework sequences is disclosed by U.S. Patent No. 5,821,337 to Carter et al, and by U.S. Patent No. 5,859,205 to Adair et al. These references disclose specific Kabat residue positions in the framework, which, in a humanized antibody may require substitution with the correspondent mouse amino acid to preserve avidity.
[0167] Another method of humanizing antibodies, referred to as "framework shuffling", relies on generating a combinatorial library with nonhuman CDR variable regions fused in frame into a pool of individual human germline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). The libraries are then screened to identify clones that encode humanized antibodies which retain good binding.
[0168] The choice of human variable regions, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (framework region) for the humanized antibody (Sims et al., J. Immunol., 151 :2296, 1993; Chothia et al., J. Mol. Biol., 196:901 , 1987). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. (U.S.A.), 89:4285, 1992: Presta et al., J. Immunol., 151 :2623, 1993).
[0169] The choice of nonhuman residues to substitute into the human variable region can be influenced by a variety of factors. These factors include, for example, the rarity of the amino acid in a particular position, the probability of interaction with either the CDRs or the
Figure imgf000059_0001
antigen, and the probability of participating in the interface between the light and heavy chain variable domain interface. (See, for example, U.S. Patent Nos. 5,693,761 , 6,632,927, and 6,639,055). One method to analyze these factors is through the use of three-dimensional models of the non-human and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, nonhuman residues can be selected and substituted for human variable region residues in order to achieve the desired antibody characteristic, such as increased affinity for the target antigen(s).
[0170] Methods for making fully human antibodies have been described in the art. By way of example, a method for producing a TAA antibody or antigen-binding fragment thereof comprises the steps of synthesizing a library of human antibodies on phage, screening the library with TAA or an antibody-binding portion thereof, isolating phage that bind TAA, and obtaining the antibody from the phage. By way of another example, one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with TAA or an antigenic portion thereof to create an immune response, extracting antibody-producing ceils from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage. Recombinant anti-TAA antibodies of the invention may be obtained in this way. [0171] Again, by way of example, recombinant human anti-TAA antibodies of the invention can also be isolated by screening a recombinant combinatorial antibody library.
Preferably the library is a scFv phage display library, generated using human VL and VH CDNAS prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the
Figure imgf000060_0001
Stratagene SurfZAP™ phage display kit, catalog no. 240612). There also are other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271 , WO 92/20791 , WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology, 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85, 1992; Huse et al., Science, 246:1275-1281, 1989; McCafferty et al., Nature, 348:552-554, 1990; Griffiths et al., EMBO J., 12:725-734, 1993; Hawkins et al., J. Mol. Biol., 226:889-896, 1992; Clackson et al., Nature, 352:624-628, 1991 ; Gram et al., Proc. Natl. Acad. Sci. (U.S.A ), 89:3576-3580, 1992; Garrad et al., Bio/Technology, 9:1373-1377, 1991 ; Hoogenboom et al., Nuc. Acid Res., 19:4133-4137, 1991 ; and Barbas et al., Proc. Natl. Acad. Sci. (U.S.A.), 88:7978-7982, 1991), all incorporated herein by reference.
[0172] Human antibodies are also produced by immunizing a non-human, transgenic animal comprising within its genome some or all of human immunoglobulin heavy chain and light chain loci with a human IgE antigen, e.g., a XenoMouse™ animal (Abgenix, Inc./Amgen, Inc.- Fremont, Calif.). XenoMouse™ mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 , 1994 and U.S. Pat. Nos. 5,916,771 , 5,939,598, 5,985,615, 5,998,209, 6,075,181 , 6,091 ,001 , 6,114,598, 6,130,364, 6,162,963 and 6,150,584. XenoMouse™ mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies. In some embodiments, the XenoMouse™ mice contain approximately 80% of the human antibody V gene repertoire through introduction of megabase sized, germline configuration fragments of the human heavy chain loci and kappa light chain loci in yeast artificial chromosome (YAC). In other embodiments, XenoMouse™ mice further contain approximately all of the human lambda light chain locus. See Mendez et al , Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J Exp. Med., 188:483-495, 1998; and WO 98/24893.
[0173] In various embodiments, the ADCs of the present invention utilize an antibody or antigen-binding fragment thereof is a polyclonal antibody, a monoclonal antibody or antigen- binding fragment thereof, a recombinant antibody, a diabody, a chimerized or chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof,
Figure imgf000061_0001
a fully human antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen- binding fragment thereof, a single chain antibody, an Fv, an Fd, an Fab, an Fab', or an F(ab')2, and synthetic or semi-synthetic antibodies.
[0174] In various embodiments, the ADCs of the present invention utilize an antibody or antigen-binding fragment that binds to a TAA with a dissociation constant (KD) of, e.g., at least about 1x10'3 M, at least about 1x10'4 M, at least about 1x10'5 M, at least about 1x10’8 M, at least about 1x1 O'7 M, at least about 1x1 O'8 M, at least about 1x10'9 M, at least about 1x10'10 M, at least about 1x1 O'11 M, or at least about 1x1 O'12 M. In various embodiments, the fusion molecules of the present invention utilize an antibody or antigen-binding fragment that binds to a TAA with a dissociation constant (KD) in the range of, e.g., at least about 1x10'3 M to at least about 1x1 O'4 M. at least about 1x10-4 M to at least about 1x10‘5 M, at least about 1x10-s M to at least about 1x1 O'6 M, at least about 1x10‘6 M to at least about 1x1 O’7 M, at least about 1x10‘7 M to at least about 1x10"8 M, at least about 1x1 O’8 M to at least about 1x10"® M, at least about 1x1 O’9 M to at least about 1x10' 10 M, at least about 1x1 O’10 M to at least about 1x1 O’11 M, or at least about 1x10-11 M to at least about 1x1 O'12 M.
[0175] In various embodiments, the ADCs of the present invention utilize an antibody or antigen-binding fragment that cross-com petes for binding to the same epitope on the TAA as a reference antibody which comprises the heavy chain variable region and light chain variable region set forth in the references and sequence listings provided herein.
[0176] In various embodiments, antibodies contemplated for use in the ADCs of the present invention include but are not limited to LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101 , anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 or KC4 (both anti- mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM5), MN- 15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p). Immu 31 (an anti-alpha- fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti- PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti- PSM.A), G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab (anti-
Figure imgf000062_0001
CD52), bevacizumab (antl-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-mucin) ;trastuzumab (anti-ErbB2); and anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremeiimumab (PFIZER). Such antibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084: 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491 ; 7,612,180; 7,642,239; and U.S. Patent Application Pub!. Nos 20050271671; 20060193865; 20060210475; 20070087001 ; the Examples section of each incorporated herein by reference.) Specific known antibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No. 7,109,304), hlMMU-31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat No. 7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541 ,440), hR1 (U.S. patent application Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No. 7,541,440), AB-PG1- XG1-026 (U.S. patent application Ser. No. 11/983,372, deposited as ATCC PTA-4405 and PTA- 4406) and D2/B (WO 2009/130575) the text of each recited patent or application is incorporated herein by reference.
[0177] In various embodiments, the ADCs of the present invention include at least one antibody or fragment thereof that binds to Her2.
[0178] In another aspect, the present invention features bispecific molecules comprising an anti-Her2 antibody, or antigen-binding fragment thereof, of the invention. An antibody of the invention, or antigen-binding fragment thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other
Figure imgf000063_0001
binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results. In various embodiments, the invention includes bispecific molecules capable of binding both to FcyR or FcaR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing Her2. In such embodiments, the bispecific molecules target Her2 expressing cells to effector cell and trigger Fc receptor-mediated effector cell activities, e.g., phagocytosis of a Her2 expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion Methods of preparing the bispecific molecules of the present invention are well known in the art.
[0179] In various embodiments, another functional molecule (e.g., another antibody or ligand for a receptor) which is linked to the anti-Her2 antibody, can be selected from the group consisting of: agonistic, antagonistic, or blocking antibodies to signaling molecules such as Trop-2, Her-3, EGFR, IGF-R, c-Met, EphA2, EphB2, and MUC16; agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecoles (immune checkpoints) such as PD-1, PD-L1, OX-40, CS137, GITR, LAG3, TIM-3, and VISTA; CD3 found on T cells.
[0180] Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). In various embodiments, antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807, 1981 ; by "polydoma" techniques (see, e.g., U.S. Patent No. 4,474,893); or by recombinant DNA techniques.
[0181] In various embodiments bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes at least one of which is a tumor associate antigen. In various embodiments the antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having a different specificity.
Drug Moiety
Figure imgf000064_0001
[0182] In the ADCs of the present invention, any agent that exerts a therapeutic effect on cancer cells or activated immune ceils can be used as the warhead conjugated to an anti- target antigen antibody. Useful classes of cytotoxic or immunosuppressive agents include, for example, antitubulin agents (e.g., auristatins and maytansinoids), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and-carboplatin), anthracyclines, antibiotics, antifolates, anti metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, RNA polymerase inhibitors (e.g., amatoxins such as a-amanitin), kinase inhibitors, vinca alkaloids, oligonucleotides, or the like.
[0183] Individual cytotoxic or immunosuppressive agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5-fiuordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6- mercaptopunne, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP- 16 and VM-26.
[0184] In various embodiments, the therapeutic drug moiety is a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, a-amanitin, and mitoxantrone. In various embodiments, the cytotoxic agent is a conventional chemotherapeutic such as, for example, doxorubicin,
Figure imgf000065_0001
paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. In addition, potent agents such as CC-1065 analogues, calicheamicin, maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can be used in the ADCs of the present invention.
[0185] Techniques for conjugating therapeutic agents to proteins, and in particular to antibodies, are well-known. (See, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs in Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89/12624.)
[0186] Linkers in ADCs may have significant impacts on the biological activities. For example, in vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation method in developing safe and efficacious ADCs for cancer therapy.
[0187] Some embodiments of the invention relate to camptothecin payloads linked to antibodies via a cleavable linker. In this example, antibody was reduced by adding antibody to TCEP (Tris (2-carboxy ethyl) phosphine) dissolved in a pH-adjusted PBS EDTA buffer. The antibody/TCEP solution was incubated at 37°C for 2-3 hrs. The reduced antibodies were buffer exchanged into conjugation reaction buffer. The camptothecin payload dissolved in DMSO was added to a solution of reduced monoclonal antibody at payload/antibody ratio of 7 ■ 30:1 in order to achieve different drug to antibody ratios (DARs). The payload/antibody solution was incubated for 1-2 hr at 20°C while the reduced antibody was conjugated to the payload via the maleimide group. After conjugation was completed, the reaction mixture was desalted and concentrated to yield the ADCs. The biochemical properties of the resulting ADCs were
Figure imgf000066_0001
characterized using size-exclusion chromatography high pressure liquid chromatography (SEC- HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to confirm drug loading (DAR). The final ADC products are comprised of four or six or seven payload-linker molecules. The cysteine conjugation method used in the conjugation process produces more homogenouse ADCs compared to lysine conjugation method.
[0188] In ADCs, high drug loading, e.g., drug ratio >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. Typically, drug moieties conjugated to an antibody during a conjugation reaction are less than the theoretical maximum. The drug loading also referred as the Drug-Antibody ratio (DAR) is the average number of drugs per antibody. In the case of antibody IgG 1 and lgG4 isotypes, where the drugs are bound to cysteines after partial antibody reduction, drug loading may range from 1 to 8 drugs (D) per antibody, i.e., where 2, 4, 5, 6, and 8 drug moieties are covalently attached to the antibody. In the case of an antibody lgG2 isotype, where the drugs are bound to cysteines after partial antibody reduction, drug loading may range from 1 to 12 drugs (D) per antibody, i.e. , where 2, 4, 6, 8, 10, and 12 drug moieties are covalently attached to the antibody. Compositions of ADCs include collections of cell binding agents, e.g., antibodies, conjugated with a range of drugs, from 1 to 8 or 1 to 12. The average number of drugs per antibody in preparations of ADCs from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis. The ADCs of the present invention has much higher solubility compared to those ADCs containing SMCC- DM1 or vc-MMAE payload, therefore allow more drugs to be conjugated to the antibody i.e., DAR > 7.
Pharmaceutical Compositions
[0189] In another aspect, the present invention provides a pharmaceutical composition comprising an ADC as described herein, with one or more pharmaceutically acceptable excipient(s). The pharmaceutical compositions and methods of uses described herein also encompass embodiments of combinations (co-administration) with other active agents, as
Figure imgf000067_0001
detailed below. The ADCs provided herein can be formulated by a variety of methods apparent to those of skill in the art of pharmaceutical formulation. Such methods may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all GMP regulations of the U.S. Food and Drug Administration.
[0190] Generally, ADCs of the invention are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s), or carriers. Such pharmaceutically acceptable excipients and carriers are well known and understood by those of ordinary skill and have been extensively described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990) The pharmaceutically acceptable carriers may be included for purposes of modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Such pharmaceutical compositions may influence the physical state, stability, rate of in viva release, and rate of in viva clearance of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine): antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide): solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20,
Figure imgf000068_0001
polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles: diluents; excipients and/or pharmaceutical adjuvants.
[0191] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present invention, compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the therapeutic composition may be formulated as a lyophilizate using appropriate excipients such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage.
[0192] The pharmaceutical compositions of the invention are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a patient and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like. In various embodiments, the pharmaceutical composition is formulated for parenteral administration via a route selected from, e.g., subcutaneous injection, intraperitoneal injection, intramuscular Injection, intrasternal injection,
Figure imgf000069_0001
intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.
[0193] When parenteral administration is contemplated, the therapeutic pharmaceutical compositions may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired ADC in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical formulations suitable for injectable administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Other parentally administrate formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
[0194] Any method for formulating and administering peptides, proteins, antibodies, and immunoconjugates accepted in the art may suitably be employed for administering the ADCs of the present invention.
Dosing
[0195] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses
Figure imgf000070_0001
may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The term "dosage unit form," as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
[0196] The precise dose of ADC to be employed in the methods of the present invention will depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. It is to be noted that dosage values may include single or multiple doses, and that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Further, the dosage regimen with the compositions of this disclosure may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the subject, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses intra-subject dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
[0197] For administration to human subjects, the total monthly dose of the ADCs of the invention can be in the range of 0.002-500 mg per patient, 0.002-400 mg per patient, 0.002-300 mg per patient, 0.002-200 mg per patient, 0.002-100 mg per patient, 0.002-50 mg per patient, 0.006-500 mg per patient, 0.006-400 mg per patient, 0.006-300 mg per patient, 0.006-200 mg per patient, 0.006-100 mg per patient, 0.006-50 mg per patient, 0.02-500 mg per patient, 0.02-
Figure imgf000071_0001
400 mg per patient, 0.02-300 mg per patient, 0.02-200 mg per patient, 0.02-100 mg per patient, 0.02-50 mg per patient, 0.06-500 mg per patient, 0.06-400 mg per patient, 0.06-300 mg per patient, 0.06-200 mg per patient, 0.06-100 mg per patient, 0.06-50 mg per patient, 0.2-500 mg per patient, 0.2-400 mg per patient, 0.2-300 mg per patient, 0 2-200 mg per patient, 0.2-100 mg per patient, 0.2-50 mg per patient, 0.6-500 mg per patient, 0.6-400 mg per patient, 0.6-300 mg per patient, 0.6-200 mg per patient, 0.6-100 mg per patient, or 0.6-50 mg per patient, 2-500 mg per patient, 2-400 mg per patient, 2-300 mg per patient, 2-200 mg per patient, 2-100 mg per patient, 2-50 mg per patient, 6-500 mg per patient, 6-400 mg per patient, 6-300 mg per patient, 6-200 mg per patient, 6-100 mg per patient, or 6-50 mg per patient, depending, of course, on the mode of administration. The total monthly dose can be administered in single or divided doses and can, at the physician's discretion, fall outside of the typical ranges given herein. [0198^ An exemplary, non-limiting weekly dosing range for a therapeutically effective amount of the ADCs of the invention can be about 0.0001 to about 0.9 mg/kg, about 0.0001 to about 0.8 mg/kg, about 0.0001 to about 0.7 mg/kg, about 0.0001 to about 0.6 mg/kg, about 0.0001 to about 0.5 mg/kg, about 0.0001 to about 0.4 mg/kg, about 0.0001 to about 0.3 mg/kg, about 0.0001 to about 0.2 mg/kg, about 0.0001 to about 0.1 mg/kg, about 0.0003 to about 0.9 mg/kg, about 0.0003 to about 0.8 mg/kg, about 0.0003 to about 0.7 mg/kg, about 0.0003 to about 0.6 mg/kg, about 0.0003 to about 0.5 mg/kg, about 0.0003 to about 0.4 mg/kg, about 0.0003 to about 0.3 mg/kg, about 0.0003 to about 0.2 mg/kg, about 0.0003 to about 0.1 mg/kg, about 0.001 to about 0.9 mg/kg, about 0.001 to about 0.8 mg/kg, about 0.001 to about 0.7 mg/kg, about 0.001 to about 0.6 mg/kg, about 0.001 to about 0.5 mg/kg, about 0.001 to about 0.4 mg/kg, about 0.001 to about 0.3 mg/kg, about 0.001 to about 0.2 mg/kg, about 0.0001 to about 0.1 mg/kg, about 0.003 to about 0.9 mg/kg, about 0.003 to about 0.8 mg/kg, about 0.003 to about 0.7 mg/kg, about 0.003 to about 0.6 mg/kg, about 0.003 to about 0.5 mg/kg, about 0.003 to about 0.4 mg/kg, about 0.003 to about 0.3 mg/kg, about 0 003 to about 0.2 mg/kg, about 0.003 to about 0.1 mg/kg, about 0.01 to about 0.9 mg/kg, about 0.01 to about 0.8 mg/kg, about 0.01 to about 0.7 mg/kg, about 0.01 to about 0.6 mg/kg, about 0.01 to about 0.5 mg/kg, about 0.01 to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.03 to about 0.9 mg/kg, about 0.03 to about 0.8 mg/kg, about 0.03 to about 0.7 mg/kg, about 0.03 to about 0.6 mg/kg, about 0.03 to about 0.5 mg/kg,
Figure imgf000072_0001
about 0.03 to about 0.4 mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.1 to about 0.9 mg/kg, about 0.1 to about 0.8 mg/kg, about 0.1 to about 0.7 mg/kg, about 0.1 to about 0.6 mg/kg, about 0.1 to about 0.5 mg/kg, about 0.1 to about 0.4 mg/kg, about 0.1 to about 0.3 mg/kg, about 0.1 to about 0.2 mg/kg, about 0.1 to about 0.1 mg/kg, about 0.3 to about 0.9 mg/kg, about 0.3 to about 0.8 mg/kg, about 0.3 to about 0.7 mg/kg, about 0.3 to about 0.6 mg/kg, about 0.3 to about 0.5 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.3 to about 0.3 mg/kg, about 0.3 to about 0.2 mg/kg, about 0.3 to about 0.1 mg/kg
[0199] to various embodiments, single or multiple administrations of the pharmaceutical compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the ADCs disclosed herein to effectively treat the patient. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
[0200] The dosing frequency of the administration of the ADC pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The patient can be treated at regular intervals, such as weekly or monthly, until a desired therapeutic result is achieved, or treated with a loading dose followed by maintenance dose at regular intervals. Exemplary dosing frequencies include, but are not limited to: once weekly without break: once weekly, every other week; once every 2 weeks; once every 3 weeks; weakly without break for 2 weeks, twice weekly without break for 2 weeks, twice weekly without break for 3 weeks, twice weekly without break for 4 weeks, twice weekly without break for 5 weeks, twice weekly without break for 6 weeks, twice weekly without break for 7 weeks, twice weekly without break for 8 weeks, monthly; once every other month; once every three months; once every four months; once every five months; or once every six months, or yearly.
[0201] Toxicity and therapeutic index of the pharmaceutical compositions of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio
Figure imgf000073_0001
between toxic and therapeutic effective dose is the therapeutic index and it can be expressed as the ratio LDS0/EDS0. Compositions that exhibit iarge therapeutic indices are generally preferred.
Therapeutic Methods of Use
[0202] In one aspect, the present invention relates to a method of treating a proliferative disease (such as cancer) in an individual, comprising administering to the individual a therapeutically effective amount of an ADC. Importantly, the ADCs and methods described herein can be used to effectively treat cancers, including recurrent, resistant, or refractory cancers, at surprisingly low doses.
[0203] In various embodiments, the methods of the present invention are useful in treating certain cellular proliferative diseases. Such diseases include, but are not limited to, the following: a) proliferative diseases of the breast, which include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma, lobular carcinoma in situ and metastatic breast cancer; b) proliferative diseases of lymphocytic cells, which include, but are not limited to, various T cell and B cell lymphomas, non-Hodgkins lymphoma, cutaneous T cell lymphoma, Hodgkins disease, and lymphoma of the central nervous system; (c) multiple myeloma, chronic neutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, juvenile myelomonocytic leukemia, refractory anemia with ringed sideroblasts and without ringed sideroblasts, refractory cytopenia (myelodysplastic syndrome) with multilineage dysplasia, refractory anemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, myelodysplastic syndrome with t(9;12)(q22;p12), and myelogenous leukemia (e.g., Philadelphia chromosome positive (t(9;22)(qq34;q11)); d) proliferative diseases of the skin, which include, but are not limited to, basal cell carcinoma, squamous cell carcinoma, malignant melanoma and Kaposi’s sarcoma; e) leukemias, which include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia, f) proliferative diseases of the digestive tract, which include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach (gastric), pancreatic cancer,
Figure imgf000074_0001
pancreatic cancer-islet cell, rectal, small-intestine and salivary gland cancers; g) proliferative diseases of the liver, which include, but are not limited to, hepatocellular carcinoma, cholangiocarcinoma, mixed hepatocellular cholangiocarcinoma, primary liver cancer and metastatic liver cancer; h) proliferative diseases of the male reproductive organs, which include, but are not limited to, prostate cancer, testicular cancer and penile cancer; i) proliferative diseases of the female reproductive organs, which include, but are not limited to, uterine cancer (endometrial), cervical, ovarian, vaginal, vulval cancers, uterine sarcoma and ovarian germ cell tumor; j) proliferative diseases of the respiratory tract, which include, but are not limited to, small cell and non-small cell lung carcinoma, bronchial adema, pleuropulmonary blastoma and malignant mesothelioma; k) proliferative diseases of the brain, which include, but are not limited to, brain stem and hyptothalamic glioma, cerebellar and cerebral astrocytoma, medullablastoma, ependymal tumors, oligodendroglial, meningiomas and neuroectodermal and pineal tumors; I) proliferative diseases of the eye, which include, but are not limited to, intraocular melanoma, retinoblastoma, and rhabdomyosarcoma; m) proliferative diseases of the head and neck, which include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancers, and lip and oral cancer, squamous neck cancer, metastatic paranasal sinus cancer; n) proliferative diseases of the thyroid, which include, but are not limited to, thyroid cancer, thymoma, malignant thymoma, medullary thyroid carcinomas, papillary thyroid carcinomas, multiple endocrine neoplasia type 2A (MEN2A), pheochromocytoma, parathyroid adenomas, multiple endocrine neoplasia type 2B (MEN2B). familial medullary thyroid carcinoma (FMTC) and carcinoids; o) proliferative diseases of the urinary tract, which include, but are not limited to, bladder cancer; p) sarcomas, which include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma; q) proliferative diseases of the kidneys, which include, but are not limited to, renal cell carcinoma, clear cell carcinoma of the kidney; and renal cell adenocarcinoma; r) precursor B-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia), B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B- cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of MALT type, nodal marginal zone B-cell lymphoma, follicular lymphoma,
Figure imgf000075_0001
mantle-cell lymphoma, diffuse large B-cel! lymphoma, mediastinal large B-cell lymphoma, primary effusion lymphoma and Burkitt's lymphoma/Burkitt cell leukemia; (s) precursor T- lymphoblastic lymphoma/leukemia (precursor T-cell acute lymphoblastic leukemia), T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell lymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type, enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides/Sezary syndrome, anaplastic large-cell lymphoma, T/null cell, primary cutaneous type, peripheral T-cell lymphoma, not otherwise characterized, angioimmunoblastic T-cell lymphoma, anaplastic large-cell lymphoma, T/null cell, and primary systemic type; (t) nodular lymphocyte-predominant Hodgkin's lymphoma, nodular sclerosis Hodgkin's lymphoma (grades 1 and 2), lymphocyte-rich classical Hodgkin’s lymphoma, mixed cellularity Hodgkin's lymphoma, and lymphocyte depletion Hodgkin's lymphoma; and (u) AML with t(8;21)(q22;q22), AML1(CBF-alpha)/ETO, acute promyelocytic leukemia (AML with t(15; 17)(q22;q11-12) and variants, PML/RAR-alpha), AML with abnormal bone marrow eosinophils (inv(16)(p13q22) or t(15;16)(p13;qT1), CBFb/MYH11. times.), and AML with 11q23 (MLL) abnormalities, AML minimally differentiated, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroid leukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelosis with myelofibrosis.
[0204] In various embodiments, the proliferative disease is a cancer selected from the group consisting of: B cell lymphoma; a lung cancer (small cell lung cancer and non-small cell lung cancer); a bronchus cancer; a colorectal cancer; a prostate cancer; a breast cancer; a pancreas cancer; a stomach cancer; an ovarian cancer; a urinary bladder cancer; a brain or central nervous system cancer; a peripheral nervous system cancer; an esophageal cancer; a cervical cancer; a melanoma; a uterine or endometrial cancer; a cancer of the oral cavity or pharynx; a liver cancer; a kidney cancer; a biliary tract cancer; a small bowel or appendix cancer; a salivary gland cancer; a thyroid gland cancer; a adrenal gland cancer; an osteosarcoma; a chondrosarcoma; a liposarcoma; a testes cancer; and a malignant fibrous histiocytoma; a skin cancer; a head and neck cancer; lymphomas; sarcomas; multiple myeloma; and leukemias.
Figure imgf000076_0001
[0205] In various embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an ADC, wherein the ADC is administered to the individual at a weekly dosage selected from the group consisting of .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg. In various embodiments, the ADC is administered to the individual at a dosage (e g., at a weekly dosage) included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.03 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.1 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg. In various embodiments, the ADC is administered to the individual at a dosage (e.g., at a weekly dosage) of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg. In various embodiments, the cancer expresses the TAA of the ADC of the present invention. In various embodiments, the cancer is a non -TAA expressing cancer in the tumor microenvironment of a TAA expressing cancer.
[0206] In various embodiments, the methods may inhibit or prevent the growth or proliferation of TAA expressing or non-TAA expressing tumor cells in an individual, such as for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. As a result, where the tumor is a solid tumor, the modulation may reduce the size of the solid tumor by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
[0207] The inhibition of the tumor cell proliferation can be measured by cell-based assays, such as bromodeoxyuridine (BRDU) incorporation (Hoshino et al., Int. J. Cancer 38,
Figure imgf000077_0001
369, 1986; Campana et a!., J. Immunol. Meth. 107:79, 1988; [3H]-thymidine incorporation (Chen, J., Oncogene 13: 1395-403, 1996; Jeoung, J., J. Biol. Chem. 270:18367-73, 1995; the dye Alamar Blue (available from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998). The anchorage independent growth of cancer cells is assessed by colony formation assay in soft agar, such as by counting the number of cancer cell colonies formed on top of the soft agar (see Examples and Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989).
[020S] The inhibition of tumor cell growth in a subject may be assessed by monitoring the cancer growth in a subject, for example in an animal model or in human subjects. One exemplary monitoring method is tumorigenicity assays. In one example, a xenograft comprises human cells from a pre-existing tumor or from a tumor cell line. Tumor xenograft assays are known in the art and described herein (see, e.g., Ogawa et al., Oncogene 19:6043-6052, 2000). In another embodiment, tumorigenicity is monitored using the hollow fiber assay, which is described in U.S. Patent No. 5,698,413, which is incorporated herein by reference in its entirety. [0209] The percentage of the inhibition is calculated by comparing the tumor cell proliferation, anchorage independent growth, or tumor cell growth under modulator treatment with that under negative control condition (typically without modulator treatment). For example, where the number of tumor cells or tumor cell colonies (colony formation assay), or PRDU or pH]-thymidine incorporation is A (under the treatment of modulators) and C (under negative control condition), the percentage of inhibition would be (C-A)/Cx100%.
[0210] Examples of tumor cell lines derived from human tumors and available for use in the in vitro and in vivo studies include, but are not limited to, leukemia cell lines (e.g., CCRF- CEM, HL-60(TB), K-562, MOLT-4, RPM1-8226, SR, P388 and P388/ADR, H292, MV-4-11); non-small cell lung cancer cell lines (e.g., A549/ATCC, EKVX, HOP-62, HOP- 92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522 and LXFL 529); small cell lung cancer ceil lines (e.g., DMS 114 and SHP-77); colon cancer cell lines (e.g., COLO 205, HOC-2998, HCT-116, HCT-15. HT29, KM12, SW-620, DLD-1 and KM20L2); central nervous system (CNS) cancer cell lines (e.g., SF-268, SF-295, SF-539, SNB-19, SNB-75, U251 , SNB-78 and XF 498); melanoma cell lines (e.g., LOX I MVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257,
Figure imgf000078_0001
UACC-62, RPMI-7951 and M19-MEL): ovarian cancer cell lines (e.g., IGROV1 , OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8 and SK-OV-3); renal cancer cell lines (e.g., 786- 0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, RXF-631 and SN12K1); prostate cancer cell lines (e.g., PC-3 and DU-145); breast cancer cell lines (e.g., MCF7, NCI/ADR-RES, MDA-MB- 231/ATCC, HS 578T, MDA-MB-435, BT-549, T-47D and MDA-MB-468); and thyroid cancer cell lines (e.g., SK-N-SH).
[0211] In another aspect, the present invention relates to a method of activating or stimulating an non-TAA expressing immune cell located in the tumor microenvironment of a TAA expressing tumor, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an ADC; wherein the ADC is administered to the individual at a dosage (e.g., at a weekly dosage) included in any of the following ranges: about 0.0001 to about 0.0003 mg/kg, about 0.0003 to about 0.001 mg/kg, about 0.001 to about 0.003 mg/kg, about 0.003 to about 0.01 mg/kg, about 0.01 to about 0.03 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.1 to about 0.3 mg/kg, about 0.3 to about 0.4 mg/kg, about 0.4 to about 0.5 mg/kg, about 0.5 to about 0.6 mg/kg, about 0.6 to about 0.7 mg/kg, about 0.7 to about 0.8 mg/kg, and about 0.8 to about 0.9 mg/kg. In various embodiments, the ADC is administered to the individual at a weekly dosage selected from the group consisting of about .0001 mg/kg, of about .0003 mg/kg, of about .001 mg/kg, of about .003 mg/kg, of about .01 mg/kg. of about .03 mg/kg, of about 0.1 mg/kg, of about 0.2 mg/kg, of about 0.3 mg/kg, of about 0.4 mg/kg, of about 0.5 mg/kg, of about 0.6 mg/kg, of about 0.7 mg/kg, of about 0.8 mg/kg, and of about 0.9 mg/kg. In various embodiments, the ADC is administered to the individual at a dosage (e.g., at a weekly dosage) of no greater than about any of: .0001 mg/kg, .0003 mg/kg, .001 mg/kg, .003 mg/kg, .01 mg/kg, .03 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, and 0.9 mg/kg.
[0212] In various embodiments, the methods described herein may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders, such approaches including, but not limited to chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation. For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant
Figure imgf000079_0001
of other conventional cancer therapy. The present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through use of the fusion molecules described herein.
[0213] A wide array of conventional compounds has been shown to have anti-neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant T-cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti -neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.
[0214] When the ADC disclosed herein is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such fusion molecule may enhance the therapeutic effect of the anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic agent. This allows decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti-neoplastic agent in resistant T-cells. In various embodiments, a second anti-cancer agent, such as a chemotherapeutic agent, will be administered to the patient. The list of exemplary chemotherapeutic agent includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fluradabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics, as well as combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, the dosages of such chemotherapeutic agents include, but is not limited to, about any of 10
Figure imgf000080_0001
mg/m2, 20 mg/m2, 30 mg/m2, 40 mg/m2, 50 mg/m2, 60 mg/m2, 75 mg/m2, 80 mg/m2, 90 mg/m2, 100 mg/m2, 120 mg/m2, 150 mg/m2, 175 mg/m2, 200 mg/m2, 210 mg/m2, 220 mg/m2, 230 mg/m2, 240 mg/m2, 250 mg/m2, 260 mg/m2, and 300 mg/m2.
Combination Immunotherapy
[0215] In another aspect, the present invention relates to combination therapies designed to treat a proliferative disease (such as cancer) in an individual, comprising administering to the individual: a) a therapeutically effective amount of an ADC, and b) immunotherapy, wherein the combination therapy optionally provides increased effector cell killing of tumor cells, i.e. , a synergy exists between the ADC and the immunotherapy when co- administered.
[0216] In various embodiments, the proliferative disease is a cancer selected from the group consisting of: B cell lymphoma; a lung cancer (small cell lung cancer and non-small cell lung cancer); a bronchus cancer; a colorectal cancer; a prostate cancer; a breast cancer; a pancreas cancer; a stomach cancer; an ovarian cancer; a urinary bladder cancer; a brain or central nervous system cancer; a peripheral nervous system cancer; an esophageal cancer; a cervical cancer; a melanoma; a uterine or endometrial cancer: a cancer of the oral cavity or pharynx; a liver cancer; a kidney cancer; a biliary tract cancer: a small bowel or appendix cancer; a salivary gland cancer; a thyroid gland cancer; a adrenal gland cancer; an osteosarcoma; a chondrosarcoma; a liposarcoma; a testes cancer; and a malignant fibrous histiocytoma; a skin cancer: a head and neck cancer; lymphomas; sarcomas; multiple myeloma; and leukemias.
[0217] In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody- drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1 , CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec 7, Siglec 8, Siglec 9, Siglec 15 and VISTA; treatment using
Figure imgf000081_0001
bispecific T ceil engaging antibodies (BITE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-a, IFN-p and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and/or TCR transgenic); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using vaccine such as BCG.
[0218] In various embodiments, there is provided a combination therapy method of treating a proliferative disease in an individual, comprising administering to the individual a) an effective amount of an ADC; and b) immunotherapy; wherein the combination therapy provides increased effector cell killing. In various embodiments, the immunotherapy is treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules. In various embodiments, the immunotherapy is treatment using chimeric antigen receptor (CAR)-T cells. In various embodiments, the immunotherapy is treatment using CAR-NK cells. In various embodiments, the immunotherapy is treatment using bispecific T cell engaging antibodies (BITE®). In various embodiments, the cancer expresses the TAA of the ADC of the present invention. In various embodiments, the cancer is a non-TAA expressing cancer in the tumor microenvironment of a TAA expressing cancer. In various embodiments, the immunotherapy will target a TAA that is different than the TAA targeted by the ADC.
Kits
[0219] In certain embodiments, this invention provides for kits for the treatment of cancer and/or in an adjunct therapy. Kits typically comprise a container containing an ADC of the present invention. The ADC can be present in a pharmacologically acceptable excipient. The kits may optionally include an immunotherapy cancer agent.
Figure imgf000082_0001
[0220] In addition, the kits can optionally include instructional materials disclosing means of use of the ADC and/or immunotherapy to treat a cancer. The instructional materials may also, optionally, teach preferred dosages, counter-indications, and the like.
[0221] The kits can also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, and additionally comprise means for disinfecting a wound, for reducing pain, for attachment of a dressing, and the like.
[0222] While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials
[0223] The following examples are provided to more fully illustrate the invention but are not construed as limiting the scope thereof.
Example 1
Synthesis of Compound 5
Figure imgf000083_0001
[0224] HATU (2.85g,7.5mmol) was added to a solution of 6-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1-yl) hexanoic acid (1.06g, 5mmol), (S)-tert- butyl 2-amino-3-methylbutanoate hydrochloride (1.1g , 5.25mmol) in DMF (15 mL). DIPEA (1.1g, 8.5 mmol) was added to the mixture, which was stirred overnight at room temperature. The mixture was diluted with DCM, which was washed with aqueous sodium bicarbonate and brine. The organic layer was dried and concentrated by evaporation to give a residue, which was purified with chromatograph on a
Figure imgf000083_0002
silica gel column (eluent: EtOAc/Hexane = 0-75%) to give 1.1 g of t-Butyl ester as an oil. This oil was dissolved in DCM-TFA (1 :1 ; 20 mL) at RT. After 4 h the solution was rotavapored and dried under high vacuum overnight to give the title compound 1 as a light-yellow solid (0.93g, 60.0% yield). MS: m/z =311.1 [M+H], 1H NMR (500 MHz, DMSO-d6) 6: 7.88 (d, 7 - 8.5 Hz, 1 H), 7.00 (s, 2H), 4.20 - 4.10 (m, 1 H), 3.37 (t, 7 - 7.0 Hz, 2H), 2.14 (ddq, J = 21.2, 14.1 , 7.2 Hz, 2H), 2.06 - 1.96 (m, 1 H), 1.57-1.41 (m, 4H), 1.24 - 1.14 (m, 2H), 0.86 (d, J = 6.7 Hz, 6H).
Figure imgf000084_0001
[0225] HATU (1.48g, 3.9mmol) was added to a solution of compound 1 (0.93g, 3mmol), (S)-tert-butyl 2-aminopropanoate hydrochloride (0.54mg 3mmol) in DMF (20 mL). DIPEA (0.58g, 4.5mmol) was added to the mixture and it was stirred overnight at room temperature. The mixture was diluted with DCM, which was washed with aqueous sodium bicarbonate and brine. The organic layer was dried and concentrated by evaporation to give a residue, which was purified with chromatograph on a silica gel column (eluent: EtOAc/Hexane ~ 0-75%) to give the intermediate (0.8 g) as an oil. 1H NMR (500 MHz, DMSO-d6) 3: 8.23 (d, J ~ 6.7 Hz, 1 H), 7.74 (d, J = 9.1 Hz, 1 H), 7.00 (s, 2H), 4.23 - 4.16 (m, 1H), 4.10 (dd, J = 14.0, 7.0 Hz, 1 H), 3.37 (t, 7 = 7.0 Hz, 2H), 2.18 — 2.05 (m, 2H), 1.93 (dq, 7 = 13.3, 6.6 Hz, 1 H), 1.47 (dd, J - 14.1 , 6.9 Hz, 4H), 1.38 (s, 9H), 1.23 (d, 7 - 7.2 Hz, 3H), 1.20 - 1.15 (m, 2H), 0.86 (d, J - 6.7 Hz, 3H), 0.82 (d, J ~ 6.6 Hz, 3H). This oil was dissolved in DCM-TFA (v/v=1 : 1 ; 20 mL). After 4 h the solution was rotavapored and dried under high vacuum overnight to give the title compound 2 (0.4 g, 34.9% yield) as a light-yellow solid. MS: m/z =382.1 (M+1).
Figure imgf000084_0002
[0226] To a solution of 2 (200mg, 0.52 mmol) and tert-butyl 2-(aminooxy)acetate (77mg, 0.52 mmol) in DMF (10mL) were added HATU (259mg, 0.68 mmol) and DIEA (138pL, 0.79 mmol). The mixture was stirred overnight. The mixture was diluted with DCM (40mL), which was washed with aqueous sodium bicarbonate (3x10mL) and brine. The organic layer was dried and concentrated by evaporation to give a residue, which was purified with chromatograph on a silica gel column (eluent: MeOH/DCM = 1-5%) to give the title compound 3 (0.15 g, 56%) as an oil.
Figure imgf000085_0001
[0227] Compound 3 (0.15g, 0.29 mmol) was dissolved in DCM-TFA mixture (v/v=1:1; 10 mL). After stirring for 4 hrs, the solution was evaporated, and the residue was dissolved in 2 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product, which was filtered and washed with methyl tert-butyl ether (5mL). The solid was dried under high vacuum to give the title compound 4 (0.1 g, 75 % yield).
Figure imgf000085_0002
[0228] To a solution of 4 (100mg, 0.22 mmol) and N-hydroxysuccinimide (32mg, 0.282 mmol) in DCM (5mL) and DMF (2mL) was added EDCI (54mg, 0.282 mmol). The resulting mixture was stirred for 3hrs. Exatecan mesylate (50mg, 0.094 mmol) was added, followed by
Figure imgf000085_0003
addition of Di PEA (49pL, 0.282 mmol). The mixture was diluted with DCM (50mL) and the mixture was washed with 1 N HCI solution (10mL), saturated aqueous NaHCOs solution and brine. The organic layer was dried over anhydrous Na£SO4, filtered, and evaporated to give a residue, which was purified by HPLC (eluent: ACN/H2O = 10-80%) to give title compound 5 (41mg, 50%) as a solid. MS: m/z-872.1 (M+1).
Example 2
Synthesis of Compound 20
Figure imgf000086_0001
[0229] To a stirring solution of tert-butyl N-hydroxy-N-methylcarbamate (5g, 34mmol) in THF (50mL) at OoC was added NaH (1.36g, 60%, 34mmol) under argon. After stirring at 0°C for 30 mins, ethyl bromoacetate (6.8g, 40.8mmol) was added. The mixture was warmed to rt and stirred for 4hrs. The mixture was diluted with ethyl acetate (50mL) and the resulting mixture was washed with water (3x1 OmL) and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent: ethyl acetate/hexane = 20-100%) to give title compound 6 (3.7g, 47%) as an oil.
Figure imgf000086_0002
[0230] To a solution of S (800mg, 3.4 mmol) in THF (/ mL) was added a solution of
NaOH (278mg, 6.95 mmol) in water (2.5 mL). The mixture was stirred for 4hrs. The mixture was
Figure imgf000086_0003
diluted with ethyl acetate (20mL) and the resulting mixture was washed with 1 N HCI, water and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to give the title compound 7 (0.4g, 57%) as an oil.
Figure imgf000087_0001
[0231] Compound 7 (3g, 14.6 mmol) was treated with 4 N HCI in dioxane (12mL, 48 mmol) and the resulting mixture was stirred for 3 hrs. Removal of the volatiles afforded crude compound 8 (1.5g, 98%).
Figure imgf000087_0002
[0232] To a solution of compound 8 (1.5g, 14.3 mmol) in a mixture of THF and H2O (20mL, v/v = 1 :1) were added K2CO3 (2.96g, 21.4 mmol) and allyl chloroformate (2.06g, 17.1 mmol). The resulting mixture was stirred for 2 hrs. The mixture was acidified with 1 N HCI solution to pH ~ 6. THF was evaporated under vacuum and the aqueous solution was extracted with DCM (3x1 OmL), washed with water and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give the title compound 9 (1.2g, 44%) as a solid.
Figure imgf000087_0003
[0233] To a solution of exatecan mesylate (300mg, 0.56 mmol) in DMF (8mL) were added compound 9 (213 mg, 1.13mmol), HATU (257mg, 0.68 mmol) and DIPEA (200pL, 1.12 mmol). The mixture was stirred for 2hrs. The mixture was diluted with DCM (30mL) and washed with NaHCOs solution (2x1 OmL) and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the title compound 10 (200mg, 58%) as a solid.
Figure imgf000088_0001
[0234] To a solution of compound 10 (200mg, 0.33mmol) in DCM (10 mL) were added Pd(PPh3)4 (19mg, 0.016 mmol) and pyrrolidine (47mg, 0.66mmol). The mixture was stirred for 15 mins. The reaction was quenched with 15 mL of brine, and the mixture was extracted with DCM (2x 15mL). The organic layers were dried over anhydrous NaaSCU, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the title compound 11 (160mg, 93%) as a solid.
Figure imgf000088_0002
[0235] To a solution of 12 (0.25g, 0.66mmol) and 4,4'-dinitrodiphenyl carbonate (0.40g, 1.32 mmol) in DMF (6mL) was added DI PEA (0.18 mL, 0.99 mmol). The mixture was stirred overnight. The mixture was diluted with DCM (50mL) and the resulting mixture was washed with water (3x 10mL) and brine. The organic layer was dried over anhydrous NazSCXs, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=:5-50%) to provide the title compound 13 (200mg, 56%) as a white solid.
Figure imgf000089_0001
[0236] To a solution of 11 (20mg, 0.038 mmol) and 13 (42mg, 0.076 mmol) in DMF (3 mL) was added DI PEA (15 pL, 0.076 mmol). The mixture was stirred overnight The mixture was loaded to HPLC column for purification (eluent: ACN/H2O=5-80% for 25 mins) to afford the title compound 14 (30mg, 85%) as a solid.
Figure imgf000089_0002
14 15
[0237] To a solution of compound 14 (30mg, 0.032mmol) in DCM (5 mL) were added
Pd(PPh3)4 (1.87 mg, 0.0016mmol) and pyrrolidine (4.6mg, 0.064mmol). The mixture was stirred
Figure imgf000089_0003
for 15 mins. The reaction was quenched with 5 mL of brine, and the mixture was extracted with DCM (2x 10mL). The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by HPLC (eluent: ACN/H2O=5-80% for 25 mins) to provide the title compound 15 (20mg, 74%) as a solid.
Figure imgf000090_0001
[0238] To a solution of 15 (20mg, 0.023 mmol) and N-succinimidyl 6- maieimidohexanoate (15mg, 0.046 mmol) in DMF (5 mL) was added DIEA (10pL, 0.046 mmol). The mixture was stirred overnight. The mixture was loaded to HPLC column for purification (eluent: ACN/H2O~5-80% for 25 mins) to afford the title compound 16 (10mg, 42%) as a solid. MS: m/z=1035.4 (M+1).
Exampie 3
Synthesis of Compound 17
Figure imgf000090_0002
[0239] To a solution of 2 (30mg, 0.057 mmol) and 11 (33mg, 0.086 mmol) in DMF (3 mL) and DCM 3mL) was added DIC (14.5mg, 0.115 mmol), pyridine (9mg, 0.115mmol) and HOAt (9.4mg, 0.069mmol). The mixture was stirred overnight. The mixture was loaded to HPLC column for purification (eluent: ACN/H2O=5-80% for 25 mins) to afford 17 (10mg, 20%) as a solid. MS: m/z=886.3 (M+1).
Example 4
Synthesis of Compound 20
Figure imgf000091_0001
[0240] Compound 2 (150 mg, 0.39 mmol) and 4-aminobenzyl alcohol (68 mg, 0.55 mmol) were dissolved in anhydrous DCM (9 ml) and anhydrous MeOH (3ml). EEDQ (190 mg, 0.78 mmol) was added to the mixture, which was stirred overnight at room temperature. The reaction mixture was evaporated under vacuum to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the title compound 18 (150 mg, 78.4%). MS: m/z =487.2 (M+H).
Figure imgf000091_0002
[0241] To a mixture of compound 18 (150mg, 0.3 mmol) in 4mL of DMF was added bis p-nitrophenyl carbonate (0.19g, 0.6mmol) and the reaction lasted for 16 h at room temperature, the reaction mixture was evaporated under vacuum to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the title compound 19 (150 mg, 74.6%). MS: m/z = 652.2 (M+1).
Figure imgf000091_0003
Figure imgf000092_0001
[0242] To a solution of compound 19 (19.6mg, 0.03mmol) and compound 11 (10.2mg, 0.02mmol) in DMF (1.5 mL) was added DIPEA (5.2mg, 0.04mmol). The mixture was stirred at room temperature for 1 hr. The reaction mixture was purified by preparative HPLC to give the title compound 20 (7mg, 34%) as a solid. MS: m/z =1021.4 (M+1).
Example 5
Synthesis of Compound 30
Figure imgf000092_0002
[0243] L-Phe-Gly-OH (1g, 4.5 mmol) was dissolved in a mixture of THF (9mL) and water (9mL). To it were added K2CO3 (0.93g, 6.7 mmol) and allyl chioroformate (0.65g, 5.4 mmol).
The mixture was stirred overnight. 1 N HCI solution was added to the mixture to acidify to pH = 6. The resulting mixture was concentrated under vacuum to give a residue, which was dissolved in DCIWMeOH (v/v=3:1 , 20mL). The mixture was filtered and evaporated to give crude title compound 21.
Figure imgf000092_0003
Figure imgf000093_0001
[0244] To a solution of the crude 21 in DCMZ MeOH (v/v == 3:1, 20mL) were added 4- aminobenzyl alcohol (0.78g, 6 3 mmol) and EEDQ (2 23g, 9mmol). The mixture was stirred overnight and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH'DCM-3-10%) to provide the title compound 22 (500 mg, 27%).
Figure imgf000093_0002
[0245] To a mixture of compound 22 (200mg, 0.48mmol) in 5mL of DMF was added bis p-nitrophenyi carbonate (296mg, 0.97mmol) and the reaction lasted for 24 hrs at room temperature. DCM (30mL) was added to the reaction mixture and washed with water (3x5mL) and brine. The organic layer was dried over anhydrous NaaSO^ filtered, and evaporated under vacuum to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the title compound 23 (200 mg, 71.4%) as a solid.
Figure imgf000093_0003
[0246] To a solution of compound 23 (220 mg, 0.78mmol) and compound 11 (100mg, 0.19mmol) in DMF (1.5 mL) was added DIPEA (65pL, 0.38mmol). The mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC (eluent: ACN/H2O:=5”80% for 25 mins) to give the title compound 24 (50mg, 27%) as a solid.
Figure imgf000094_0001
24 25
[0247] To a solution of compound 24 (50mg, 0.052mmol) in DCM (5 mL) were added Pd(PPhs)4 (3 mg, 0.0026mmol) and pyrrolidine (7.4mg, O.lmmol). The mixture was stirred for 15 mins. The reaction was quenched with 5 mL of brine, and the mixture was extracted with DCM (2x 10mL). The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by HPLC (eluent: ACN/HzO=5-80% for 25 mins) to provide the title compound 25 (40mg, 88%) as a solid.
Figure imgf000094_0002
[0248] To a solution of 6-maleimidohexanoic acid (2.11g, 10 mmol) in DMF (20mL) were added tert-butyl glycinate (1.31g, 10mmol), HATU (4.56g, 12mmol) and DIPEA (3.51mL, 20mmol). The mixture was stirred for 2hrs. The mixture was diluted with ethyl acetate (100mL) and washed with NaHCOs solution (2x1 OmL), water (3x1 OmL) and brine. The organic layers
Figure imgf000094_0003
were dried over anhydrous NasSCh, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the title compound 26 (1.8g, 56%).
Figure imgf000095_0001
26 27
[0249] To a solution of compound 26 (1.8g, 3.08 mmol) in DCM (10mL) was added TFA (10mL). The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 27 (1g, 68%) as a white solid.
Figure imgf000095_0002
[0250] To a solution of compound 27 (1g, 3.73 mmol) in DMF (10mL) were added tert- butyl glycinate (0.5g, 3.73mmol), HATU (1.7g, 4.47mmol) and DIPEA (1.30mL, 7.46mmol). The mixture was stirred for 2hrs. The mixture was diluted with ethyl acetate (100mL) and washed with NaHCOs solution (2x1 OmL), water (3x1 OmL) and brine. The organic layers were dried over anhydrous NasSCU, filtered, and evaporated to give the crude title compound 28.
Figure imgf000095_0003
[0251] To a solution of crude compound 28 in DCM (10mL) was added TFA (10mL).
The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 29 (0.5g, 42% from 27) as a white solid.
Figure imgf000096_0001
[0252] To a solution of compound 29 (74mg, 0.22mmol) and compound 25 (100mg, O.Hmmol) in DMF (3mL) were HATU (52mg, 0.137mmol) and DIPEA (0.04mL, 0.23mmol). The mixture was stirred for 2hrs. The mixture was purified by HPLC (eluent: ACN/H2O = 5-80% for 25 mins) to provide the title compound 30 (13mg, 10%) as a solid. MS: m/z=1083.4 (M+1).
Example 6
Synthesis of Compound 39 (BI-P-353)
FmocHN - FmooHN
Figure imgf000096_0003
Figure imgf000096_0002
Figure imgf000096_0004
[0253] To a solution of Fmoc-Gly-Gly-OH (3.54g, 10mmol) in DMF (50mL) were added CU(OAC)2 (0.67g, 3.7mmol), HOAc (1.3mL, 22.76mmol) and Pb(OAc)4 (5.08g, 11.46mmol). The resulting mixture was heated to 60°C for 30mins, and then cooled to rt The mixture was diluted with EtOAc (150mL) and washed with water (3x 60mL) and brine The mixture was dried over
Figure imgf000096_0005
anhydrous Na2SO4, filtered, and evaporated to give a residue, which was passed through a column (eluent: EtOAc/n-Heptane = 0-75%) to provide the title compound 31 (2g, 54.3%). MS: m/z =369.1 (M+1).
Figure imgf000097_0001
[0254] To a solution compound 31 (370mg, 1mmol) and 3-mercaptopropanoic acid (212mg, 2mmoi) in DCM (12mL) was added 3 mL of TFA. The resulting mixture was stirred for 30mins. The mixture was evaporated to give a residue, which was dissolved in DCM. The mixture was washed with water and brine. The mixture was dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: EtOAc/n- Heptane = 30%-100%) to give the title compound 32 (200 mg, 48%). MS: m/z =415.1 (M+1).
Figure imgf000097_0002
[0255] To a solution of compound 32 (53.8mg, 0.13mmol) in 5mL of DCM and 2mL of DMF were added HOSu (35mg, 0.3mmol) and EDCI (57.5mg, 0.3mmol). The mixture was stirred for 3hrs. To a solution of exatecan mesylate (53.1 mg, 0.1 mmol) and DIEA (38.8mg, 0.3mmol) in DMF (1mL) was added the above mixture. The resulting mixture was stirred overnight. 50mL of DCM was added and the resulting mixture was washed with 1 N HCI, sat. aqueous NaHCOs solution and brine. The mixture was dried over anhydrous NasSCh, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=1%-10%) to give of the title compound 33 (55mg, 51%). MS: m/z =832.2 (M+1).
Figure imgf000097_0003
Figure imgf000098_0001
[0256] To a solution of compound 33 (50mg, 0.060mmol) in DMF (1mL) was added piperidine (20pL,0.02mmol). The mixture was stirred for 2hrs and then evaporated to give a reisdue, which was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 34 (30 mg, 82%) as a yellow solid. MS: m/z =610.2 (M+1).
Figure imgf000098_0002
[0257] HATU (0.49g, 1 ,3mmo!) was added to a solution of Fmoc-Gly-Gly-OH (0.35g,
1mmol), (S)-terf-butyl 2-amino-3-phenylpropanoate (0.22mg , 1mmol) in DMF (15 mL). DIPEA
(0.26g, 2mmol) was added to the mixture and stirred overnight at room temperature. The mixture was diluted with ethyl acetate, washed with aqueous sodium bicarbonate and brine. The organic layer was dried and concentrated by evaporation to give a residue, which was passed through a column (eluent: EtOAc/n-Heptane = 10-50%) to give the tile compound 35 (0.4 g, 72%) as a white solid. MS: m/z =558.2 (M+1),
Figure imgf000098_0003
[025S] Compound 35 (0.4g, 0.71 mmol) was treated with TFA/DCM (1 :1 , 10mL) for 2hrs.
Figure imgf000098_0004
Evaporation of the mixture gave an oil. Ether was added to crash out the product as a white solid. Filtered and washed with ether to give the title compound 36 (0.25g, 70%). MS: m/z =502.2 (M+1).
Figure imgf000099_0001
[0259] HATU (38.0mg,0.1mmol) was added to a solution of compound 34 (30mg,0.05mmol), compound 36 (32.1mg , 0.064mmol) in DMF (3 mL). DIPEA (19.1mg, 0.15 mmol) was added to the mixture and stirred overnight at room temperature. The mixture was diluted with DCM, washed with aqueous sodium bicarbonate and brine. The organic layer was dried over anhydrous feSCU, filtered, and concentrated by evaporation to give a residue, which was purified by preparative HPLC (eluent: ACN/HzO = 10-80% for 25mins) to give the title compound 37 (30 mg, 55%) as a yellow solid. MS: m/z =1093.3 (M+1).
Figure imgf000099_0002
[0260] To a solution of compound 37 (30mg, 0.027mmoi) in DMF (3mL) was added
DBU (8.3mg, 0.054mmol). The mixture was stirred for 1h and then evaporated to give the crude product. The crude product was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 38 (15 mg, 64%) as a yellow solid. MS: m/z =871.3 (M+1).
Figure imgf000099_0003
Figure imgf000100_0001
[0261] To a solution of 2,5-dioxQpyrrolidin"1--yl-6-(2,5"dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanoate (13.3mg, 0.043mmol) and compound 38 (15 mg, 0.017mmol) in DMF (3 mL) were added DIPEA (4.4mg, 0.034mmol) and HOAt (1mg, 0.007mmol). The mixture was stirred at room temperature for 5 hours. The reaction mixture was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 39 (8mg, 43.6%) as a yellow solid. MS: m/z =1064.4(M+1).
Example I
Synthesis of Compound 45 (BI-P352)
Figure imgf000100_0002
[0262] To a solution of compound 35 (370mg, 1 mmol) and 2-mercaptoacetic acid (180mg,
2mmol) in DCM (12mL) was added 3 mL of TFA. The resulting mixture was stirred for 30mins. The mixture was evaporated to give a residue, which was dissolved in DCM. The mixture was washed with water and brine. The mixture was dried over anhydrous Na^SCk filtered, and evaporated to give a residue, which was purified by silica gel chromatography (ethyl acetate/n- Heptane = 30%-100%) to give the title compound 40 (200 mg, 50%). MS: m/z =401.1 (M+1).
Figure imgf000100_0003
[0263] To a solution of compound 40 (52mg, 0.13mmol) in 5mL of DCM and 2mL of DMF were added HOSu (35mg, 0.3mmol) and EDCI (57.5mg, 0.3mmol). The mixture was stirred for 3hrs. To a solution of exatecan mesylate (53.1 mg, 0.1 mmol) and DIEA (38.8mg, O.Smmol) in DMF (1mL) was added the above mixture. The resulting mixture was stirred overnight 50mL of DCM was added and the resulting mixture was washed with 1 N HCI, sat. aqueous NaHCO3 solution and brine. The mixture was dried over anhydrous NajSCh, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (MeOH/DCM=1%-10%) to give the title compound 41 (50 mg, 61 %). MS: m/z =818.2 (M+1).
Figure imgf000101_0001
41 42
[0264] To a solution of compound 41 (50mg, O.OSOmmol) in DMF (1mL) was added piperidine (20pL). The mixture was stirred for 2hrs and then evaporated to give a residue, which was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 42 (20 mg, 56%) as a yellow solid. MS: m/z =596.2 (M+1).
Figure imgf000101_0002
[0265] HATU (38.0mg,0.1mmol) was added to a solution of compound 42 (20mg , 0.033mmol) and compound 36 (33 mg, 0.066mmol) in DMF (2 mL). DIPEA (8.5mg, 0.066 mmol) was added to the mixture. The mixture was stirred for 2hrs at room temperature. The mixture was diluted with DCM, washed with water and brine. The organic layer was dried anhydrous NasSCk filtered, and evaporated to give a residue, which was purified preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 43 (15 mg, 42%) as a yellow solid. MS: m/z =1079.3 (M+1).
Figure imgf000102_0001
[0266] To a solution of compound 43 (15mg, 0.013mmol) in DMF (2mL) was added piperidine (20pL). The mixture was stirred for 1 hr and then evaporated to give the crude product, which was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 44 (8mg, 72%) as a yellow solid. MS: m/z =857 3 (M+1).
Figure imgf000102_0002
[0267] A solution of 2,5-dioxopyrrolidin-1-yl 6-(2,5~dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanoate (8.6mg, 0.028mmol) and compound 44 (8mg, 0.009mmol) in DMF (2 mL) at room
Figure imgf000102_0003
temperature were treated with DIPEA (2.4mg, 0.018mmol), HOAt(1mg, 0.007mmol). The mixture was stirred at room temperature for 2 hrs. The reaction mixture was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 45 (3mg, 30.6%) as a yellow solid. MS: m/z =1050.3(M+1).
Example 8
Synthesis of Compound 46 (Bi-340)
Figure imgf000103_0001
[0268] HATU (43.0mg, 0.113mmoi) was added to a solution of exatecan mesylate (50mg , 0.094mmol) and 2-methyithioacetic add (20 mg, 0.192mmol) in DMF (3mL). DIPEA
(35pL, 0.2mmol) was added to the mixture. The mixture was stirred for 2hrs at room temperature. The mixture was diluted with DCM, then washed with NaHCOs solution, water and brine. The organic layer was dried anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 46 (30 mg, 60%) as a solid.
Exampie 9
Synthesis of Compound 47 (Bi-341)
Figure imgf000103_0002
Figure imgf000104_0001
[0269] HATU (43.0mg, 0.113mmol) was added to a solution of exatecan mesylate
(50mg , 0.094mmol) and 2-methylthiopropanoic acid (23 mg, 0.192mmol) in DMF (3mL). DIPEA
(35pL, 0.2mmol) was added to the mixture. The mixture was stirred for 2hrs at room temperature. The mixture was diluted with DCM, then washed with NaHCCA solution, water and brine. The organic layer was dried anhydrous NaaSCU, filtered, and evaporated to give a residue, which was purified by preparative HPLC (eluent: ACN/HsO = 10-80% for 25mins) to give the title compound 47 (30 mg, 58%) as a solid.
Example 10
Synthesis of Compound 56
Figure imgf000104_0002
[0270] HATU (160mg, 0.42mmol) was added to a solution of compound 11 (146mg ,
0.28mmol) and Fmoc-Gly-OH (100 mg, 0.33mmol) in DMF (3mL). DIPEA (122pL, 0.70mmol) was added to the mixture. The mixture was stirred for 20 mins at room temperature. The mixture was diluted with DCM, then washed with NaHCOs solution, water and brine. The organic layer was
Figure imgf000104_0003
dried anhydrous Na2SCu, filtered, and evaporated to give a residue, which was purified by flash chromatograpy on silica gel (eluent: MeOH/DCM = 3-15%) to give the title compound 48 (120 mg, 53%) as a solid.
Figure imgf000105_0001
[0271] To a solution of compound 48 (120mg, 0.149mmol) in DMF (4mL) was added piperidine (800pL). The mixture was stirred for 1 hr and then evaporated to give the crude product, which was purified by preparative HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 49 (50mg, 58%) as a solid. LRMS: m/z-580.2 [M+1 ]+. Molecular Formula: C29H30FN5O7; Molecular Weight: 579.58.
Figure imgf000105_0002
50
[0272] To a solution of 6-maleimidohexanoic acid (2.11g, 10 mmol) in DMF (20 mL) were added tert-butyl glycinate (1.31g, 10 mmol), HATU (4.56g, 12 mmol) and DIPEA (3.51 mL, 20 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with ethyl acetate (100 mL) and washed with NaHCOs solution (2x10 mL), water (3x10 mL) and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give a residue, which was purified by silica gel chromatography (eluent: MeOH/DCM=3-10%) to provide the desired compound 50 (1.8 g, 56%).
Figure imgf000105_0003
Figure imgf000106_0001
[0273] To a solution of compound 50 (1.8 g, 3.08 mmol) in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 51 (1 g, 68%) as a white solid.
Figure imgf000106_0002
[0274] To a solution of compound 51 (1 g, 3.73 mmol) in DMF (10 mL) were added tert- butyl glycinate (0.5 g, 3.73 mmol), HATU (1.7 g, 4.47 mmol) and DIPEA (1.30 mL, 7.46 mmol). The mixture was stirred for 2 hrs. The mixture was diluted with ethyl acetate (100 mL) and washed with NaHCOs solution (2x10 mL), water (3x10 mL) and brine. The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give the crude compound 52.
Figure imgf000106_0003
[0275] To a solution of crude compound 52 in DCM (10 mL) was added TFA (10 mL). The mixture was stirred for 2 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of ethyl acetate. Methyl tert-butyl ether was added to precipitate
Figure imgf000106_0004
the product. The solid was filtered and washed with some methyl tert-butyl ether to give compound 53 (0.5 g, 42% from 51) as a white solid.
Figure imgf000107_0001
[0276] To a solution of compound 53 (0.5 g, 1 .53 mmol) in DMF (15 mL) were added H-
L-Phe-OtBu (0.4 g, 1.53 mmol), HATU (0.87 g, 2.30 mmol) and DI PEA (0.49 g, 3.84 mmol). The mixture was stirred for 2hrs. The mixture was diluted with ethyl acetate (60 mL) and washed with NaHCOs solution (2x50 mL), water (20 mL) and brine. The organic layers were dried over anhydrous Na2SC>4, filtered, and evaporated to give a residue, which was purified by flash chromatography on silica gel (eluent EtOAc/hexane = 5-50%) to give the title compound 54 (0.4
Figure imgf000107_0002
[0277] To a solution of 54 (0.4 g, 0.75 mmol) in DCM (20mL) was added TFA (10 mL). The mixture was stirred for 3 hrs. The volatiles were removed under vacuum and the resulting residue was dissolved in 1 mL of DCM. Ether was added to precipitate the product. The solid was filtered and washed with ether to give compound 55 (0.15 g, 42%) as a white solid. LRMS: m/z=473.22 [M+1]*. Molecular Formula: C23H28N4O7; Molecular Weight: 472.49.
Figure imgf000107_0003
Figure imgf000108_0001
[0278] HATU (66mg, 0.172mmol) was added to a solution of compound 49 (50mg ,
0.086mmol) and 55 (61mg, 0.129mmol) in DMF (3mL). DIPEA (45pL 0.258mmol) was added to the mixture. The mixture was purified by HPLC (eluent: ACN/H2O = 10-80% for 25mins) to give the title compound 56 (40 mg, 45%) as a solid. LRMS: m/z=1034.4
Figure imgf000108_0002
Molecular Formula: C52H56FN9O13; Molecular Weight: 1034.05.
Example 11
Generation of Anti- Her2-A DCs
[0279] The anti-Her2 antibody, Herceptin, was conjugated to camptothecin derivatives to form ADCs and evaluated for their ability to inhibit the growth of multiple cancer cell lines expressing different levels of Her2. Camptothecin derivative stabilizes the topoisomerase I complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.
[0280] Some embodiments of the invention relate to camptothecin derivatives linked to antibodies via a cleavable linker. In this example, Herceptin was reduced by adding antibody to
Figure imgf000108_0003
TCEP (Tris (2-carboxyethy!) phosphine) dissolved in a pH-adjusted PBS EDTA buffer. The antibody/TCEP solution was incubated at 37°C for 2 hrs. At the end of the reduction the excessive TCEP was removed by ultrafiltration using an amicon ultra filter. The reduced antibodies were buffer exchanged into conjugation reaction buffer. The solution of camptothecin payloads in DMA was added to a solution of reduced anti-Her2 monoclonal antibody at payload/antibody ratio of 7 - 30:1 to achieve different drug to antibody ratios (DARs). The payload/antibody solution was incubated for 2 hr at 25°C while the reduced antibody was conjugated to the payload via the thiol maleimide ligation After conjugation was completed, the reaction mixture was desalted and concentrated to yield anti-Her2 ADCs. The biochemical properties of the resulting ADCs were characterized using size-exclusion chromatography high pressure liquid chromatography (SEC-HPLC) to determine purity and aggregation content, and by using hydrophobic interaction chromatography HPLC (HIC-HPLC) to confirm drug loading (DAR). The conjugation procedure is illustrated in Figure 2. The final ADC products are comprised of >seven BI-P601 molecules. The cysteine conjugation method used in the conjugation process produces more homogeneous ADCs compared to lysine conjugation method.
Example 12
/n Vitro Cytotoxicity of Anti-Her2 ADCs in Cancer Cell Lines Expressing Different Her2 Expression Levels
[02811 Anti-Her2 ADCs were prepared by conjugation of Herceptin with different camptothecin derivatives with similar DARs, and were tested against BT-474 breast cancer cells and N87 gastric cancer cells representing high Her2 expression, and BxPC-3 pancreatic cancer cells with low/none level of Her2. In vitro cytotoxicity assay was performed. Free camptothecin derivatives were also tested in the same assay. Briefly, all cell lines were cultured in a suitable culture medium at 37°C in a humidified incubator atmosphere of 5% CO2. Cells were plated in 96-well flat bottom plates. Cell seeding number ranged from 500 cells/ /100 ul/ well to 6,000 cells/100 pl/well. Cells were allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. ADCs or free camptothecin derivatives were prepared from stock solution and diluted
Figure imgf000109_0001
into appropriated working concentration 24 hours after cell seeding. A serial ten-fold dilution for seven points was performed with culture medium. The final concentrations were ranging from 10,000 nM to 0.001 nM. The cells were incubated with ADCs for 5 days. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot#PL701) was added to the wells for 1-4 hours at 37°C and the absorbance at 450 nm was measured using a Microplate Reader (SpectraMax M5, Molecular Devices) and SoftMax Pro5.4.1 software. Dose-response curves were generated and IC50 was calculated using GraphPad Prism 7 three-parameter curve fitting.
[0282] Figure 3 - Figure 6 show the representative killing curves of free camptothecin derivatives and ADCs containing payload BI-P352, BI-P353 in BT-474 (Figure 3), NCI-N87 (Figure 4), SK-BR-3 (Figure 5) and BxPC-3 (Figure 6) cells. Overall, ADCs containing various camptothecin derivative payloads demonstrated specific and potent in vitro killing activities in Her2-expressingcells.
[0283] Table 2 and Table 3 summarize the IC50 values from in vitro cytotoxicity assays. Results demonstrate that Herceptin-352 and Herceptin-353 induced potent cytotoxicity against BT-474, SK-BR-3 and NCI-N87 tumor cells that have high Her2 expression, with IC50 in the sub nM range. In the Her2 negative BxPC-3 cells, IC50 of both ADCs was around 100 nM. On the other hand, the free payload BI-340 and BI-341 was able to kill all three cell lines with similar IC50s. These in vitro cytotoxicity results suggest a wide therapeutic window for the BI-340 and BI-341 containing ADCs.
Table 2
Figure imgf000110_0002
Figure imgf000110_0001
Table 3
Figure imgf000111_0002
Example 13
Plasma Stability of Herceptin-BI-P353
[0284] To assess the in vitro plasma stability of ADC, Herceptin-BI-P353 (0.1 mg/ml) was incubated in biank human plasma at 37°C for up to 96 hours. At each time point, samples were drawn and the remaining amount of total antibody (naked + conjugated) and conjugated antibody (ADC) were measured using a quantitative sandwich enzyme linked immunoassay (ELISA). In brief, to measure total antibody, human Her2 protein was coated onto the microplate to capture Herceptin antibodies. After removing the unbound antibodies, the bound total Herceptin antibody was detected with horseradish peroxidase (HRP) conjugated goat anti- human IgG Fc specific polycolonal antibody. For the determination of Herceptin-BI-P353 ADC only, a mouse anti-DXd antibody was added as the secondary antibody. The concentration of Herceptin- BI-P353 ADC was then detected with HRP conjugated goat anti-mouse lgG(H+L) antibody. Figure xx shows the percentage of remaining total antibody and Herceptin-BI-P353 at different time points. The trend for Herceptin-BI-P353 was similar to that of total antibody. Both of them remained at 88% after 96 hours. These results indicate that Herceptin-BI-P353 is stable in human plasma.
Figure imgf000111_0001
Example 14 in Vivo Characterization of Herceptin-BI-P353
[0285] The anti-tumor activities of Herceptin-BI-P353 ADC was assessed using Hem- positive NCI-N87 xenograft model in mouse subjects. Five million cells were harvested from culture flasks and implanted subcutaneously into the right flank of 6- to 7-week-old BALB/c nude mice. Dosing started when tumors were established. Herceptin-BI-P353 and Herceptin-Dxd was administered at 2.5 and 5.0 mg/kg weekly for a total of 3 doses. Tumors were measured twice a week throughout the course of the experiments, with tumor volume calculated using the following formula: tumor volume (mm3) = (length x width2)/2.
[0286] Figure 8A shows the results of the in vivo NCI-N87 xenograft study. As shown, the administration of Herceptin-353 at both dose levels resulted in partial tumor regression, similar to the effects induced by Herceptin-DXd. Both ADCs had no obvious effect on mouse body weigh change compared to vehicle control as show in Figure 8B.
[0287] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present invention. While the articles and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the invention. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, it should be understood that although the present invention has been
Figure imgf000112_0001
specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
Figure imgf000113_0001

Claims

What is claimed is:
1. An antibody drug conjugate (ADC) comprising an antibody chemically linked to a camptothecin analog represented by the following formula (I):
Ab-(L-D)n (I) or a pharmaceutically acceptable salt thereof; wherein n is about 1 to about 8; wherein Ab is an antibody or antigen binding fragment thereof; wherein D is a camptothecin analog; and wherein L is a bivalent linker.
2. An ADC having the formula I according to claim 1, wherein the camptothecin analog drug moiety has the structure:
Figure imgf000114_0001
wherein Ri is a hydrogen atom or a C1-C6 alkyl; wherein X is selected from -C(=O)-(CH2)n1-S-, ”C("O)-(CH2)n1-O-NR2-, -C(=O)-(CH2)n1-
N(OR2)-, -C(=O)-(CH2)n1-NR2-, -S(=O)2-CH2~(CH2)n1-S-, ~S(=O)2-CH2-(CH2)n1-O-, -S(=O)2-CH2- (CH2)n1-O-NR2-,-S(-O)2-CH2-(CH2)n1-N(OR2)-1 S(=O)2-CH2-(CH2)n1-N(R2)-, -S(-O)2-NH-CH2- (CH2)n1-S-, -S(=O)2-NH-CH2-(CH2)n1-O-, -C(=O)-O-(CH2)n2-S-i -C(=O)-O-(CH2)n2-O-, -C(=O)-O- (CH2)n2-O-NR2-, “C(=O)-O“(CH2)n2-N(OR2)", -C(=O)-O-(CH2)n2-N(R2)-, -C(=O)-NH-(CH2)n2-S-, - C(=O)-NH-(CH2)n2-O-, -C(=O)-NH-(CH2)n2-O-NR2-, -C(=O)-NH-(CH2)n2-N(OR2)~, and -C(=O)-
NH-(CH2)n2-N(R2)-; wherein L is a bivalent linker comprising a peptide moiety of 2-4 amino acid represented by the following formula (II):
L!-L2“L3 wherein L1 is a linker attached to the antibody comprising a reactive functional group selected from maleimide, bromoacetyl, iodoacetyl, thiol, amino, alkyl bromide, aikyl iodide, carboxyl, and NHS ester; wherein L2 is 2-4 AA peptide moiety; wherein L3 is a self-immolative moiety connected to the drug moiety that is:
Figure imgf000115_0001
and wherein R2 is hydrogen atom or C1-C6 alkyl; n1 is 1 , 2, 3, 4 or 5; and wherein n2 is 2, 3,
4 or 5,
3. An ADC having the formula I according to any one of claims 1-2, wherein Ab is capable of binding to a tumor associated antigen (TAA) selected from the group consisting of Trop-2, Her2, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21 , CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD123, CD127, CD134, CD137, CD138, CD146, CD147, CD152, CD154, CD174, CD195, CD200, CD205, CD212, CD223, CD227, CD253, CD272, CD274, CD276, CD278, CD279, CD309, CD319, CD326, CD340, DR6, Kv1.3, 5E10, MUC1, uPA, MAGES, MUC16, KLK3, K-ras, Mesothelin, p53, Survivin, G250, PSMA, Endoplasmin, BCMA, GPNMB, EphA2, EphB2, TMEFF2, Integrin beta 6, 5T4, CA9, IGF-1 R, Axl, B7H3, B7H4, CDH6, HAVCR1 , STEAP-1, STEAP-2, UPK2, CLDN18, CLDN6, CLDN9, c-Met, MICA, UV-1 , ROR1, ADAMS, Stn, DLK-1 and CEACAM-5.
Figure imgf000115_0002
4. An ADC having the formula I according to any one of claims 1-3, wherein Ab is an anti- Her2 antibody or a binding fragment thereof.
5. An ADC having the formula I according to any one of claims 1-2, wherein L has a structure selected from:
Figure imgf000116_0001
Figure imgf000117_0001
6. An ADC according to claim 2, wherein L2 is a dipeptide, tripeptide or tetrapeptide.
7. An ADC according to claim 6, wherein L2 is selected from: gly-gly, gly-gly-gly, phe-lys, val-ala, val-cit, gly-gly-phe-gly, val-cit-gly, val-gln-gly, val-giu-gly, phe-lys-gly, glu-val-ala, glu- val-cit, p-ala-gly-phe-giy, and gly-gly-phe-gly-gly.
8. An ADC according to any one of claims 1-2, wherein the camptothecin analog drug moiety has the structure of compound BI-P353.
9. A pharmaceutical composition comprising an ADC of any of claims 1-8.
Figure imgf000117_0002
10. A method for treating a cancer comprising administering to a subject in need thereof a pharmaceutical composition according to claim 9.
11. A method for treating cancer comprising administering to a subject in need thereof a pharmaceutical composition according to claim 9 in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, iPS induced CAR-T or iPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG), optionally wherein a synergy exists between the ADC constructs and the immunotherapy when co-administered.
12. A method according to any one of claims 10-11, wherein the cancer is selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head-neck cancer, and rhabdomyosarcoma.
13. A method according to any one of claims 10-12, wherein the subject has a resistant or refractory cancer.
14. A method of making an antibody drug conjugate, comprising reacting compound Bl- P353 with an antibody, thereby obtaining the antibody drug conjugate.
Figure imgf000118_0001
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