DOSAGE SCHEMES AND DOSAGE FORMS TO INHIBIT TARGETED TGF-BETA

专利摘要:
the present invention relates in general to bw-independent dosing regimens and dosage forms of a bifunctional protein targeting the programmed cell death binding human protein 1 (pd-l1) and the factor transforming growth factor (tgfß).
公开号:BR112019013924A2
申请号:R112019013924-9
申请日:2018-01-05
公开日:2020-02-11
发明作者:Dussault Isabelle；El Bawab Samer；Vugmeyster Yulia；Khandelwal Akash
申请人:Merck Patent Gmbh；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for “DOSAGE REGIMES AND DOSAGE FORMS FOR INHIBITION OF TARGETED TGF-BETA”.
CROSS REFERENCE WITH RELATED REQUIREMENTS [0001] This application claims the benefit of and priority for US patent application number 62 / 443,698, filed on January 7, 2017, and for US patent application number 62 / 581,978, registered on 6 November 2017, whose contents are incorporated by each one by reference in their entirety for all purposes.
FIELD OF THE INVENTION [0002] The present invention relates in general to BW-independent dosing regimens and dosage forms of a bifunctional protein targeting the Programmed Cell Death Link 1 human protein ( PD-L1) and the Transforming Growth Factor β (TGFp).
BACKGROUND [0003] Programmed death axis 1 (PD-1) / PD-L1 is an important mechanism for tumor immune evasion. Effector T cells, which chronically detect antigen, assume a depleted phenotype, marked by expression of PD-1, a state under which tumor cells are involved by positive regulation of PD-L1. Additionally, in the tumor microenvironment, myeloid cells, macrophages, parenchymal cells and T cells positively regulate PD-L1. Blocking the axis restores the effector function in these T cells.
[0004] The publication of United States patent application number 20150225483 A1, incorporated herein, in this patent application, by reference, describes a bifunctional fusion protein that combines an anti-cellulite death antibody
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2/127 programmed home 1 (PD-L1) with the soluble extracellular domain of tumor growth factor beta receptor type II (TGFpRIl) as a TGFp neutralizing "Trap" (TGFfi neutralizing "Trap"), in a single molecule . Specifically, the protein is a heterotetramer, consisting of the two anti-PDL1 immunoglobulin light chains, and the two heavy chains comprising the anti-PD-L1 heavy chain fused genetically by means of a flexible glycine-serine linker to the extracellular domain of human TGFpRIl (see Fig. 1). This anti-PD-L1 / TGFp Trap molecule is designed to target two important mechanisms of immunosuppression in the tumor microenvironment. United States patent application publication number 20150225483 A1 describes the administration of the Trap molecule in doses based on the patient's weight.
SUMMARY OF THE INVENTION [0005] The present invention provides improved dosage regimens for the administration of bifunctional proteins targeting PD-L1 and TGFp. Specifically, BW-independent dosing regimens and related dosage forms involving the administration of at least 500 mg of the bifunctional protein administered at various dosing frequencies can be used as an anti-tumor and anti-cancer therapy. The dosage regimen independent of body weight ensures that all patients, regardless of their body weight, will have adequate exposure to the drug at the tumor site.
[0006] The bifunctional protein of the present invention (anti-PD-L1 / TGFp Trap molecule, (anti-PD-L1 / ΤΰΡβ Trap molecule)) includes a first and a second polypeptide. The first polypeptide includes: (a) at least one variable region of an antibody heavy chain that binds to the Pro Cell Death Link human protein
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3/127 gram 1 (PD-L1); and (b) the Human Transforming Growth Factor β Receptor II (TGFpRIl), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΰΕβ) (e.g., a soluble fragment). The second polypeptide includes at least one variable region of an antibody light chain that binds PD-L1, in which the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site. which binds to PD-L1 (for example, any of the antibodies or antibody fragments described herein, in this patent application). As the bifunctional protein of the present invention binds to two targets, (1) PD-L1, which is largely bound to the membrane, and (2) ΤΰΡβ, which is soluble in the blood and in the interstitium, the dosing regimen regardless of body weight, it requires a dose that is effective not only to inhibit PD-L1 at the tumor site, but that is also sufficient to inhibit ΤΰΡβ.
[0007] In one aspect, the invention provides for the treatment of cancer or the inhibition of a tumor, for example, non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer (e.g., pretreated colorectal cancer ( CRC)), ovarian cancer, glioblastoma, gastric cancer (for example, pre-treated refractory or refractory Stage IV gastric cancer), biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head or neck, and squamous carcinoma of the head or neck.
[0008] The invention also features a bifunctional protein described above for use in the treatment of cancer or for use in inhibiting tumor growth. The cancer or tumor can be selected from colorectal (for example, pretreated colorectal cancer (CRC)), breast, ovarian, pancreatic, gastric (for example, recurrent non-resectable or refractory Stage IV gastric cancer) , in
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4/127 prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, vesical, neuroendocrine, head and neck, liver, nasopharynx, testicular, small cell lung cancer, lung cancer non-small cells, melanoma, basal cell skin cancer, squamous cell skin cancer, protruding dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes. The use may additionally include the administration of radiation or the administration of a chemotherapeutic agent, a biological agent, or a vaccine.
[0009] The invention also features a method of stimulating local TGFp depletion. The method includes administering a protein described above, where the protein binds TGFp in solution, binds PD-L1 on a cell surface, and loads the bound TGFp into the cell (for example, a cancer cell).
[0010] The invention also features a method of inhibiting the phosphorylation of SMAD3 in a cell (e.g., a cancer cell or an immune cell), the method including exposing the cell in the tumor microenvironment to a protein described above.
[0011] The invention also features a method of inhibiting tumor growth or treating cancer. The method includes exposing the tumor to a protein described above. The method may additionally include exposing the tumor to radiation or to a chemotherapeutic agent, a biological agent, or a vaccine. In certain modalities, the tumor or cancer is selected from colorectal (for example, pretreated colorectal cancer (CRC)), breast, ovarian, pancreatic, gastric (for example, recurrent or refractory non-resectable Stage IV gastric cancer -treated), prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, vesical, neuroendocrine, head and neck, liver, nasopharynx, testicular, cannula
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5/127 small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, protuberant dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma , and myelodysplastic syndromes.
[0012] By "TGFpRII" or "TGFp Receptor II" is meant a polypeptide having the sequence of Isoform A of the Type 2 Receptor of wild-type human TGFp (for example, the amino acid sequence of NCBI Reference Sequence (RefSeq) Access No NP_001020018 (SEQ ID NO. 8)), or a polypeptide having the wild-type human TGFp Receptor Type 2 Isoform B sequence (e.g., the NCBI amino acid sequence RefSeq Accession No. NP_003233 (SEQ ID NO. 9) ) or having a sequence substantially identical to the amino acid sequence of SEQ ID NO. 8 or SEQ ID NO. 9. TGFpRIl can retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99% of activity binding to TGFp from the wild-type sequence. The expressed TGFpRI1 polypeptide lacks the signal sequence.
[0013] An "TGFpRIl fragment capable of binding to TGFp" indicates any portion of NCBI RefSeq Accession No. NP_001020018 (SEQ ID NO. 8) or NCBI RefSeq Accession No. NP_003233 (SEQ ID NO. 9), or a sequence substantially identical to SEQ ID NO. 8 or SEQ ID NO. 9 that has a minimum of 20 (for example, a minimum of 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, or 200) amino acids in length that conserves at least part of TGFp binding activity (for example, at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95 %, or 99%) of the wild receptor or the corresponding wild fragment. Typically the said fragment is a soluble fragment. An example of a similar fragment is an extracePetition domain 870190062642, of 7/4/2019, p. 93/234
6/127 lular TGFpRII having the sequence of SEQ ID NO: 10.
[0014] By "substantially identical" is indicated a polypeptide showing at least 50%, desirably 60%, 70%, 75%, or 80%, more desirably 85%, 90%, or 95%, and most desirably 99% of amino acid sequence identity with a reference amino acid sequence. The length of comparison strings will generally be at least 10 amino acids, desirably at least 15 ontiguous amino acids, more desirably at least 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 ontiguous amino acids, and most desirably the entire length of the amino acid sequence.
[0015] By "patient" is meant a human being or a non-human animal (for example, a mammal). "Patient", "individual," "patient who needs it," and "individual who needs it" are used interchangeably in this invention, and refer to a living organism suffering from or prone to a disease or condition that may be treated by administration using the methods and compositions provided in this invention.
[0016] The terms "treat," "treating," or "treatment," and other grammatical equivalents as used in this invention, include alleviating, mitigating, improving or preventing a disease, condition or symptoms, preventing additional symptoms, improving, or preventing the underlying metabolic causes of symptoms, inhibit the disease or condition, for example, stop the development of the disease or condition, alleviate the disease or condition, cause the disease or condition to regress, alleviate a condition caused by the disease or condition, or stop the symptoms of the disease or condition, and are intended to include prophylaxis. The terms additionally include achieving a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit indicates eradication or improvement of the underlying disorder being treated. In addition, a tera benefit
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7/127 is obtained with the eradication or improvement of one or more of the physiological symptoms associated with the underlying disorder in such a way that an improvement in the patient is observed, despite the fact that the patient may still be afflicted with the underlying disorder.
[0017] By "cancer" is meant a collection of cells multiplying in an abnormal way. As used herein, in this patent application, the term "cancer" refers to all types of cancer, neoplasm, malignant or benign tumors found in mammals, including leukemia, carcinomas, and sarcomas. Examples of cancers include breast cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer, glioblastoma. Additional examples include brain cancer, lung cancer, non-small cell lung cancer, melanoma, sarcomas, prostate cancer, cervical cancer, stomach cancer, head and neck cancers, uterine cancer, mesothelioma, metastatic bone cancer , medulloblastoma, Hodgkin's disease, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macrobulinemia, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, cancer , cancer of the genitourinary tract, malignant hypercalcemia, endometrial cancer, cortical adrenal cancer, and endocrine and exocrine pancreas neoplasms.
[0018] From beginning to end of the description and claims of this specification, the word "understand" and other forms of the word, such as "comprising" and "understand", mean including, but not limited to, and are not intended to exclude , for example, other components.
[0019] By "co-administering" it is indicated that a composition described here, in this patent application, is administered at the same time, just before, or shortly after the administration of additional therapies. THE
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The protein and composition of the present invention can be administered alone or can be co-administered with a second, third, or fourth therapeutic agent (s) to a patient. Co-administration means including simultaneous or sequential administration of the protein or composition individually or in combination (more than one therapeutic agent).
[0020] The term "one" is not intended to limit as a singular. In certain embodiments, the term "one" can refer to a plural form. As used throughout this invention, the forms "one," "one," and "o" "a," include reference in the plural unless the context clearly dictates otherwise. Thus, for example, a reference to "a composition" includes a plurality of similar compositions, as well as a single composition.
[0021] A "reconstituted" formulation is a formulation which has been prepared by dissolving a lyophilized formulation in an aqueous vehicle such that the bifunctional molecule is dissolved in the reconstituted formulation. The reconstituted formulation is suitable for intravenous (IV) administration to a patient who needs it.
[0022] The term "about" refers to any minimal change in the concentration or amount of an agent that does not change the effectiveness of the agent in the preparation of a formulation and in the treatment of a disease or disorder. In modalities, the term “about” can include ± 15% of a specified numerical value or data point.
[0023] Intervals can be expressed in this invention as from "about" a particular value, and / or even "about" another particular value. When a similar range is expressed, another aspect includes from that one particular value and / or even the other particular value. Similarly, when values are expressed as approximations, using the antecedent "about," it must be en
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9/127 tended that the particular value forms another aspect. It should be further understood that the end points of each of the intervals are significant both in relation to the other end point, and independently of the other end point. It should also be understood that there are a number of values disclosed in this invention, and that each value is also disclosed as "about" that particular value in addition to the value itself. It should also be understood that from the beginning to the end of the application, the data is provided in a number of different formats and that this data represents end points and starting points and intervals for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it should be understood that they are considered disclosed also greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 as well as between 10 and 15. It should also be understood that each unit is also disclosed between two particular units. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
[0024] An "isotonic" formulation is a formulation which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsmol / KgFhO. The term hypertonic is used to describe a formulation with an osmotic pressure above the osmotic pressure of human blood. Isotonicity can be measured using a vapor pressure or an ice freezing type osmometer, for example.
[0025] The term "buffering agent" refers to one or more components that, when added to an aqueous solution, have the ability to protect the solution against changes in pH when acid or alkali is added, or after dilution with a solvent. Beyond
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10/127 of phosphate buffers, buffers of glycinate, carbonate, citrate and the like can be used, in which case sodium, potassium or ammonium ions can serve as a counterion.
[0026] An "acid" is a substance that produces hydrogen ions in aqueous solution. A "pharmaceutically acceptable acid" includes inorganic and organic acids which are non-toxic in concentration and in the manner in which they are formulated.
[0027] A "base" is a substance that produces oxyhydryl ions in aqueous solution. "Pharmaceutically acceptable bases" include inorganic and organic bases which are non-toxic in concentration and in the manner in which they are formulated.
[0028] A "lyoprotectant" is a molecule which, when combined with a protein of interest, prevents or reduces the chemical and / or physical instability of the protein after freeze-drying and subsequent storage.
[0029] A "condom" is an agent that reduces bacterial action and can optionally be added to the formulations here, in this patent application. The addition of a condom can, for example, facilitate the production of a multipurpose (multiple dose) formulation. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butyl and benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 pentanol, and m-cresol.
[0030] A "surfactant" is an active molecule on the surface containing both a hydrophobic moiety (eg, alkyl chain) and a hydrophilic moiety (eg, carboxyl and
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11/127 carboxylate). Surfactant can be added to the formulations of the invention. Surfactants suitable for use in the formulations of the present invention include, but are not limited to, polysorbates (e.g., polysorbates 20 or 80); poloxamers (for example, poloxamer 188); sorbitan esters and derivatives; Triton; sodium laurel sulfate; octyl glycoside sodium; lauryl-, myristyl-, linoleyl-, or stearylsulfobetadine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropylbetaine (for example, lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl taurate; and the MONAQUAT ™ series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68 etc.).
[0031] Other embodiments and details of the invention are presented below here, in this patent application.
BRIEF DESCRIPTION OF THE DRAWINGS [0032] FIG. 1 is a schematic drawing of an anti-PD-L1 / TGFp Trap molecule including an anti-PD-L1 antibody fused to two extracellular domains (ECD) TGFp Receptor II via a 4Gly (SEQ ID) linker NO: 11).
[0033] FIG. 2 shows a graph of a two-step ELISA demonstrating that the anti-PD-L1 / TGFp Trap simultaneously binds both PD-L1 and TGFp.
[0034] FIG. 3 is a graph showing that anti-PD-L1 / TGFp Trap induces a dramatic increase in IL-2 levels. [0035] FIG. 4A is a graph showing in vivo depletion of TGFpi in response to anti-PD-L1 / TGFp Trap. Line charts represent naive, isotype control, and three different doses, as indicated in the legend. FIG. 4B is a graph showing the
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12/127 TGFp2 in vivo depletion in response to anti-PD-L1 I TGFp trap. Line charts represent naive, isotype control, and three different doses, as indicated in the legend. FIG. 4C is a graph showing in vivo depletion of TGFp3 in response to anti-PD-L1 / TGFp Trap. Line charts represent naive, isotype control, and three different doses, as indicated in the legend. FIG. 4D is a graph showing that the occupation of PD-L1 by anti-PD-L1 / TGFp Trap supports a receptor binding model in the EMT-6 tumor system.
[0036] FIG. 5 is a graph showing the anti-tumor efficacy of anti-PD-L1 / TGFp trap (anti-PD-L1 (mut) / TGFP) control in a Detroit 562 xenograft model.
[0037] FIG. 6A is a scatter plot showing the relationship between clearance (CL) and body weight (BW). The line represents the regression line showing the relationship between CL and BW. FIG. 6B is a scatter plot showing the relationship between volume of distribution (V) and body weight. The line represents the regression line demonstrating the relationship between V and BW.
[0038] FIG. 7A is a box plot of Cmédia distribution for an entire population for a fixed dosage (1200 mg) versus a mg / kg based dose (17.65 mg / kg) in a simulated population of 68 kg median body weight . FIG. 7B is a distribution box plot of the AUC exposure for an entire population for a fixed dosage (1200 mg) versus a mg / kg based dosage (17.65 mg / kg) in a simulated population of 68 kg body weight median. FIG. 7C is a box plot of the Cmínima distribution for an entire population for a fixed dosage (1200 mg) versus a mg / kg (17.65 mg / kg) based dosage in a simulated population of 68 kg median body weight . FIG. 7D is a box plot of the Cmax distribution for an entire population for
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13/127 a fixed dose (1200 mg) versus a dose based on mg / kg (17.65 mg / kg) in a simulated population of 68 kg median body weight.
[0039] FIG. 7E is a box plot of Cmédia distribution for an entire population for a fixed dosage (500 mg) versus a mg / kg based dose (7.35 mg / kg) in a simulated population of 68 kg median body weight . FIG. 7F is a distribution box plot of the AUC exposure for an entire population for a fixed dose (500 mg) versus a mg / kg based dose (7.35 mg / kg) in a simulated population of 68 kg body weight median. FIG. 7G is a box plot of the Cmínima distribution for an entire population for a fixed dosage (500 mg) versus a mg / kg based dose (7.35 mg / kg) in a simulated population of 68 kg median body weight . FIG. 7H is a box plot of the Cmax distribution for an entire population for a fixed dose (500 mg) versus a dose based on mg / kg (7.35 mg / kg) in a simulated population of 68 kg median body weight .
[0040] FIG. 8A is a box plot of Cmédia distribution across body weight quartiles for a fixed dosage (1200 mg) versus a mg / kg based dose (17.65 mg / kg) in a simulated population of 68 kg in weight average body. FIG. 8B is a box plot of exposure distribution (AUC) across body weight quartiles for a fixed dosage (1200 mg) versus a mg / kg (17.65 mg / kg) based dosage in a simulated population of 68 kg of median body weight. FIG. 8C is a box plot of Cmínima distribution by quartiles of body weight for a fixed dosage (1200 mg) versus a dosage based on mg / kg (17.65 mg / kg) in a simulated population of 68 kg in weight average body. FIG. 8D is a box plot of Cmax distribution through
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14/127 body weight quartiles for a fixed dose (1200 mg) versus a dose based on mg / kg (17.65 mg / kg) in a simulated population of 68 kg median body weight.
[0041] FIG. 8E is a box plot of Cmédia distribution by quartiles of body weight for a fixed dosage (500 mg) versus a dosage based on mg / kg (7.35 mg / kg) in a simulated population of 68 kg in weight average body. FIG. 8F is a box plot of exposure distribution (AUG) across body weight quartiles for a fixed dosage (500 mg) versus a mg / kg based dose (7.35 mg / kg) in a simulated population of 68 kg of median body weight. FIG. 8G is a box plot of Cmínima distribution through quartiles of body weight for a fixed dosage (500 mg) versus a dosage based on mg / kg (7.35 mg / kg) in a simulated population of 68 kg in weight average body. FIG. 8H is a box plot of Cmax distribution by quartiles of body weight for a fixed dosage (500 mg) versus a dosage based on mg / kg (7.35 mg / kg) in a simulated population of 68 kg in weight average body.
[0042] FIG. 9A is the quality of the fit of the scatterplot for the Pharmacokinetic-Efficacy model showing the predicted vs. tumor volume. the observed tumor volume. FIG. 9B is the quality of the fit of the scatterplot for the PharmacokineticsEffectiveness model showing conditional weighted residues (GWRES) vs. time after the dose.
[0043] FIGS. 10A to 10C are graphs showing the predicted PK and PD-L1 (“RO”) occupation of anti-PD-L1 / TGFp Trap molecules at doses and schedules associated with tumor regression in mice. FIG. 10A is a graph showing the predicted plasma concentration vs. time. FIG. 10B is a graph showing the predicted occupancy of PD-L1 receptors vs.
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12/157 time in peripheral blood mononuclear cells (PBMC). FIG. 10C is a graph showing the predicted occupancy of PD-L1 receptors vs. tumor time.
[0044] FIGS. 11A to 11C are graphs showing the predicted PK and PD-L1 (“RO”) occupation of anti-PD-L1 / TGFp Trap molecules at doses and schedules associated with tumor stasis in mice. FIG. 11A is a graph showing the predicted plasma concentration vs. time. FIG. 11B is a graph showing the predicted occupancy of PD-L1 receptors vs. time in peripheral blood mononuclear cells. FIG. 11C is a graph showing the predicted occupancy of PD-L1 receptors vs. tumor time.
[0045] FIGS. 12A to 12B are distribution box plots of the simulated exposure (FIG. 12A: Cmédia; FIG. 12B. Cmínima) for the entire population for various dosing regimes in a simulated population of 68 kg median body weight.
[0046] FIG. 13 is a spider graph showing that patients with previously progressive disease (both with primary refractory and acquired resistant disease) have achieved important disease stabilization. It was observed that patients with disease response and disease stabilization have a range of treatments prior to the start of this study, and even had a range of treatments immediately prior to the start of the test, suggesting clinical activity of anti-PD-L1 / TGFp in a heterogeneous population of patients with exposure to previous PDx (filled triangle: individual out of treatment; filled diamond: first occurrence of a new lesion).
[0047] FIG. 14 shows a histogram of the efficacy of an anti-PD-L1 / TGFp trap molecule in patients treated with anti-PD-1 / PD-L1 treatment. The effectiveness of the anti-PD-L1 /
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TGFp trap was observed in some patients identified as refractory (black bars) and resistant (white bars) to the previous anti-PD-1 / PD-L1 population (a value of about zero (0) or a negative value of the percentage of change in the sum of diameters indicates effectiveness).
DETAILED DESCRIPTION
Independent Body Weight Dosing Regimen [0048] Independent body weight dosing regimes involving the administration of at least 500 mg of the bifunctional antiPD-L1 / TGFp Trap molecules described here, in this patent application, were developed, informed by the results a variety of preclinical and clinical evaluations of the molecules. Two studies investigated the safety, tolerability, and pharmacokinetics of the molecules, and included assessments of the target occupation of PD-L1 on peripheral blood mononuclear cells obtained from the blood of treated patients and measurements of the concentrations of TGFpl, TGFp2, and TGFp3. These evaluations were based on data from a total of 350 individuals (dose escalation cohorts of 1.3, 10 and 20 mg / kg in solid tumors, and expansion cohorts of 3 mg / kg, 10 mg / kg, 500 mg, and 1200 mg in selected tumor types). From the cut-off date data at the time of the analysis, the median duration of treatment was approximately 28 days.
Pharmacokinetic / Efficacy Model (Mouse Model) [0049] Experiments were also conducted to determine the efficacy of the anti-PD-L1 / TGFp Trap molecule in a tumor model. Results of the effectiveness of EMT-6 xenografts were used to establish the Pharmacokinetic / Efficacy model. The pharmacokinetic model established in mice was used to simulate plasma exposure to anti-PD-L1 / TGFp trap for the settings of the efficacy experiment. The parameters estiPetição 870190062642, from 07/04/2019, p. 104/234
17/127 measurements are reported in Table 1. The estimated KC50 value was
55.3 pg / ml. This value represents the mean plasma concentrations at which 50% if the maximum anti-tumor activity of the anti-PD-L1 / TGFp Trap molecule can be achieved.
[0050] Basic model diagnostic diagrams did not disclose any model specification errors. The model's predictions have the ability to capture tumor volume distributions (FIG. 9A). Conditional weighted residues are normally distributed with a mean 0 and variance 1 without a trend (FIG. 9B). The Pharmacokinetics / Efficacy model was then used to simulate tumor growth inhibition (TGI) using the predicted concentration-time profiles of humans at different doses.
[0051] Table 1: Pharmacokinetic model parameters / mouse efficacy for anti-PD-L1 molecule / TGFp trap in mice with EMT-6 xenograft
Parameters Estimate Std CV% % IIV Kg (h ~ ’> 0.068 0.0005 0.82 40 Ktr (h ¹ ) 0.055 0.0024 4.4 76 KC50 (ng / mL) 55324.6 522.3 4.4 232 Km 2 0.09 1 93 Baseline (mm ³ ) 88.3 0.87 1 47
Response Analysis Based on PD-L1 Occupation (in a Mouse Model) [0052] Using efficacy experiments, responses in mice were analyzed and classified either by tumor regression or by tumor stasis, and the occupation of PK and PD-L1 (RO) receivers based on the integrated PK / RO model. The approach demonstrated that a plasma concentration of the anti-PD-L1 / TGFp Trap molecule between 40 and 100 pg / mL associated with an occupation of PD-L1 receptors (PD-L1 RO) above
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95% tumor to achieve tumor regression (FIGs. 9A-9B). The plasma concentration of the anti-PD-L1 / TGFp Trap molecule between 10 and 40 pg / mL associated with an occupation of PD-L1 receptors above 95% on the periphery is required to achieve tumor stasis (FIGs. 10A to 10C).
[0053] Analysis of response and predicted PK / RO in mice leads to FIGs. 11- to 11C, which summarize the PK / RO / Efficacy for the anti-PD-L1 / TGFp Trap molecule in mice. 95% occupancy of PD-L1 receptors is obtained at a plasma concentration of 40 pg / mL with an expected / estimated tumor growth inhibition (TGI) of only about 65%. Increasing the concentration above 40 pg / mL results in an additional increase in inhibition of tumor growth. 95% inhibition of tumor growth is obtained at an average plasma concentration of about 100 pg / mL.
[0054] Based on the population pharmacokinetic model described below, a fixed dose (NT: flat dose) of at least 500 mg administered once every two weeks is required to maintain an average concentration of around 100 pg / mL, while whereas a fixed dose of about 1200 mg administered once every two weeks is required to maintain a minimum of about 100 pg / ml. In certain embodiments, about 1200 mg to about 3000 mg (for example, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, etc.) of a protein product of the present invention (e.g., anti-PD-L1 / TGFP Trap) is administered to an individual. In certain embodiments, about 1200 mg of anti-PDL1 / TGFp Trap molecule is administered to an individual once every two weeks. In certain embodiments, about 1800 mg
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19/127 anti-PD-L1 molecule I TGFp trap is administered to an individual once every three weeks.
[0055] In embodiments, about 1200 mg to about 3000 mg (for example, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, etc.) of the protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1 is administered to the subject.
[0056] In certain embodiments, about 1200 mg of the protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1 is administered to an individual once every two weeks. In certain embodiments, about 1800 mg of the protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1 is administered to an individual a every three weeks.
Pharmacokinetic Analysis (PK) Sampling in Humans [0057] Serum samples for analysis of pharmacokinetic data (PK) were collected before the start of the first dose and at the following time points after the first dose: on Day 1 immediately after the infusion and 4 hours after the start of the infusion; on Day 2 at least 24 hours after the end of Day 1 infusion; and on Days 8 and 15. All subsequent dosing occasions selected pre-dose, end of infusion and 2 to 8 hours after the end of infusion, samples were collected on days 15, 29, 43. For later time points in days 57, 71 and 85, pre-dose samples were or should be collected
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20/127 of those followed by PK sampling once every 6 weeks PK up to 12 weeks, then PK sampling once every 12 weeks. In the expansion phase, sparse PK sampling was conducted.
Establishment of Independent Body Weight Dosage Regime [0058] Informed by clinical and preclinical data, a new independent body weight dosing regime was created for the administration of anti-PD-L1 / TGFp Trap molecules, to obtain less variability in exposure, reduce dosing errors, reduce the time needed to prepare doses, and reduce drug waste compared to mg / kg dosage, thereby facilitating favorable treatment results. According to one modality, a fixed dose of at least 500 mg can be administered, regardless of the patient's body weight. According to another modality, a fixed dose of at least 1200 mg can be administered, regardless of the patient's body weight. Typically, a similar dose would be administered repeatedly, such as once every two weeks or once every 3 weeks, for example.
[0059] The PK data from the “Sampling of PK Analysis in Humans” described above were used to produce a population pharmacokinetic model and to perform simulations of possible dosing regimens. A modeling method, known as the full approach model, described in Gastonguay, M., Full Covariate Models as an Alternative to Methods Relying on Statistical Significance for Inferences about Covariate Effects: A Review of Methodology and 42 Case Studies, ( 2011) p. 20, Abstract 2229, to the population model data obtained from the simulations to obtain parameters having the following characteristics: 2-compartment PK model with linear elimination, IIV over CL, V1, and V2, additive
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12/21 combined and proportional residual error, complete covariate model on CL and V1. The following baseline covariables were included in the final model: age, weight, sex, race, albumin, CRP, platelet count, eGFR, liver failure, ECOG score, tumor size, tumor type, and previous treatment with biological agents. The following estimates of typical parameter estimates of the pharmacokinetics of the protein of the present invention (for example, anti-PD-L1 / TGFp Trap) were obtained: clearance (CL) 0.0177 L / h (6.2%), volume central distribution (V1) 3.64 L (8.81%), peripheral distribution volume (V2) 0.513 L (25.1%), and intercompartmental clearance (Q) 0.00219 L / h (17.8% ). The interpatient variability was 22% for CL, 20% for V1, and 135% for V2. Body weight was a relevant covariate in both CL and V1. To corroborate the fixed dosing approach, the impact of the dosing strategy on the variability of the exposure of the protein of the present invention (for example, anti-PD-L1 / TGFp Trap) was explored. Specifically, simulations were performed to compare exposure distribution using a 1200 mg fixed-dose approach once every two weeks versus a body weight-adjusted dosing approach of or 17.65 mg / kg once every two weeks ( corresponding to 1200 mg once every two weeks for a 68 kg individual or 15 mg / kg once every two weeks (corresponding to 1200 mg for an 80 kg individual) .Additionally, simulations were performed to compare the exposure distribution using a fixed 500 mg dosing approach once every two weeks versus a 7.35 mg / kg body weight adjusted dosing approach once every two weeks (corresponding to 500 mg once every two weeks for a 68 kg individual) In addition, simulations were performed to assess the following fixed doses once every three weeks
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22/127 nas: 1200 mg, 1400, mg, 1600 mg, 1800 mg, 2000 mg, 2200 mg, 2400 mg, 2600 mg, 2800 mg, 3000 mg.
[0060] The following methodology was used for simulations: N = 200 sets of parameter estimates were extracted from the normal multivariate distribution of parameter estimates, using the variance-covariance matrix of the final PK model. For each parameter estimate, 200 IIV estimates were extracted from the $ OMEGA multivariate normal distribution, resulting in a total of 40,000 (200 x 200) individuals. The original data set (N = 380) was re-sampled with replacement to generate 40,000 sets of corresponding covariables and steady-state exposure metrics (AUG, Cmédia, Cmínima and Cmáx) were generated for each dosing regimen.
[0061] Simulations showed that across a broad spectrum of body weight, the variability in exposure is slightly greater for body weight based dosing compared to fixed dosing. An example of distribution of exposure at 17.65 mg / kg and a fixed dose of 1200 mg, or 7.35 mg / kg and a fixed dose of 500 mg for a median body weight of 68 kg is shown in FIGs. 7A and 7B, respectively. Simulations also showed the opposite trend in exposure distributions across weight quartiles across the patient population: underweight patients are more exposed with fixed dosage, whereas overweight patients are more exposed with dosage adjusted by body weight.
[0062] An example of exposure distribution across quartiles weighing 17.65 mg / kg and a fixed dose of 1200 mg or 7.35 mg / kg and a fixed dose of 500 mg for a median body weight of 68 kg is shown in FIGs. 8A and 8B, respectively.
Establishment of Dose / Effective Dosage Regime and Exposure in Humans: Preliminary dose-response and exposure response in Non-Small Cell Lung Cancer from 2 ^to
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Nail (2L NSCLC) after dosing once every 2 weeks (q2w) of anti-PD-L1 / TGFp Trap [0063] In one aspect, dose-response and exposure-response assessments are based on data from 80 subjects who were administered either 500 mg or 1200 mg of anti-PD-L1 / TGFp Trap once every 2 weeks (q2w) (n = 40 per cohort) in the treatment of 2L non-small cell lung cancer. The dose-response and exposure-response of the individuals were evaluated. From the cut data at the time of the analysis, a total of 17 individuals remained on treatment with a median follow-up of 35.2 (range, 1.3 to 47.3) weeks. The overall response rate (ORR) not confirmed by the researcher was 25.0% (ORR 500 mg, 22.5%; ORR 1200 mg, 27.5%), with 9 partial responses (PR) seen in 500 mg, and 1 complete response (CR) and 10 PRs at 1200 mg. Clinical activity was observed through levels of PD-L1 expression with ORR of 71.4% to 1200 mg noted in patients with tumor expression of PD-L1> 80% (7 patients). The most common treatment-related adverse events (EARTs) were itching (18.8%), maculopapular rash (17.5%), decreased appetite (12.5%), and asthenia (11.3%). Grade> 3 treatment-related adverse events occurred in 20 patients (25%). There were no treatment-related deaths.
[0064] For the exposure-response assessment, the population pharmacokinetic model described above was used to predict the first cycle of exposures based on the dosage and covariate information of these 80 patients. Specifically, AUG and Cminima were predicted after a single dose for each individual using Bayes empirical estimates of population pharmacokinetic parameters (Tables 2 and 3).
[0065] Table 2: Summary of AUCo-336h, predicted by cohort
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Cohort Median (mg * h / L) Percentile from 2.5 to 97.5 (mg * h / L) 500 mg q2w 27346 14561 - 59193 1200 mg q2w 64981 47449 - 84799
AUCo -336h, sd = AUG for a period of 0 to 336 hours after a single dose, as predicted using a population pharmacokinetic model.
[0066] Table 3: Summary of Cmínima, sd predicted by cohort
Cohort Median (pg / mL) 2.5 to 97.5 percentile (pg / mL) 500 mg q2w 31.82 9.803 to 100.6 1200 mg q2w 78.76 51.25 to 136.2
Cmínima, sd - Cmínima after a single dose, as predicted using a population pharmacokinetic model.
[0067] Predicted exposure data were combined for cohorts of 500mg q2w and 1200mg q2w to calculate a response rate for each quartile of predicted exposure, as shown in Table 4 and Table 5 below. These preliminary data suggest that 1200 mg q2w may provide a more favorable efficacy profile compared to 500 mg q2w. In addition, these data suggest that the exposure range obtained with 1200 mg q2w dosing regimen is associated with response (by RECIST v1.1) in 2L non-small cell lung cancer and that this exposure range can be used to design alternative dosing regimes as shown in the example below (Fig. 12).
[0068] Table 4: Response rate observed by AUCo-336h, sd in individuals with 2L non-small cell lung cancer treated with either 500 mg or 1200 mg of anti-PD-L1 / TGFp trap once every 2 weeks
AUC- Quartile336h, sd Number of respondents in a 500 mg cohort Number of responders in a 1200 mg cohort Total number of respondents per quartile Total Response Rate 0 to 25% 2/20 0/0 2/20 10%
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25 to 50% 5/18 0/2 5/20 25% 50 to 75% 1/1 4/19 5/20 25% 75 to 100% 3/1 6/19 6/20 30%
AUCo -336h, sd = AUG for 0 to 336 hours after a single dose, as predicted using a population pharmacokinetic model.
[0069] Table 5: Response rate observed by Cmínima, sd in individuals with 2L non-small cell lung cancer treated with either 500 mg or 1200 mg of anti-PD-L1 / TGFp trap once every 2 weeks ( n = 40 per dose group).
Quartín da Cmínima, sd Number of respondents in a 500 mg cohort Number of responders in a 1200 mg cohort Total number of respondents Rate ofanswerTotal 0 to 25% 1/20 0/0 1/20 5% 25 to 50% 5/15 0/5 5/20 25% 50 to 75% 1/3 3/17 4/20 20% 75 to 100% 1/2 7/18 8/20 40%
Cmínima, sd - Cmínima after a single dose, as predicted using a population pharmacokinetic model.
Establishing Dosing Regimen with Multiple Dosing Frequencies [0070] Data regimes with varying dosing frequencies have been created to enable less frequent administration and / or to allow coordination of dosing schedules with concomitant medications. Specifically, the preliminary population pharmacokinetic modeling and simulation methodology described above were used to simulate exposures for various dosing regimes and to compare regimes based on exposure.
[0071] Based on these simulations, a fixed dose of at least 500 mg administered once every two weeks is required to maintain an average concentration of around 100 pg / mL for a typical individual, whereas a fixed dose is required approximately 1200 mg given once every two weeks to maintain a Cmí
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26/127 minimum of about 100 pg / ml.
[0072] Based on simulations for Cmédia, 1200 mg once every two weeks is equivalent to 1800 mg once every three weeks (Fig. 12A), whereas for Cminima, 1200 mg once every two weeks is equivalent to 2800 mg once every three weeks (Fig. 12B). And for Cmédia, 500 mg once every two weeks is equivalent to 750 mg once every three weeks; for Cminima, 500 mg once every two weeks is equivalent to 1,167 mg once every three weeks.
TGFg as a Cancer Target [0073] The present invention allows for localized reduction in TGFp in a tumor microenvironment by capturing TGFp using a soluble cytokine receptor (TGFpRIl) tied to an antibody portion targeting a checkpoint receptor ) immune cell found on the outer surface of certain tumor cells or immune cells. An example of an antibody portion of the invention for an immune checkpoint protein is anti-PD-L1. This bifunctional molecule, sometimes referred to in this document as an “antibody-Cytokine Trap,” is effective precisely because the anti-receptor antibody and the Cytokine Trap are physically linked. The resulting advantage (in relation, for example, to the administration of the antibody and the receptor as separate molecules) is partly because cytokines function predominantly in the local environment through autocrine and paracrine functions. The antibody portion directs the Cytokine Trap to the tumor microenvironment where it can be most effective, neutralizing the autocrine or paracrine local immunosuppressive effects. In addition, in cases where the antibody target is internalized after binding to the antibody, an effective mechanism for clearance of the cytokine / cytokine receptor complex is provided. It has been shown to internalize
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27/127 antibody-mediated targeting for PD-L1, and anti-PD-L1 / TGFp Trap has been shown to have a similar internalization rate to anti-PD-L1. This is a distinct advantage over the use of an anti-TGFp antibody because first, an anti-TGFp antibody may not be completely neutralizing; and second, the antibody can act as a vehicle extending the cytokine half-life.
[0074] In fact, as described below, treatment with anti-PD-L1 / TGFp Trap causes a synergistic antitumor effect due to the simultaneous blocking of the interaction between PD-L1 on tumor cells and PD-1 on immune cells , and the neutralization of TGFβ in the tumor microenvironment. To be limited by theory, this is presumably due to a synergistic effect obtained from the simultaneous blocking of the two main immune escape mechanisms, and in addition, to the depletion of TGFp in the tumor microenvironment by a single molecular entity. This depletion is achieved by (1) targeting anti-PD-L1 tumor cells; (2) binding of autocrine / paracrine TGFp in the tumor microenvironment by the TGFp Trap; and (3) destruction of bound TGFβ through PD-L1 receptor-mediated endocytosis. Furthermore, TGFpRIl fused to the C-terminal of Fc (IgG crystallization fragment) was several times more potent than the TGFpRIl-Fc that places TGFpRIl at the N-terminal of Fc.
[0075] TGFp has been a somewhat questionable target in cancer immunotherapy due to its paradoxical roles like Jekyll and molecular cancer Hyde (Bierie et al., Nat. Rev. Cancer, 2006; 6: 506-20). Like some other cytokines, TGFp activity is dependent on the stage of development and the context. In fact, TGFp can act as either a tumor stimulator or a tumor suppressor, affecting tumor initiation, progression and metastasis. The mechanisms underlying this dual role of TGFp remain uncertain (Yang et al., Trends Immunol. 2010; 31: 220-227). Although it was
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28/127 postulated that Smad-dependent signaling mediates the inhibition of TGFp signaling growth, while Smad-independent pathways contribute to its tumor-stimulating effect, there are also data showing that Smad-dependent pathways are involved in tumor progression (Yang et al., Cancer Res. 2008; 68: 9107-11).
[0076] Both the TGFβ ligand and the receptor have been studied intensively as therapeutic targets. There are three isoforms of ligands, TGFpl, 2 and 3, all of which exist as homodimers. There are also three TGFp receptors (TGFpR), which are called TGFpR type I, II and III (López-Casillas et al., J Cell Biol. 1994; 124: 557-68). TGFpRI is the signal chain and cannot bind ligand. TGFpRIl binds the ligand TGFpl e 3, but not TGFp2, with high affinity. The TGFpRIl / TGFp complex recruits TGFpRI to form the signaling complex (Won et al., Cancer Res. 1999; 59: 1273-7). TGFpRIH is a positive regulator of TGFp binding to its signaling receptors and binding all 3 isoforms of TGFp with high affinity. On the cell surface, the TGFp / TGFpRIH complex binds TGFpRII and then recruits TGFpRI, which displaces TGFpRIH to form the signaling complex.
[0077] Although the three different TGFβ isoforms all signal through the same receptor, it is known that they have differential expression patterns and non-overlapping functions in vivo. The knockout mice of three different TGF-β isoforms have different phenotypes, indicating numerous unbalanced functions (Bujak et al., Cardiovasc Res. 2007; 74: 184-95). While TGFpl null mice have defects of 4 hematopoiesis and vasculogenesis and TGFp3 null mice have pulmonary development and defective palatogenesis, TGFp2 null mice have several developmental abnormalities, the most prominent being multiple deformi.
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29/127 cardiac disorders (Bartram et al., Circulation. 2001; 103: 2745-52; Yamagishi et al., Anat Rec. 2012; 295: 257-67). In addition, TGFp is implicated to play an important role in repairing myocardial damage after ischemia and reperfusion injury. In an adult heart, cardiomyocytes secrete TGFp, which acts as an autocrine to maintain the spontaneous beat rate. Importantly, 70 to 85% of the TGFp secreted by cardiomyocytes is TGFp2 (Roberts et al., J Clin Invest. 1992; 90: 2056-62). Despite concerns about cardiotoxicity created by treatment with TGFpRI kinase inhibitors, the present applicants observed a lack of toxicity, including cardiotoxicity, for anti-PD-L1 / TGFp trap in monkeys.
[0078] Therapeutic approaches to neutralize TGFp include the use of extracellular TGFp receptor domains as traps for soluble receptors and neutralizing antibodies. From the Receptor Trap (N.T .: Receptor Trap) approach, soluble TGFpRIH may seem the obvious choice once it binds to all three TGFp ligands. However, TGFpRIH, which naturally occurs as a 280 to 330 kD glucosaminoglcano glycoprotein (GAG), with an extracellular domain of 762 amino acid residues, is a very complex protein for biotherapeutic development. Soluble TGFpRIH devoid of GAG can be produced in insect cells and has been shown to be a potent TGFp neutralizing agent (Vilchis-Landeros et al, Biochem J 355: 215, 2001). The two separate binding domains (the endoglobin-related and the uromodulin-related) of TGFpRIH can be expressed independently, but it has been shown to have affinities 20 to 100 times less than that of soluble TGFpRIH, and greatly reduced neutralizing activity (Mendoza et al., Biochemistry. 2009; 48: 1175565). On the other hand, the TGFpRIl extracellular domain has only
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136 amino acid residues in length and can be produced as a glycosylated protein of 25 to 35 kD. It has been further demonstrated that recombinant soluble TGFpRI1 binds to TGFp1 with a 200 pM Kd, which is reasonably similar to the 50 pM Kd for full-length TGFpRI1 on cells (Lin et al., J Biol Chem. 1995; 270: 2747-54). Soluble TGFpRIl-Fc has been tested as an anticancer agent and has been shown to inhibit the growth of murine malignant mesothelioma established in a tumor model (Suzuki et al., Clin. Cancer Res., 2004; 10: 5907-18). Since TGFpRII does not bind to TGFp2, and TGFpRIII binds to TGFp1 and 3 with less affinity than TGFpRII, a fusion protein of the TGFpRIII endogline domain and TGFpRII extracellular domain has been produced in bacteria and has been shown to inhibit TGFpl signaling. and 2 in cell-based assays more effectively than either TGFpRII or RIH (Verona etal., Protein Eng Des Sei. 2008; 21: 463-73).
[0079] Yet another approach to neutralizing all three isoforms of TGFp ligands is to screen for a panneutralizing anti-TGFp antibody, or an anti-receptor antibody that blocks the receptor to bind to TGFpl, 2 and 3. GC1008, a human antibody specific for all isoforms of TGFp, were in a Phase l / ll study in patients with advanced malignant melanoma or renal cell carcinoma (Morris et al., J Clin Oncol 2008; 26: 9028 (Meeting abstract)). Although the treatment was considered safe and well tolerated, only limited clinical efficacy was observed, and therefore it was difficult to interpret the importance of anti-TGFp therapy without further characterizing the immunological effects (Flavell et a!., Nat Rev Immunol. 2010; 10 : 554-67). There were also antibodies specific to TGFβ isoforms tested in the clinic. Metelimumab, an antibody specific for TGFpl was tested in a Phase 2 clinical study as a treatment to prevent excessive healing in the postoperative period for
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12/31 glaucoma; and Lerdelimumab, an antibody specific for TGFp2, was found to be safe but ineffective to improve healing after eye surgery in a Phase 3 study (Khaw et al., Ophthalmology 2007; 114: 1822-1830). Anti-TGFpRIl antibodies that block receptor binding to all three TGFp isoforms, such as the anti-human TGFpRIl TR1 antibody and the mouse anti-TGFpRIl MT1 antibody, also showed some therapeutic efficacy against primary tumor growth and metastasis in models in mouse (Zhong et al., Clin Cancer Res. 2010; 16: 1191-205). However, in a recent Phase I study of the TR1 antibody (LY3022859), dose escalation beyond 25 mg (fixed dose) was considered unsafe due to uncontrolled release of cytokines, despite prophylactic treatment (Tolcher et al., Cancer Chemother Pharmacol 2017; 79: 673-680). To date, the vast majority of studies on anti-cancer treatment targeting TGFp, including small molecule inhibitors of TGFp signaling which are often quite toxic, are mostly in the preclinical stage and the antitumor efficacy obtained has been limited (Calone et al., Exp Oncol. 2012; 34: 9-16; Connolly et al., Int J Biol Sei. 2012; 8: 964-78).
[0080] The TGFp Trap antibody of the invention is a bifunctional protein containing at least a portion of a human TGFp Receptor II (TGFpRIl) that is capable of binding to TGFp. In certain embodiments, the TGFβ Trap polypeptide is a soluble portion of the human TGFβ Type 2 Receptor Isoform A (SEQ ID NO: 8) that is capable of binding to TGFβ. In certain embodiments, the TGFp Trap polypeptide contains at least amino acids 73 to 184 of SEQ ID NO: 8. In certain embodiments, the TGFp Trap polypeptide contains amino acids 24 to 184 of SEQ ID NO: 8. In certain embodiments modalities, the TGFp Trap polypeptide is a soluble portion of the Type 2 Receptor Isoform B of
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Human TGFp (SEQ ID NO: 9) which is capable of binding to TGFp. In certain embodiments, the TGFp Trap polypeptide contains at least amino acids 48 to 159 of SEQ ID NO: 9. In certain embodiments, the TGFp Trap polypeptide contains amino acids 24 to 159 of SEQ ID NO: 9. embodiments, the TGFp Trap polypeptide contains amino acids 24 to 105 of SEQ ID NO: 9.
Mechanisms of Action [0081] The approach to targeting T cell inhibition inspection points for de-inhibition with therapeutic antibodies is an area of intense investigation (for a review, see Pardoll, Nat Rev Cancer. 2012; 12: 253- 264). In one approach, a portion of antibody or antigen-binding fragment targets the receptor proteins from T cell inhibition checkpoints on the T cell, such as, for example: CTLA-4, PD-1, BTLA , LAG3, TIM-3, or LAIR1. In another approach, the antibody portion targets counter-receptors on antigen presenting cells and tumor cells (which co-opt some of these counter-receptors for their own immune evasion), such as, for example: PD-L1 (B7- H1), B7-DC, HVEM, TIM-4, B7-H3, or B7-H4.
[0082] The invention contemplates anti-TGFp antibody traps that target, through their portion of antibody or antigen-binding fragment thereof, inspection points for inhibition of T cells for disinhibition. To this end, applicants have tested the anti-tumor efficacy of combining a TGFp Trap (Τ (3Εβ Trap) with antibodies targeting various receptor proteins for T cell inhibition checkpoints, such as anti-PD-1, anti-PD -L1, anti-TIM-3 and anti-LAG3.
[0083] Programmed death axis 1 (PD-1) / PD-L1 is an important mechanism for tumor immune evasion. Effector T cells that
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33/127 chronically detect antigen assume an exhausted phenotype marked by expression of PD-1, a state under which tumor cells are involved by upward regulation of PD-L1. Additionally, in the tumor microenvironment, myeloid cells, macrophages, parenchymal cells and T cells upwardly regulate PD-L1. The blockade of the axis restores the effector function in these T cells. Anti-PD-L1 / TGFp Trap also binds to TGFp (isoforms 1,2, and 3), which is an inhibitory cytokine produced in the tumor microenvironment by cells including apoptotic netrophils , myeloid-derived suppressor cells, T cells and tumor. Inhibition of TGFp by soluble TGFpRIl reduced malignant mesothelioma in a manner that was associated with increases in the antitumor effects of CD8 + T cells. It has been shown that the absence of TGFpl produced by activated CD4 + T cells and Treg cells inhibits tumor growth, and protects mice against spontaneous cancer. Therefore, TGFp appears to be important for tumor immune evasion.
[0084] TGFp has growth inhibitory effects on normal epithelial cells, acting as a regulator of epithelial cell homeostasis, and acts as a tumor suppressor during initial carcinogenesis. As tumors progress to malignancy, the effects of inhibiting TGFp growth on the tumor are lost through mutation in one or more signaling components of the TGFp pathway or through oncogenic reprogramming. After loss of sensitivity to inhibit TGFp, the tumor continues to produce high levels of TGFp, which then serve to stimulate tumor growth. The TGFp cytokine is overexpressed in several types of cancer with correlation with the tumor stage. Many types of cells in the tumor microenvironment produce TGFβ, including the tumor cells themselves, immature myeloid cells, regulatory T cells, and stromal fibroblasts; these cells collectively ge
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34/127 had a large TGFp reservoir in the extracellular matrix. TGFp signaling contributes to tumor progression by promoting metastasis, stimulating angiogenesis, and suppressing innate and adaptive antitumor immunity. As a largely immunosuppressive factor, TGFp directly down-regulates the effector function of activated cytotoxic T cells and NK cells and potently induces the differentiation of naive CD4 + T cells into the immunosuppressive regulatory T cell (Treg) phenotype. In addition, TGFp polarizes macrophages and netrophils to a wound healing phenotype that is associated with the production of immunosuppressive cytokines. As a therapeutic strategy, neutralizing TGFp activity has the potential to control tumor growth by restoring effective antitumor immunity, blocking metastasis, and inhibiting angiogenesis.
[0085] Combining these pathways, PD-1 or PD-L1, and TGFp, is attractive as an anti-tumor approach. Blocking PD-1 and concomitant TGFp can restore pro-inflammatory cytokines. Anti-PD-L1 / TGFp Trap includes, for example, an extracellular domain of the human TGFp receptor TGFpRIl covalently joined via a glycine / serine linker to the C terminal of each anti-PD-L1 IgG antibody heavy chain 1 totally human. Given the emerging picture for the PD-1 / PD-L1 class, in which the responses are evident but with room for an increase in the effect size, it is assumed that the co-targeting of a complementary immun modulation step will improve the tumor response . A similar TGF targeting agent, fresolimumab, which is a monoclonal antibody targeting TGFpl, 2 and 3, showed initial evidence of tumor response in a Phase I study in individuals with melanoma.
[0086] In certain embodiments, the present invention provides experiments, which demonstrated that the TGFpRIl portion
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35/127 anti-PD-L1 / TGFp Trap (the Trap control “anti-PDL-1 (mut) / TGFp Trap”) caused antitumor activity. For example, after subcutaneous implantation in a Detroit 562 human pharyngeal carcinoma model, anti-PDL-1 (mut) / TGFp Trap caused a dose-dependent reduction in tumor volume when administered at 25 pg, 76 pg, or to 228 pg (FIG. 5).
[0087] In certain embodiments, the present invention provides experiments, which demonstrated that the protein of the present invention simultaneously bound both PD-L1 and TGFp (FIG. 2).
[0088] In certain embodiments, the present invention provides experiments, which demonstrated that the protein of the present invention (e.g., anti-PD-L1 / TGFP Trap) inhibited PD-L1 and TGFp-dependent signaling in vitro. In certain embodiments, the present invention provides experiments, which demonstrated that the protein of the present invention enhanced the effector function of T cells in vitro by blocking PD-L1-mediated immune inhibition as measured by an IL-2 induction test. after stimulation with superantigen (FIG. 3). At approximately 100 ng / ml, the protein of the present invention induced a dramatic increase in IL-2 levels in vitro (FIG. 3).
[0089] In certain embodiments, the present invention provides experiments, which demonstrated that the protein of the present invention (e.g., anti-PD-L1 / TGFP Trap) caused depletion of TGFp from the blood in vivo. Treatment of EMT-6 breast cancer cells implanted orthotopically in JH mice with 55 pg, or 164 pg, or 492 pg of the protein of the present invention resulted in efficient and specific depletion of TGFpl (FIG. 4A), TGFP2 (FIG. 4B ), and TGFP3 (FIG. 4C). In addition, the present invention provides experiments, which demonstrated that the protein of
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36/127 the present invention occupied the target PD-L1, corroborating the notion that the protein of the present invention fits into a receptor binding model in the EMT-6 tumor system (FIG. 4D).
[0090] In certain embodiments, the present invention provides experiments, which demonstrated that the protein of the present invention specifically bound, efficiently, and simultaneously to PD-L1 and TGFp, had potent antitumor activity in a variety of mouse models, suppressed tumor growth and metastasis, as well as prolonged survival and conferred long-lasting protective antitumor immunity.
[0091] In a first-in-human phase I dose escalation study, in addition to monitoring the pharmacokinetics of the anti-PD-L1 / TGFp trap molecule, the mechanism of action was investigated, particularly against TGFp cytokines.
[0092] Patients were treated with an anti-PD-L1 / TGFp Trap molecule intravenously administered at 5 dose levels of about 0.3, about 1, about 3, about 10, or about 20 mg / kg once every two weeks. Pharmacokinetic analyzes of the samples were performed until day 85. The occupation of the target PD-L1 was measured in CD3 + PBMCs by cytometry blood flow from the patient collected in the pre-dose, Day 2 (D2), D15, and D43. In addition, blood levels of TGFpi at 3 and proinflammatory cytokines were measured at these time points with an additional time point at D8 using multiple analytically validated Luminex and ECLIA based immunoassays. In one aspect, patients can be treated with an anti-PD-L1 / TGFp Trap molecule intravenously administered at 6 dose levels, including the dose levels described above, at a dose of about 30 mg / kg or about 40 mg / kg every two weeks. Pharmacokinetic analyzes of patients treated at 6 dose levels can be performed alongside
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37/127 tir samples for up ^to 6 · after the dose. The occupation of target PD-L1 can also be measured in CD3 + PBMCs by blood flow cytometry from a patient collected in the pre-dose, Day 2 (D2), D15, D43, and even D85. In addition, blood levels of TGFpi at 3 and proinflammatory cytokines can be measured at these time points with an additional time point, for example, at D8, using validated multiple Luminex and ECLIA based immunoassays analytically.
[0093] The results indicated that PK exposure to the anti-PD-L1 / TGFp Trap molecule during the first cycle increased in a dose-approximately manner between 3 to 20 mg / kg, without significant accumulation within the first 85 days of treatment. There was about 80% occupancy of target PD-L1 at 3 mg / kg to 20 mg / kg which was maintained from the beginning to the end of the dosing interval. There was additionally a small (1.7 times in D2) but significant IFNy induction at 0.3 to 20 mg / kg (p = 0.001, n = 19). The levels of TGFpi, TGFP2, and TGFP3 in the blood were reduced by a minimum of 99%, 92%, and 91%, respectively, at all time points to dose levels of 1 to 20 mg / kg. At the lowest dose of 0.3 mg / kg, TGFp1-3 levels were depleted in D2 and D8, but not in D15. In addition, there was additionally a strong correlation between drug PK levels and TGFp uptake. Therefore, complete capture of TGFp1-3 was achieved at a drug dose level of 1 mg / kg or above.
Anti-PD-L1 Antibodies [0094] The invention can include any anti-PD-L1 antibody, or antigen-binding fragment thereof, described in the art. Anti-PD-L1 antibodies are commercially available, for example, the 29E2A3 antibody (Biolegend, Cat. No. 329701). Antibodies can be monoclonal, chimeric, humanized, or human. Fragments of
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38/127 antibodies include Fab, F (ab ') 2, scFv and Fv fragments, which are described in further detail below.
[0095] Examples of antibodies are described in International PCT Patent Publication No. WO 2013/079174. These antibodies can include a heavy chain variable region polypeptide including a sequence of HVR-H1, HVR-H2, and HVR-H3, where:
(a) the HVR-H1 sequence is X1YX2MX3 (SEQ ID NO: 21);
(b) the HVR-H2 sequence is SIYPSGGX4TFYADX5VKG (SEQ ID NO:
22);
(c) the HVR-H3 sequence is IKLGTVTTVX ₆ Y (SEQ ID NO: 23); additionally where: X1 is K, R, T, Q, G, A, W, Μ, I, or S; X2 is V, R, K, L, M, or I; X ₃ is Η, T, N, Q, A, V, Y, W, F, or M; X ₄ is F or I; X ₅ is S or T; Xe is E or D.
[0096] In one mode, X1 is Μ, I, or S; X2 is R, K, L, M, or I; X3 is F or Μ; X4 is F or I; X5 is S or T; Xe is E or D.
[0097] In another modality X1 is Μ, I, or S; X2 is L, M, or I; X3 is F or Μ; X4 is I; X5 is S or T; Xe is D.
[0098] In yet another modality, X1 is S; X2 is I; X3 is Μ; X4 is I; X5 is T; Xe is D.
[0099] In another aspect, the polypeptide additionally includes heavy chain variable region structure sequences juxtaposed between the HVRs according to the formula: (HC-FR1) - (HVR-H1) - (HCFR2) - (HVR-H2 ) - (HC-FR3) - (HVR-H3) - (HC-FR4).
[00100] In yet another aspect, structure sequences are derived from human consensus structure sequences or human germline structure sequences.
[00101] In an additional aspect, at least one of the structure sequences is as follows:
HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 24);
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HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO: 25);
HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 26);
HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 27).
[00102] In an even further aspect, the heavy chain polypeptide is additionally combined with a variable region light chain including an HVR-L1, HVR-L2, and HVR-L3, where:
(a) the HVR-L1 sequence is TGTX ₇ X ₈ DVGX ₉ YNYVS (SEQ ID NO: 28);
(b) the HVR-L2 sequence is X10VX11X12RPS (SEQ ID NO: 29);
(c) the HVR-L3 sequence is SSX13TX14X15X16X17RV (SEQ ID NO: 30); additionally where: X7 is N or S; X ₈ is T, R, or S; X9 is A or G; X10 is E or D; X11 is I, N or S; X12 is D, H or N; X13 is F or Y; X14 is N or S; X15 is R, T or S; X16 is G or S; X17 is I or T.
[00103] In another modality, X7 is N or S; X ₈ is T, R, or S; X9 is A or G; X10 is E or D; Xn is N or S; X12 is N; X13 is F or Y; Xu is S; X15 is S; X16 is G or S; X17 is T.
[00104] In yet another modality, X7 is S; X ₈ is S; X9 is G; X10 is D; X11 is S; X12 is N; X13 is Y; Xi ₄ is S; X15 is S; Xi ₆ is S; X17 is T.
[00105] In a still additional aspect, the light chain additionally includes variable region light chain juxtaposed structure sequences between the HVRs according to the formula: (LC-FR1MHVRL1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4).
[00106] In a still further aspect, light chain structure sequences are derived from human consensus structure sequences or human germline structure sequences.
[00107] In a still further aspect, the light chain structure sequences are light chain sequences.
[00108] In an additional aspect, at least one of section 870190062642, of 07/04/2019, p. 127/234
40/127 structure structure is as follows:
LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO: 31);
LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO: 32);
LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 33);
LC-FR4 is FGTGTKVTVL (SEQ ID NO: 34).
[00109] In another embodiment, the invention provides an anti-PD-L1 antibody or antigen binding fragment including a heavy chain variable region and a light chain sequence, where:
(a) the heavy chain includes an HVR-H1, HVR-H2, and HVR-H3, in which additionally: (i) the HVR-H1 sequence is X1YX2MX3 (SEQ ID NO: 21); (ii) the HVR-H2 sequence is SIYPSGGX4TFYADX5VKG (SEQ ID NO: 22); (iii) the HVR-H3 sequence is IKLGTVTTVX ₆ Y (SEQ ID NO:
23), and;
(b) the light chain includes an HVR-L1, HVR-L2, and HVR-L3, in which additionally: (iv) the HVR-L1 sequence is TGTX7X8DVGX9YNYVS (SEQ ID NO: 28); (v) the HVR-L2 sequence is X10VX11X12RPS (SEQ ID NO: 29); (vi) the HVR-L3 sequence is SSX13TX14X15X16X17RV (SEQ ID NO: 30); where: X1 is K, R, T, Q, G, A, W, Μ, I, or S; X ₂ is V, R, K, L, M, or I; X ₃ is Η, T, N, Q, A, V, Y, W, F, or M; X ₄ is F or I; X ₅ is S or T; X ₆ is E or D; X ₇ is N or S; Xe is T, R, or S; X9 is A or G; X10 is E or D; X11 is I, N, or S; X12 is D, H, or N; X13 is F or Y; X14 is N or S; X15 is R, T, or S; X16 is G or S; X17 is I or T.
[00110] In a modality, X1 is Μ, I, or S; X2 is R, K, L, M, or I; X3 is F or M; X ₄ is F or I; X5 is S or T; Xe is E or D; X7 is N or S; Xe is T, R, or S; X9 is A or G; X10 is E or D; Xn is N or S; X12 is N; X13 is F or Y; Xu is S; X15 is S; X16 is G or S; X17 is T.
[00111] In another modality, X1 is Μ, I, or S; X2 is L, M, or I; X3 is F or M; X ₄ is I; X ₅ is S or T; X ₆ is D; X ₇ is N or S; X ₈ is T, R, or S; X ₉ is A
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41/127 or G; X10 is E or D; Xn is N or S; X12 is N; X13 is F or Y; Xu is S; X15 is S; X16 is G or S; X17 is T.
[00112] In yet another modality, X1 is S; X2 is I; X3 is Μ; X4 is I; X5 is T; X ₆ is D; X ₇ is S; X ₈ is S; X ₉ is G; X10 is D; Xu is S; X12 is N; X13 is Y; Xu is S; X15 is S; X16 is S; X17 is T.
[00113] In an additional aspect, the heavy chain variable region includes one or more structure sequences juxtaposed between HVRs such as: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) (HVR-H3) - (HC-FR4), and the light chain variable regions include one or more structure sequences juxtaposed between HVRs such as: (LC-FR1 MHVR-L1) - (LC- FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LCFR4).
[00114] In a still further aspect, the structure sequences are derived from human consensus structure sequences or human germline sequences.
[00115] In a still further aspect, one or more heavy chain structure sequences is as follows:
HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:
24);
HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO: 25);
HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 26);
HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 27).
[00116] In a still further aspect, the light chain structure sequences are light chain sequences.
[00117] In a still further aspect, one or more of the light chain structure sequences is as follows:
LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO: 31);
LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO: 32);
LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID
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NO: 33);
LC-FR4 is FGTGTKVTVL (SEQ ID NO: 34).
[00118] In a still further aspect, the heavy chain variable region polypeptide, antibody, or antibody fragment additionally includes at least one Ch1 domain.
[00119] In a more specific aspect, the heavy chain variable region polypeptide, antibody, or antibody fragment additionally includes a Ch1 domain, a Ch2 domain, and a Ch3 domain. [00120] In a still further aspect, the variable region light chain, antibody, or antibody fragment additionally includes a Cl domain.
[00121] In a still further aspect, the antibody additionally includes a Ch1 domain, a Ch2 domain, a Ch3 domain, and a Cl domain.
[00122] In a still further specific aspect, the antibody additionally includes a human or murine constant region.
[00123] In a still further aspect, the human constant region is selected from the group consisting of lgG1, lgG2, lgG2, lgG3, lgG4.
[00124] In a still further specific aspect, the human or murine constant region is IgG 1.
[00125] In yet another embodiment, the invention relates to an anti-PD-L1 antibody including a heavy chain variable region and a light chain sequence, where:
(a) the heavy chain includes an HVR-H1, an HVR-H2, and an HVRH3, having at least 80% general sequence identity with SYIMM (SEQ ID NO: 35), SIYPSGGITFYADTVKG (SEQ ID NO: 36), and IKLGTVTTVDY (SEQ ID NO: 37), respectively, and (b) the light chain includes an HVR-L1, an HVR-L2, and an HVR-L3, having at least 80% general sequence identity with TGTSS
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DVGGYNYVS (SEQ ID NO: 38), DVSNRPS (SEQ ID NO: 39), and SSYTSSSTRV (SEQ ID NO: 40), respectively.
[00126] In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
[00127] In yet another embodiment, the invention relates to an anti-PD-L1 antibody including a heavy chain variable region and a light chain sequence, where:
(a) the heavy chain includes an HVR-H1, an HVR-H2, and an HVRH3, having at least 80% general sequence identity with MYMMM (SEQ ID NO: 41), SIYPSGGITFYADSVKG (SEQ ID NO: 42), and IKLGTVTTVDY (SEQ ID NO: 37), respectively, and (b) the light chain includes an HVR-L1, an HVR-L2, and an HVR-L3, having at least 80% general sequence identity with TGTSSDVGAYNYVS (SEQ ID NO: 43), DVSNRPS (SEQ ID NO: 39), and SSYTSSSTRV (SEQ ID NO: 40), respectively.
[00128] In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
[00129] In an even further aspect, in the antibody or antibody fragment according to the invention, compared to the sequences of HVR-H1, HVR-H2, and HVR-H3, at least those amino acids remain unchanged which are highlighted by underlined as follows:
(a) in HVR-H1 SYIMM (SEQ ID NO: 35), (b) in HVR-H2 SIYPSGGITFYADTVKG (SEQ ID NO: 36), (c) in HVR-H3 IKLGTVTTVDY (SEQ ID NO: 37);
and additionally where, compared to the sequences of HVRL1, HVR-L2, and HVR-L3 at a minimum those amino acids remain unchanged which are highlighted by an underscore as follows:
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44/127 (a) HVR-L1 TGTSSDVGGYNYVS (SEQ ID NO: 38) (b) HVR-L2 DVSNRPS (SEQ ID NO: 39) (c) HVR-L3 SSYTSSSTRV (SEQ ID NO: 40).
[00130] In another aspect, the heavy chain variable region includes one or more structure sequences juxtaposed between the HVRs such as: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) (HC-FR4), and the light chain variable regions include one or more structure sequences juxtaposed between HVRs such as: (LC-FR1) (HVR-L1) - (LC -FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4).
[00131] In yet another aspect, the structure sequences are derived from human germline sequences.
[00132] In a still further aspect, one or more heavy chain structure sequences is as follows:
HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 24);
HC-FR2 is WVRQAPGKGLEWVS (SEQ ID NO: 25);
HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 26);
HC-FR4 is WGQGTLVTVSS (SEQ ID NO: 27).
[00133] In a still further aspect, the light chain structure sequences are derived from a light chain sequence.
[00134] In a still further aspect, one or more of the light chain structure sequences is as follows:
LC-FR1 is QSALTQPASVSGSPGQSITISC (SEQ ID NO: 31);
LC-FR2 is WYQQHPGKAPKLMIY (SEQ ID NO: 32);
LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 33);
LC-FR4 is FGTGTKVTVL (SEQ ID NO: 34).
[00135] In a still further specific aspect, the antibody adiPetição 870190062642, of 07/04/2019, p. 132/234
45/127 internationally includes a human or murine constant region.
[00136] In a still further aspect, the human constant region is selected from the group consisting of lgG1, lgG2, lgG2, lgG3, lgG4.
[00137] In certain embodiments, the invention relates to an anti-PD-L1 antibody including a heavy chain variable region and a light chain sequence, where:
(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMVWRQAPGKGLEWVSSIYPSGGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS (SEQ ID NO: 44) is a light chain with a string of at least
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSN RPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 45).
[00138] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
[00139] In certain embodiments, the invention relates to an anti-PD-L1 antibody including a heavy chain variable region and a light chain sequence, where:
(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEVWSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWG
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QGTLVTVSS (SEQ ID NO: 46), and (b) the light chain sequence has at least 85% sequence identity with the light chain sequence:
QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNR PSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 47).
[00140] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[00141] In another embodiment the antibody binds to human PD-L1, mice, or monkey cynomolgus. In a specific aspect, the antibody is capable of blocking the interaction between human, mouse, or cynomolgus monkey PD-L1 and the respective human, mouse, or monkey cynomolgus PD-1 receptors.
[00142] In another embodiment, the antibody binds to human PD-L1 with a KD of 5x10 ^-9 M or less, preferably with a KD of 2x10 ^-9 M or less, and even more preferably with a KD 1x10 ^-9 M or less.
[00143] In yet another embodiment, the invention relates to an anti-PD-L1 antibody or antigen-binding fragment thereof which binds to a functional epitope including human PD-L1 residues Y56 and D61.
[00144] In a specific aspect, the functional epitope additionally includes human PD-L1 E58, E60, Q66, R113, and M115.
[00145] In a more specific aspect, the antibody binds to a conformational epitope, including residues 54-66 and 112-122 of human PDL1.
[00146] In certain embodiments, the invention relates to an anti-PD-L1 antibody, or antigen-binding fragment thereof,
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47/127 which performs cross competition for binding to PD-L1 with an antibody according to the invention as described here, in this patent application.
[00147] In certain embodiments, the invention relates to proteins and polypeptides including any of the anti-PD-L1 antibodies described above in combination with at least one pharmaceutically acceptable carrier.
[00148] In certain embodiments, the invention relates to an isolated nucleic acid encoding a polypeptide, or a light or heavy chain variable region sequence of an anti-PD-L1 antibody, or antigen binding fragment thereof , as described here, in this patent application. In certain embodiments, the invention relates to an isolated nucleic acid encoding a light or heavy chain variable region sequence of an anti-PD-L1 antibody, in which:
(a) the heavy chain includes an HVR-H1 sequence, an HVR-H2 sequence, and an HVR-H3 sequence having at least 80% sequence identity with SYIMM (SEQ ID NO: 35), SIYPSGGITFYADTVKG (SEQ ID NO: 36), and IKLGTVTTVDY (SEQ ID NO: 37), respectively, or (b) the light chain includes an HVR-L1 sequence, HVRL2 sequence, and HVR-L3 sequence having at least 80% identity of sequence with TGTSSDVGGYNYVS (SEQ ID NO: 38), DVSNRPS (SEQ ID NO: 39), and SSYTSSSTRV (SEQ ID NO: 40), respectively. [00149] In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
[00150] In an additional aspect, the nucleic acid sequence for the heavy chain is:
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48/127 atggagttgc ctgttaggct gttggtgctg atgttctgga ttcctgctag ctccagcgag 60 gtgcagctgc tggaatccgg cggaggactg gtgcagcctg gcggctccct gagactgtct 120 tgcgccgcct ccggcttcac cttctccagc tacatcatga tgtgggtgcg acaggcccct 180 ggcaagggcc tggaatgggt gtcctccatc tacccctccg gcggcatcac cttctacgcc 240 gacaccgtga agggccggtt caccatctcc cgggacaact ccaagaacac cctgtacctg 300 cagatgaact ccctgcgggc cgaggacacc gccgtgtact actgcgcccg gatcaagctg 360 ggcaccgtga ccaccgtgga ctactggggc cagggcaccc tggtgacagt gtcctccgcc 420 tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tgg tggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcacg ggatgagctg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtcccc gggtaaa 1407 is a light chain: SEQ ID NO: 48
atggagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cttaagccag 60 tccgccctga cccagcctgc ctccgtgtct ggctcccctg gccagtccat caccatcagc 120 tgcaccggca cctccagcga cgtgggcggc tacaactacg tgtcctggta tcagcagcac 180 cccggcaagg cccccaagct gatgatctac gacgtgtcca accggccctc cggcgtgtcc 240 aacagattct ccggctccaa gtccggcaac accgcctccc tgaccatcag cggactgcag 300 gcagaggacg aggccgacta ctactgctcc tcctacacct cctccagcac cagagtgttc 360 ggcaccggca caaaagtgac cgtgctgggc cagcccaagg ccaacccaac cgtgacactg 420 ttccccccat cctccgagga actgcaggcc aacaaggcca ccctggtctg cctgatctca 480 gatttctatc caggcgccgt gaccgtggcc tggaaggctg atggctcccc agtgaaggcc 540 ggcgtggaaa ccaccaagcc ctccaagcag tccaacaaca aatacgccgc ctcctcctac 600 ctgtccctga cccccgagca gtggaagtcc caccggtcct acagctgcca ggtcacacac 660 gagggctcca ccgtggaaaa gaccgtcgcc cccaccgagt gctca 705 (SEQ ID NO: 49).
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49/127 [00151] Additional examples of anti-PD-L1 antibodies that can be used in an anti-PD-L1 / TGFp Trap are described in United States patent application publication No. US 2010/0203056. In one embodiment of the invention, the antibody portion is YW243.55S70. In another embodiment of the invention, the antibody portion is MPDL3289A.
[00152] In certain embodiments, the invention relates to a portion of anti-PD-L1 antibody including a heavy chain variable region and a light chain sequence, where:
(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGF DYWGQGTLVTVSS (light string with a string of at least%) (with a sequence of at least 85%, and
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 13) [00153]
(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGF DYWGQGTLVTVSA (SEQ ID NO: 85), and has a string of at least (85)
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50/127 sequence with the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR States. 743. [00155] In an embodiment of the invention, the anti-PD-L1 antibody is MDX-1105.
[00156] In certain embodiments, the anti-PD-L1 antibody is MEDI-4736.
Constant Region [00157] The proteins and peptides of the invention can include an immunoglobulin constant region or a fragment, analog, variant, mutant, or derivative of the constant region. In certain embodiments, the constant region is derived from a human immunoglobulin heavy chain, for example, lgG1, lgG2, lgG3, lgG4, or other classes. In certain embodiments, the constant region includes a CH2 domain. In certain embodiments, the constant region includes CH2 and CH3 domains or includes CH2-CH3 hinge. Alternatively, the constant region can include all or a portion of the hinge region, the CH2 domain and / or the CH3 domain.
[00158] In one embodiment, the constant region contains a mutation that reduces the affinity for an Fc receptor or reduces the effector function of Fc. For example, the constant region may contain a mutation that eliminates the glycosylation site within the constant region of an IgG heavy chain. In some embodiments, the constant region contains mutations, deletions, or insertions at an amino acid position corresponding to Leu234, Leu235, Gly236, Gly237, Asn297, or lgG1 Pro331 (amino acids are numbered according to
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51/127 with the European Union nomenclature). In a particular embodiment, the constant region contains a mutation at an amino acid position corresponding to Asn297 of lgG1. In alternative modalities, the constant region contains mutations, deletions, or insertions at an amino acid position corresponding to Leu281, Leu282, Gly283, Gly284, Asn344, or IgG 1 Pro378.
[00159] In some embodiments, the constant region contains a CH2 domain derived from a heavy chain of human IgG2 or IgG4. Preferably, the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain. In one embodiment, the mutation alters asparagine within the amino acid sequence Gln-Phe-Asn-Ser (SEQ ID NO: 15) within the CH2 domain of the lgG2 or lgG4 heavy chain. Preferably, the mutation changes asparagine to glutamine. Alternatively, the mutation alters both phenylalanine and asparagine within the Gln-Phe-Asn-Ser amino acid sequence (SEQ ID NO: 15). In one embodiment, the amino acid sequence Gln-Phe-Asn-Ser (SEQ ID NO: 15) is replaced with an amino acid sequence Gln-Ala-GIn-Ser (SEQ ID NO: 16). Asparagine within the GlnPhe-Asn-Ser amino acid sequence (SEQ ID NO: 15) corresponds to Asn297 of IgG1.
[00160] In another embodiment, the constant region includes a CH2 domain and at least a portion of an articulation region. The hinge region can be derived from an immunoglobulin heavy chain, for example, lgG1, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable classes. Most preferably, the hinge region is derived from a human IgG1 heavy chain. In one embodiment, the cysteine in the amino acid sequence Pro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO: 17) of the lgG1 hinge region is altered. In certain modalities, the
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52/127 Pro-Lys-Ser-Cys-Asp-Lys amino acid sequence (SEQ ID NO: 17) is replaced with a Pro-Lys-Ser-Ser-AspLys amino acid sequence (SEQ ID NO: 18). In certain embodiments, the constant region includes a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In certain embodiments, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain, whereas the hinge region is derived from an altered human IgG 1 heavy chain.
[00161] Alteration of amino acids near the junction of the Fc portion and the non-Fc portion can dramatically increase the serum half-life of the Fc fusion protein (PCT International Publication No. WO 0158957, the invention of which is incorporated by means of reference). Therefore, the junction region of a protein or polypeptide of the present invention may contain changes that, in relation to the naturally occurring sequences of an immunoglobulin and erythropoietin heavy chain, are preferably within about 10 amino acids from the point of junction. These amino acid changes can cause an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is replaced. Preferably, the C-terminal lysine residue of an IgG sequence is replaced with a non-lysine amino acid, such as alanine or leucine, to further increase the serum half-life. In another embodiment, the constant region is derived from an IgG sequence in which the amino acid sequence Leu-Ser-Leu-Ser (SEQ ID NO: 19) next to the Cterminal of the constant region is altered to eliminate potential junctional T cell epitopes . For example, in one embodiment, the Leu-Ser-Leu-Ser amino acid sequence (SEQ ID NO: 19) is replaced with an Ala-Thr-Ala-Thr amino acid sequence (SEQ ID NO:
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20). In other embodiments, the amino acids within the LeuSer-Leu-Ser segment (SEQ ID NO: 19) are replaced with other amino acids such as glycine or proline. Detailed methods of generating amino acid substitutions of the Leu-Ser-Leu-Ser segment (SEQ ID NO: 19) near the C-terminus of an lgG1, lgG2, lgG3, lgG4, or other molecule of the immunoglobulin class have been described in the Publication of United States Patent No. US 2003/0166877, the invention of which is hereby incorporated by reference.
[00162] Articulation regions suitable for the present invention can be derived from IgG1, IgG2, IgG3, IgG4, and other classes of immunoglobulins. The lgG1 hinge region has three cysteines, two of which are involved in disulfide bonds between the two immunoglobulin heavy chains. These same cysteines allow for an efficient and consistent disulfide bond formation between Fc portions. Therefore, a hinge region of the present invention is derived from IgG1, for example, from human IgG1. In some embodiments, the first cysteine within the human IgG1 hinge region is mutated to another amino acid, preferably serine. The lgG2 isotype hinge region has four disulfide bonds that tend to promote oligomerization and possibly incorrect disulfide bond during secretion in recombinant systems. A suitable joint region can be derived from an IgG2 joint; the first two cysteines are each preferentially mutated to another amino acid. The lgG4 hinge region is known to form interchain disulfide bonds ineffectively. However, a hinge region suitable for the present invention can be derived from the lgG4 hinge region, preferably containing a mutation that reinforces the correct formation of disulfide bonds between heavy chain derived moieties (Angal S, etal. ( 1993) Mol. Immunol., 30: 105-8).
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54/127 [00163] According to the present invention, the constant region can contain CH2 and / or CH3 domains and a hinge region that are derived from different antibody isotypes, i.e., a hybrid constant region. For example, in one embodiment, the constant region contains CH2 and / or CH3 domains derived from lgG2 or lgG4 and a mutant hinge region derived from lgG1. Alternatively, a mutant hinge region from another IgG subclass is used in a hybrid constant region. For example, a mutant form of the lgG4 hinge that allows efficient disulfide bonding between the two heavy chains can be used. A mutant joint can also be derived from an IgG2 joint in which the first two cysteines are each mutated to another amino acid. The assembly of similar hybrid constant regions has been described in U.S. Patent Publication No. 2003/0044423, the invention of which is hereby incorporated by reference.
[00164] In accordance with the present invention, the constant region may contain one or more mutations described here, in this patent application. Combinations of mutations in the Fc portion may have additive or synergistic effects on the prolonged serum half-life and increased in vivo potency of the bifunctional molecule. Therefore, in an example embodiment, the constant region may contain (i) a region derived from an IgG sequence in which the amino acid sequence Leu-Ser-Leu-Ser (SEQ ID NO: 19) is replaced with a sequence of amino acids Ala-Thr-Ala-Thr (SEQ ID NO: 20); (ii) a C-terminal alanine residue instead of lysine; (iii) a CH2 domain and a hinge region that are derived from different antibody isotypes, for example, a lgG2 CH2 domain and an altered lgG1 hinge region; and (iv) a mutation that eliminates the glycosylation site within the lgG2-derived CH2 domain, for example, one if
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55/127 amino acid sequence Gln-Ala-GIn-Ser (SEQ ID NO: 16) instead of the amino acid sequence Gln-Phe-Asn-Ser (SEQ ID NO: 15) within the CH2 domain derived from lgG2.
Antibody Fragments [00165] The proteins and polypeptides of the invention may also include antigen-binding antibody fragments. Examples of antibody fragments include scFv, Fv, Fab, F (ab ') 2, and single domain VHH fragments such as those of camelid origin.
[00166] Fragments of single chain antibodies, also known as single chain antibodies (scFvs), are recombinant polypeptides which typically bind to antigens or receptors; these fragments contain at least one fragment of an antibody variable heavy chain (Vh) amino acid sequence tied to at least one fragment of an antibody variable light chain (V1) sequence with or without one or more interconnecting chains. A similar linker may be a short, flexible peptide selected to ensure that the appropriate three-dimensional folding of the V1 and Vh domains occurs as soon as they are ligated in order to maintain the binding specificity of the target antibody of the entire antibody from which the antibody fragment of single chain is derived. In general, the carboxyl terminus of the V1 or Vh sequence is covalently linked by a peptide linker similar to the amino acid terminus of a complementary V1 and Vh sequence. Fragments of single chain antibodies can be generated by molecular cloning, the antibody phage display library or similar techniques. These proteins can be produced either in eukaryotic or prokaryotic cells, including bacteria.
[00167] Fragments of single chain antibodies contain sequences of amino acids having at least one of the variable regions or
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CDRs of the entire antibodies described in this specification, but some or all of the domains contained in those antibodies are missing. These constant domains are not necessary for antigen binding, but they do constitute a major portion of the structure of whole antibodies. Fragments of single chain antibodies therefore can overcome some of the problems associated with the use of antibodies containing part or all of a constant domain. For example, fragments of single chain antibodies tend to be free of unwanted interactions between biological molecules and the heavy chain constant region, or other unwanted biological activity. In addition, fragments of single chain antibodies are considerably smaller than whole antibodies and therefore may have greater capillary permeability than whole antibodies, allowing single chain antibody fragments to locate and bind to target antigen binding sites. most efficiently. In addition, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. In addition, the relatively small size of fragments of single chain antibodies makes them less likely than whole antibodies to elicit an immune response in a receptor. [00168] Antibody fragments that have the same binding characteristics or binding characteristics comparable to those of the entire antibody may also be present. The said fragments may contain one or both of the Fab fragments or the F (ab ') 2 fragment. Antibody fragments can contain all six CDRs of the entire antibody, although fragments containing less than all of the referred regions, such as three, four or five CDRs, are also functional.
Pharmaceutical Compositions [00169] The present invention also features pharmaceutical compositions
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57/127 drugs that contain a therapeutically effective amount of a protein described herein, in this patent application. The compositions can be formulated for use in a variety of drug delivery systems. One or more excipients or physiologically acceptable vehicles can also be included in the composition for appropriate formulation. Formulations suitable for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249: 1527-1533, 1990).
[00170] In one aspect, the present invention provides an intravenous drug release formulation that includes 500 mg to 2000 mg of a protein including a first polypeptide and a second polypeptide, the first polypeptide includes: (a) at least one variable region an antibody heavy chain that binds to the Programmed Cell Death Link 1 human protein (PD-L1); and (b) the human Transforming Growth Factor β Receptor II (TGFpRIl), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΰΕβ), the second polypeptide includes at least one variable region of a chain light of an antibody that binds to PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen-binding site that binds to PD-L1.
In certain embodiments, a protein product of the present invention includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1.
[00172] In certain embodiments of the present invention, the intravenous drug delivery formulation can include about 500 mg to about 2400 mg dose (e.g., about 500 mg to
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58/127 about 2300 mg, about 500 mg to about 2200 mg, about 500 mg to about 2100 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to 2400 mg, about 700 mg to 2400 mg, about 800 mg to 2400 mg, about 900 mg to 2400 mg, about 1000 mg to 2400 mg, about 1100 mg to 2400 mg, about 1200 mg to 2400 mg, about 1300 mg to 2400 mg, about 1400 mg to 2400 mg, about 1500 mg to 2400 mg, about 1600 mg to 2400 mg, about 1700 mg to 2400 mg, about 1800 mg to 2400 mg, about and 1900 mg to 2400 mg, about 2000 mg to 2400 mg, about 2100 mg to 2400 mg, about 2200 mg to 2400 mg, or about 2300 mg to 2400 mg) of a protein of the present invention (e.g. anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, the intravenous drug delivery formulation can include about 500 to about 2000 mg of a dose of a protein of the present invention (for example, anti-PD-L1 / TGFp Trap (for example, including a first polypeptide which includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide which includes the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, the intravenous drug delivery formulation can include about 500 mg of a dose of a protein product of the present invention
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59/127 with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the intravenous drug delivery formulation can include 500 mg dose of a protein of the present invention (for example, anti-PDL1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, the intravenous drug delivery formulation may include about 1200 mg of a dose of a protein product of the present invention with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the intravenous drug release formulation may include 1200 mg dose of a protein of the present invention (for example, anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, the intravenous drug delivery formulation can include about 1200 mg to about 3000 mg (for example, about 1200 mg to about 3000 mg, about 1200 mg to about 2900 mg, about 1200 mg about 2800 mg, about 1200 mg to about 2700 mg, about 1200 mg to about 2600 mg, about 1200 mg to about 2500 mg, about 1200 mg to about 2400 mg, about 1200 mg about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg,
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60/127 about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 3000 mg, about 1400 mg to about 3000 mg, about 1500 mg to about 3000 mg, about 1600 mg to about 3000 mg, about 1700 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 1900 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2100 mg to about 3000 mg, about 2200 mg to about 3000 mg, about 2300 mg to about 3000 mg, about 2400 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 2600 mg to about 3000 mg, about 2700 mg to about 3000 mg, about 2800 mg to about 3000 mg, about 2900 mg to about 3000 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, ce about 2800 mg, about 2900 mg, or about 3000 mg) of a protein product of the present invention (e.g., anti-PD-L1 / TGFP Trap). In certain embodiments, the intravenous drug delivery formulation can include about 1200 mg to about 3000 mg (for example, about 1200 mg to about 3000 mg, about 1200 mg to about 2900 mg, about 1200 mg about 2800 mg, about 1200 mg to about 2700 mg, about 1200 mg to about 2600 mg, about 1200 mg to about 2500 mg, about 1200 mg to about 2400 mg, about 1200 mg about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg at about 1300 mg,
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61/127 about 1300 mg to about 3000 mg, about 1400 mg to about 3000 mg, about 1500 mg to about 3000 mg, about 1600 mg to about 3000 mg, about 1700 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 1900 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2100 mg to about 3000 mg, about 2200 mg to about 3000 mg, about 2300 mg to about 3000 mg, about 2400 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 2600 mg to about 3000 mg, about 2700 mg to about 3000 mg, about 2800 mg to about 3000 mg, about 2900 mg to about 3000 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1500 mg, about 1600 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg) of a prot product acid with a first polypeptide comprising the amino acid sequence of SEQ ID NO: 3, and the second polypeptide comprising SEQ ID NO amino acid sequence: 1.
[00173] In certain embodiments, the intravenous drug release formulation can include about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg , about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg , about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150 mg, about 1175 mg , about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1300 mg, about 1400 mg, about 1425 mg , about 1450 mg, about
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1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about
1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about
1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about
2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, or about 2400 mg of the protein of the present invention (e.g., anti-PD-L1 / TGFp Trap).
[00174] The intravenous drug delivery formulation of the present invention can be contained in a bag, a pen, or a syringe. In certain embodiments, the bag can be connected to a channel comprising a tube and / or a needle. In certain embodiments, the formulation can be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation can be freeze-dried (lyophilized) and contained in about 12 to 60 small bottles. In certain embodiments, the formulation can be freeze-dried and about 45 mg of the freeze-dried formulation can be contained in a small bottle. In certain embodiments, about 40 mg to about 100 mg of freeze-dried formulation can be contained in a small bottle. In certain embodiments, freeze-dried formulations of 12, 27, or 45 small vials are combined until the therapeutic dose of the protein is obtained in the intravenous drug formulation. In certain embodiments, the formulation can be a liquid formulation of a protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1, and stored as about 250 mg / vial to about 2000 mg / vial (for example, about 250 mg /
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63/127 small bottle at about 2000 mg / small bottle, about 250 mg / small bottle at about 1900 mg / small bottle, about 250 mg / small bottle at about 1800 mg / small bottle, about 250 mg / vial about 1700 mg / vial, about 250 mg / vial about 1600 mg / vial, about 250 mg / vial about 1500 mg / vial, about 250 mg / vial small to about 1400 mg / small bottle, about 250 mg / small bottle to about 1300 mg / small bottle, about 250 mg / small bottle to about 1200 mg / small bottle, about 250 mg / small bottle to about 1100 mg / small bottle, about 250 mg / small bottle about 1000 mg / small bottle, about 250 mg / small bottle about 900 mg / small bottle, about 250 mg / small bottle about 800 mg / small bottle, about 250 mg / small bottle to about 700 mg / small bottle, about 250 mg / small bottle to about 600 mg / small bottle, about 250 mg / small bottle to about 500 mg / small bottle, about 250 mg / small bottle to about 400 mg / small bottle, about 250 mg / small bottle to about 300 mg / small bottle, about 300 mg / small bottle at about 2000 mg / small bottle, about 400 mg / small bottle at about 2000 mg / small bottle, about 500 mg / small bottle at about 2000 mg / small bottle, about 600 mg / small bottle about 2000 mg / small bottle, about 700 mg / small bottle about 2000 mg / small bottle, about 800 mg / small bottle about 2000 mg / bottle small, about 900 mg / small bottle to about 2000 mg / small bottle, about 1000 mg / small bottle to about 2000 mg / small bottle, about 1100 mg / small bottle to about 2000 mg / small bottle, about 1200 mg / small bottle to about 2000 mg / small bottle, about 1300 mg / small bottle about
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64/127 of 2000 mg / small bottle, about 1400 mg / small bottle to about 2000 mg / small bottle, about 1500 mg / small bottle to about 2000 mg / small bottle, about 1600 mg / small bottle to about 2000 mg / small bottle, about 1700 mg / small bottle about 2000 mg / small bottle, about 1800 mg / small bottle about 2000 mg / small bottle, or about 1900 mg / small bottle about 2000 mg / small bottle). In certain embodiments, the formulation can be a liquid formulation and stored at about 600 mg / vial. In certain embodiments, the formulation can be a liquid formulation and stored at about 1200 mg / vial. In certain embodiments, the formulation can be a liquid formulation and stored at about 1800 mg / vial. In certain embodiments, the formulation can be a liquid formulation and stored as about 250 mg / vial.
[00175] This invention provides an aqueous liquid pharmaceutical formulation including a therapeutically effective amount of the protein of the present invention (e.g., anti-PD-L1 / TGFp Trap) in a buffered solution forming a formulation.
[00176] These compositions can be sterilized by conventional sterilization techniques, or they can be filtered sterile. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the preparations will typically be between 3 and 11, most preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the aforementioned agent or agents. Solid form composition can also
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65/127 be packaged in a container for a flexible amount. [00177] In certain embodiments, the present invention provides an extended life formulation including a protein of the present invention (for example, anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the sequence of amino acids of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)), in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, di - sodium hydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.
[00178] In certain embodiments, an aqueous formulation is prepared including a protein of the present invention (for example, anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)) in a pH buffered solution. The buffer of this invention can have a pH ranging from about 4 to about 8, for example, from about 4 to about 8, from about
4.5 to about 8, from about 5 to about 8, from about 5.5 to about 8, from about 6 to about 8, from about 6, 5 to about 8, from about 7 to about 8, from about 7.5 to about 8, from about 4 to about 7.5, from about 4, 5 to about 7.5, from about 5 to about 7.5, from about 5.5 to about 7.5, from about 6 to about 7.5, to from about 6.5 to about 7.5, from about 4 to about 7, from about 4.5 to about 7, from about 5 to about 7, the from about 5.5 to about 7, from about 6 to about 7, from about 4 to about 6.5, from about 4.5 to about 6.5 , from about 5 to about 6.5, from
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66/127 about 5.5 to about 6.5, from about 4 to about 6.0, from about 4.5 to about 6.0, from about 5 to about 6, or from about 4.8 to about 5.5, or it can have a pH of about 5.0 to about 5.2. Intermediate ranges at the aforementioned pH's are also intended to be part of this invention. For example, ranges of values using a combination of any of the above mentioned values as upper and / or lower limits are intended to be included. Examples of buffers that will control the pH within this range include acetate (for example, sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.
[00179] In certain embodiments, the formulation includes a buffer system which contains citrate and phosphate to maintain the pH in a range of about 4 to about 8. In certain embodiments, the pH range can be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or in a pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3 mg / ml of citric acid (for example, 1.305 mg / ml), about 0.3 mg / ml of sodium citrate (for example, 0.305 mg / ml) , about 1.5 mg / ml of disodium phosphate dihydrate (for example, 1.53 mg / ml), about 0.9 mg / ml of sodium dihydrogen phosphate dihydrate (for example, 0, 86), and about 6.2 mg / ml sodium chloride (for example, 6.165 mg / ml). In certain embodiments, the buffer system includes about 1 to 1.5 mg / ml of citric acid, about 0.25 to about 0.5 mg / ml of sodium citrate, about 1.25 to about 1 , 75 mg / ml of disodium phosphate dihydrate, about 0.7 to about 1.1 mg / ml of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / ml of sodium chloride
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67/127 sodium. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.
[00180] A polyol, which acts as a toner and can stabilize the antibody, can also be included in the formulation. The polyol is added to the formulation in an amount which can vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of polyol added can also change with respect to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (for example, mannitol) can be added, compared to a disaccharide (such as trehalose). In certain embodiments, the polyol which can be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the concentration of mannitol can be from about 5 to about 20 mg / ml. In certain embodiments, the concentration of mannitol can be from about 7.5 to about 15 mg / ml. In certain embodiments, the concentration of mannitol can be from about 10 to about 14 mg / ml. In certain embodiments, the concentration of mannitol can be about 12 mg / ml. In certain embodiments, the sorbitol polyol can be included in the formulation.
[00181] A detergent or surfactant can also be added to the formulation. Examples of detergents include non-ionic detergents such as polysorbates (for example, polysorbates 20, 80 etc.) or poloxamers (for example, poloxamer 188). The amount of detergent added is such that it reduces the aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant which is a polysorbate. In certain embodiments, the formulation may contain polysorbate 80 or Tween 80 detergent. Tween 80 is a term used to describe monooleate of
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68/127 polyoxyethylene sorbitan (20) (see Fiedler, Lexikon der Hilfsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain between about 0.1 mg / ml and about 10 mg / ml of polysorbate 80, or between about 0.5 mg / ml and about 5 mg / ml. In certain embodiments, about 0.1% of polysorbate 80 can be added to the formulation.
Lyophilized Formulation [00182] The lyophilized formulation of the present invention includes the anti-PD-L1 / TGFp Trap molecule and a lyoprotectant. The lyoprotectant can be sugar, for example, disaccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation can also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative.
[00183] The amount of sucrose or maltose useful for stabilizing the lyophilized drug may be a weight ratio of at least 1: 2 of protein to or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1: 2 to 1: 5.
[00184] In certain embodiments, the pH of the formulation, before lyophilization, can be determined by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.
[00185] Before lyophilization, the pH of the solution containing the protein of the present invention can be adjusted between about 6 to about 8. In certain embodiments, the pH range for the lyophilized drug can be from about 7 to about 8.
[00186] In certain embodiments, some components of salts or buffers can be added in an amount of about
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69/127 from 10 mM to about 200 mM. Salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer can be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as a counterion.
[00187] In certain modalities, a “volume agent” can be added. A "bulking agent" is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (for example, it facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present invention can contain similar bulking agents.
[00188] A condom can optionally be added to the formulations here, in this patent application, to reduce bacterial action. The addition of a condom can, for example, facilitate the production of a multi-use (multi-dose) formulation.
[00189] In certain embodiments, the lyophilized medicine can be constituted with an aqueous vehicle. The aqueous vehicle of interest here, in this patent application, is an aqueous vehicle which is pharmaceutically acceptable (for example, safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization . Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (for example, phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.
[00190] In certain modalities, the lyophilized medicine
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70/127 of the present invention is reconstituted with either sterile water for injection, (SWFI, United States Pharmacopoeia) or 0.9% Sodium Chloride Injection, (0.9% Sodium Chloride Injection, United States Pharmacopeia). During reconstitution, the lyophilized powder dissolves in a solution.
[00191] In certain embodiments, the lyophilized protein product of the present invention consists of up to about 4.5 ml_ of water for injection and diluted with 0.9% saline (sodium chloride solution).
Liquid Formulation [00192] In embodiments, the protein product of the present invention is formulated as a liquid formulation. The liquid formulation can be presented in a concentration of 10 mg / mL in any small 50R USP / Ph Eur type I vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper can be made of elastomer according to the United States Pharmacopoeia (USP) and the European Pharmacopoeia (Ph Eur). In certain embodiments, the small vials can be filled with approximately 61.2 ml of the protein product solution in order to allow an extractable volume of 60 ml. In certain embodiments, the liquid formulation can be diluted with 0.9% saline. In certain embodiments, the small vials may contain approximately 61.2 ml of the protein product solution (for example, anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)) from about 20 mg / ml to about 50 mg / ml (for example, about 20 mg / ml, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL or about 50 mg / mL) to allow an extractable volume of 60 mL for
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71/127 release about 1200 mg to about 3000 mg (for example, about 1200 mg to about 3000 mg, about 1200 mg to about 2900 mg, about 1200 mg to about 2800 mg, about 1200 mg to about 2700 mg, about 1200 mg to about 2600 mg, about 1200 mg to about 2500 mg, about 1200 mg to about 2400 mg, about 1200 mg to about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 3000 mg, about 1400 mg to about 3000 mg, about 1500 mg to about 3000 mg, about 1600 mg to about 3000 mg, about 1700 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 1900 mg to about 3000 mg, about 2 000 mg to about 3000 mg, about 2100 mg to about 3000 mg, about 2200 mg to about 3000 mg, about 2300 mg to about 3000 mg, about 2400 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 2600 mg to about 3000 mg, about 2700 mg to about 3000 mg, about 2800 mg to about 3000 mg, about 2900 mg to about 3000 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg) of the protein product (for example , anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1))
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72/127 to an individual.
[00193] In certain embodiments, the small vials may contain approximately 61.2 ml of the protein product solution (protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1) from about 20 mg / mL to about 50 mg / mL (e.g., about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml or about 50 mg / ml) to allow an extractable volume of 60 ml to release about 1200 mg to about 3000 mg (for example example, about 1200 mg to about 3000 mg, about 1200 mg to about 2900 mg, about 1200 mg to about 2800 mg, about 1200 mg to about 2700 mg, about 1200 mg to about 2600 mg, about 1200 mg to about 2500 mg, about 1200 mg to about 2400 mg, about 1200 mg to about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg a about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 3000 mg, about 1400 mg to about 3000 mg, about 1500 mg to about 3000 mg, about 1600 mg to about 3000 mg, about 1700 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 1900 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2100 mg to about 3000 mg, about 2200 mg to about 3000 mg, about 2300 mg to about 3000 mg, about 2400 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 2600 mg to about 3000 mg, about 2700 mg to about 3000 mg, about 2800 mg to about 3000 mg, about 2900 mg to
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73/127 about 3000 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1800 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg) of the protein product to an individual.
[00194] In certain embodiments, the liquid formulation of the invention can be prepared as a 10 mg / ml concentration solution in combination with a sugar at stabilizing levels. In certain embodiments, the liquid formulation can be prepared in an aqueous vehicle. In certain embodiments, a stabilizer may be added in an amount not greater than the amount which may result in an undesirable or unsuitable viscosity for intravenous administration. In certain embodiments, sugar can be disaccharides, for example, sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.
[00195] In certain embodiments, the pH of the liquid formulation can be adjusted by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.
[00196] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, crop / cell clarification, purification, storage of medicated substances / drugs and during sample analysis. Deamidation is the loss of NH3 from a protein forming a succinimide intermediate that can undergo hydrolysis. The interme
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74/127 succinimide daily results in a 17 u mass decrease in the parental peptide. Subsequent hydrolysis results in an increase in mass of 18 u. Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. Thus, deamidation is typically detectable as a 1 u increase in mass. Deamidation of an asparagine results in either aspartic or isoaspartic acid. The parameters that affect the deamidation rate include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect deamidation rates. Gly and Ser following an Asn in protein sequences results in an increased susceptibility to deamidation.
[00197] In certain embodiments, the liquid formulation of the present invention can be preserved under conditions of pH and humidity to prevent deamination of the protein product.
[00198] The aqueous vehicle of interest here, in this patent application, is an aqueous vehicle which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH-buffered solution (for example, phosphate-buffered saline), sterile saline, Ringer's solution or dextrose solution.
[00199] A condom can optionally be added to the formulations here, in this patent application, to reduce the bacterial action. The addition of a condom can, for example, facilitate the production of a multi-use (multi-dose) formulation.
[00200] Intravenous (IV) formulations may be the preferred route of administration in particular cases, such as when a patient
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75/127 is in the hospital after transplantation receiving all drugs via IV. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution before administration. In certain modalities, the diluted drug for injection is isotonic and suitable for administration by intravenous infusion.
[00201] In certain modalities, some components of salts or buffers can be added in an amount of 10 mM to 200 mM. Salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer can be glycinate, carbonate, citrate buffers, in which case sodium, potassium or ammonium ions can serve as a counterion.
[00202] A condom can optionally be added to the formulations here, in this patent application, to reduce bacterial action. The addition of a condom can, for example, facilitate the production of a multi-use (multi-dose) formulation.
[00203] The aqueous vehicle of interest here, in this patent application, is an aqueous vehicle which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH-buffered solution (for example, phosphate-buffered saline), sterile saline, Ringer's solution or dextrose solution.
[00204] A condom can optionally be added to the formulations here, in this patent application, to reduce bacterial action. The addition of a condom can, for example, facilitate
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76/127 production of a multi-use (multiple-dose) formulation.
Method of Treating Cancer or Tumor Growth Inhibition [00205] In one aspect the present invention provides a method of treating cancer or inhibiting tumor growth in an individual who needs it, the method including administering to the individual a dose of at least 500 mg of a protein including a first polypeptide and a second polypeptide. The first polypeptide includes: (a) at least one variable region of an antibody heavy chain that binds to the Programmed Cell Death Link human protein 1 (PD-L1); and (b) the Human Transforming Growth Factor β Receptor II (ΤΟΕβΗΙΙ), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΟΕβ). The second polypeptide includes at least one variable region of an antibody light chain that binds PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds to PD-L1.
[00206] In certain embodiments, the method of treating cancer or inhibiting tumor growth of the present invention involves administering to a subject a protein including two peptides in which the first polypeptide includes the amino acid sequence of SEQ ID NO: 3 , and the second polypeptide includes the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the protein is an anti-PD-L1 / ΤΟΕβ Trap molecule.
[00207] In certain embodiments, the method of treating cancer or inhibiting tumor growth of the present invention involves administering to an individual a protein (e.g., an anti-PD-L1 / ΤΟΕβ trap molecule (e.g. , including a first polypeptide that includes the amino acid sequence
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77/127 of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)) at a dose of about 1200 mg to about 3000 mg (for example, about 1200 mg at about 3000 mg, about 1200 mg to about 2900 mg, about 1200 mg to about 2800 mg, about 1200 mg to about 2700 mg, about 1200 mg to about 2600 mg, about 1200 mg to about 2500 mg, about 1200 mg to about 2400 mg, about 1200 mg to about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 3000 mg, about 1400 mg to about 3000 mg, about 1500 mg to about 3000 mg, about 1600 mg to about 3000 mg, about 170 0 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 1900 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2100 mg to about 3000 mg, about 2200 mg to about 3000 mg, about 2300 mg to about 3000 mg, about 2400 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 2600 mg to about 3000 mg, about 2700 mg to about 3000 mg, about 2800 mg to about 3000 mg, about 2900 mg to about 3000 mg, about 1200, about 1300, about 1400, about 1500, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg). In certain embodiments, about 1200 mg of anti-PD-L1 I TGFp Trap molecule is administered to an individual once every two weeks. In certain modalities, about
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78/127 of 1800 mg of anti-PD-L1 molecule I TGFp trap is administered to an individual once every three weeks. In certain embodiments, about 1200 mg of a protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3 and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1 is administered to an individual a every two weeks. In certain embodiments, about 1800 mg of a protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3 and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1 is administered to an individual a every three weeks.
[00208] In certain embodiments, the dose can be about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150 mg, about 1175 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1 825 mg, about 1850 mg, 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, 2100 mg, about 2200 mg, about 2300 mg, or about 2400 mg.
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79/127 [00209] In certain modalities, the dose can be administered once every two weeks. In certain embodiments, the protein can be administered by intravenous administration, for example, with a filled pouch, filled pen, or filled syringe. In certain embodiments, the protein is administered intravenously from a 250 ml saline bag, and the intravenous infusion can be for about an hour (for example, 50 to 80 minutes). In certain embodiments, the pouch is connected to a channel comprising a tube and / or a needle.
[00210] In certain modalities, the method treats cancer or inhibits tumor growth, for example, among the following: non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer, ovarian cancer, breast cancer, cancer prostate cancer, glioblastoma, gastric cancer, biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), head or neck adenoma, squamous head or neck carcinoma, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, liver cancer, nasopharynx cancer, testicular cancer, small cell lung cancer, basal cell skin cancer, skin cancer squamous cell, protuberant dermatofibrosarcoma, Merkel cell carcinoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes. In certain embodiments, the method treats cancer of pretreated patients, for example, pretreated non-small cell lung cancer, pretreated melanoma, pretreated pancreatic cancer, pretreated colorectal cancer, pretreated ovarian cancer , pretreated breast cancer, pretreated glioblastoma, recurrent non-resectable or refractory Stage IV gastric cancer, pretreated biliary tract cancer, pretreated esophageal cancer (carcinoma
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80/127 ma squamous cell or adenocarcinoma), pre-treated head or neck adenoma, pre-treated squamous head or neck carcinoma, pre-treated prostate cancer, pre-treated kidney cancer, pre-treated cervical cancer, myeloma pre-treated, pretreated lymphoma, pretreated leukemia, pretreated thyroid cancer, pretreated endometrial cancer, pretreated uterine cancer, pretreated bladder cancer, pretreated neuroendocrine cancer, pretreated liver cancer, pretreated nasopharyngeal cancer -treated, pretreated testicular cancer, pretreated small cell lung cancer, pretreated basal cell skin cancer, pretreated squamous cell skin cancer, pretreated bulging dermatofibrosarcoma, pretreated Merkel cell carcinoma treated, pretreated glioma, pretreated sarcoma, pretreated mesothelioma, and pretreated myelodysplastic syndromes.
[00211] In certain embodiments, the tumor is an advanced solid tumor. In certain modalities, the tumor is refractory to previous treatment. In certain modalities, patients who have had advanced non-small cell lung cancer and who have previously been treated with an anti-PD-1 or anti-PD-L1 agent (“PDx therapy”) and then have documented disease progression are treated for intravenous administration of about 1200 mg anti-PD-L1 / TGFp Trap. The patient's best overall response (BOR) to previous PDx therapy has been documented. In certain modalities, patients with progressive disease (PD) after previous PDx therapy, therefore considered as primary refractories, (ie, among these patients, progression of the disease from the beginning of PDx therapy was observed without any benefit of treatment being observed). treated by intravenous administration of about 1200 mg to about 2400 mg (for example, about 1200 mg to about 2400 mg, about 1200 mg to about 2300 mg, about
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81/127
1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 2400 mg, about 1400 mg to about 2400 mg, about 1500 mg to about 2400 mg, about 1600 mg to about 2400 mg, about 1700 mg to about 2400 mg, about 1800 mg to about 2400 mg, about 1900 mg to about 2400 mg, about 2000 mg to about 2400 mg, about 2100 mg to about 2400 mg, about 2200 mg to about 2400 mg, or about 2300 mg to about 2400 mg) of antiPD-L1 / TGFp Trap. In certain modalities, patients characterized as acquired resistant, that is, the disease of the patients initially responded to previous PDx therapy, but the patients ultimately reverted to the stage of disease progression, are treated by intravenous administration of about 1200 mg to about 2400 mg of anti-PD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. The best general response (BOR) of these patients to previous PDx therapy was stable disease (SD), partial response (PR), or complete response (CR), which then experienced further disease progression. The rationale for using anti-PD-L1 / TGFp Trap in these sub-cohorts of PDx-fail (NT: failure of PDx therapy) of non-small cell lung cancer is also to neutralize TGF-β, a molecule that it is known to inhibit tumor immune activation to stimulate clinical response in patients who have been unsuccessful in responding to isolated anterior PDx therapy.
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82/127 [00212] In certain modalities, patients who have advanced non-small cell lung cancer with refractory, relapsing, or progressive disease during or after a single platinum-based chemotherapy line are treated by intravenous administration of about 1200 mg to about 2400 mg (for example, about 1200 mg to about 2400 mg, about 1200 mg to about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 2400 mg, about 1400 mg to about 2400 mg, about 1500 mg to about 2400 mg, about 1600 mg to about 2400 mg, about 1700 mg to about 2400 mg, about 1800 mg about 2400 mg, about 1900 mg to about 2400 mg, about 2000 mg to about 2400 mg, about 2100 mg to about 2400 mg, about 2200 mg to about 2400 mg, or about 2300 mg to about 2400 mg) of anti-PD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain modalities, patients who have advanced non-small cell lung cancer with refractory, relapsing or progressive disease during or after a single platinum-based chemotherapy line are treated by intravenous administration of anti-PD -L1 / TGFp trap at a dose of about 1200 mg once every 2 weeks. In certain modalities, patients who have advanced non-small cell lung cancer with refractory, relapsing, or progressive disease during or after a single line of chemotherapy
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83/127 platinum-based are treated by intravenous administration of anti-PD-L1 / TGFp Trap at a dose of about 500 mg to about 1200 mg (for example, about 500 mg to about 1000 mg , about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg ) which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain embodiments, patients who have had non-cell lung cancer small advanced with refractory, relapsing or progressive disease during or after a single platinum-based chemotherapy line are treated by intravenous administration of anti-PD-L1 / TGFp Trap at a dose of about 500 mg once a every 2 weeks.
[00213] In certain modalities, patients with heavily pretreated refractory or refractory Stage IV gastric cancer are treated by intravenous administration of about 1200 mg to about 2400 mg (for example, about 1200 mg about about 2400 mg, about 1200 mg to about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 2400 mg, about 1400 mg to about 2400 mg, about 1500 mg to about 2400 mg, about 1600 mg to about about 2400 mg, about 1700 mg to about 2400 mg, about 1800 mg to about 2400 mg, about 1900 mg to about 2400 mg, about 2000 mg to about 2400 mg, ce from 2100 mg to about 2400 mg, about 2200 mg to
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84/127 about 2400 mg, or about 2300 mg to about 2400 mg) of antiPD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain modalities, patients with recurrent or highly pretreated refractory Stage IV gastric cancer are treated by intravenous administration of anti-PD-L1 / Trap TGFp at a dose of about 1200 mg once every 2 weeks for 2 to 30 weeks. In certain modalities, treated patients received at least 3 previous anti-cancer therapies. In certain modalities, treated patients received at least 4 previous anti-cancer therapies.
[00214] In certain modalities, patients with pretreated colorectal cancer (CRC) are treated by intravenous administration of about 1200 mg to about 2400 mg (for example, about 1200 mg to about 2400 mg, about 1200 mg to about 2300 mg, about 1200 mg to about 2200 mg, about 1200 mg to about 2100 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 2400 mg, about 1400 mg to about 2400 mg, about 1500 mg to about 2400 mg, about 1600 mg to about 2400 mg, about 1700 mg to about 2400 mg, about 1800 mg to about 2400 mg, about 1900 mg to about 2400 mg, about 2000 mg to about 2400 mg, about 2100 mg to about 2400 mg, about 2200 mg at about 2400 mg, or about 2300 mg to about 2400 mg) of anti-PD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and
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85/127 a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain embodiments, patients with pretreated colorectal cancer (CRC) are treated with anti-PD-L1 / TGFp trap at a dose of about 1200 mg once every 2 weeks for 2 to 38 weeks. In certain modalities, treated patients received at least 3 previous anti-cancer therapies. Delivery Device [00215] In one aspect, the present invention provides a drug delivery device including a formulation comprising about 500 mg to about 3000 mg of a protein including a first polypeptide and a second polypeptide, the first polypeptide includes: (a) at least one variable region of an antibody heavy chain that binds to the Programmed Cell Death Link human protein 1 (PD-L1); and (b) the human Transforming Growth Factor β Receptor II (TGFpRIl), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΰΕβ), the second polypeptide includes at least one variable region of a chain light of an antibody that binds to PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen-binding site that binds to PD-L1.
[00216] In certain modalities, the device can be a bag, a pen, or a syringe. In certain embodiments, the bag can be connected to a channel comprising a tube and / or a needle.
[00217] In certain embodiments of the present invention, the drug delivery device may include about 500 mg to about 3000 mg (e.g., about 500 mg to about 3000 mg, about 500 mg to about 2900 mg, about 500 mg to about 2800 mg, about 500 mg to about 2700 mg, about 500 mg to
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86/127 about 2600 mg, about 500 mg to about 2500 mg, about 500 mg to about 2400 mg, about 500 mg to about 2300 mg, about 500 mg to about 2200 mg, about 500 mg to about 2100 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 3000 mg, about 700 mg to about 3000 mg, about 800 mg to about 3000 mg, about 900 mg to about 3000 mg, about 1000 mg to about 3000 mg, about 1100 mg to about 3000 mg, about 1200 mg to about 3000 mg, about that of 1300 mg to about 3000 mg, about 1400 mg to about 3000 mg, about 1500 mg to about 3000 mg, about 1600 mg to about 3000 mg, about 1700 mg to about 3000 mg, about 1800 mg to about 3000 mg, about 1900 mg to about 3000 mg, about 2000 mg to about 3000 mg, about 2100 mg to about 3000 mg, about 2200 mg to about 3000 mg, about 2300 mg to about 3000 mg, about 2400 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 2600 mg to about 3000 mg, about 2700 mg to about 3000 mg, about 2800 mg to about 3000 mg, or about 2900 mg to about 3000 mg) of a protein of the present invention (for example, anti-PD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1). In certain embodiments, the drug delivery device may include about
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87/127 of 500 to about 1200 mg dose of a protein of the present invention (for example, anti-PD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3 , and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1). In certain embodiments, the drug delivery device may include about 500 mg of dose of the protein of the present invention (for example, anti-PD-L1 / TGFp Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1). In certain embodiments, the drug delivery device includes about 1200 mg dose of a protein of the present invention (for example, anti-PD-L1 / TGFβ Trap, which includes a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1). In certain embodiments, the drug delivery device includes about 1200 mg or about 1800 mg dose of the protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the sequence amino acids of SEQ ID NO: 1.
[00218] In certain embodiments, the drug delivery device includes about 1200 mg dose of the protein of the present invention (e.g., anti-PD-L1 / TGFβ Trap (e.g., including a first polypeptide that includes the sequence amino acids of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, the drug delivery device may include about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about
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88/127 ca 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150 mg, about 1175 mg, about 1200 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg, about 1500 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, or about 2400 mg of the protein of the present invention (for example , anti-PD-L1 / TGFP Trap).
Kíí [00219] In one aspect, the present invention provides a kit including one or more containers collectively including a formulation from about 500 mg to about 2400 mg (e.g., about 500 mg to about 2400 mg, about 500 mg to about 2300 mg, about 500 mg to about 2200 mg, about 500 mg to about 2100 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500
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89/127 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 2400 mg, about 700 mg to about 2400 mg, about 800 mg to about 2400 mg, about 900 mg to about 2400 mg, about 1000 mg to about 2400 mg, about 1100 mg to about 2400 mg, about 1200 mg to about 2400 mg, about 1300 mg to about 2400 mg, about 1400 mg to about 2400 mg, about 1500 mg to about 2400 mg, about 1600 mg to about 2400 mg, about 1700 mg to about 2400 mg, about 1800 mg to about 2400 mg, about 1900 mg to about 2400 mg, about 2000 mg to about 2400 mg, about 2100 mg to about 2400 mg, about 2200 mg to about 2400 mg, or about 2300 mg to about 2400 mg) of a protein including a first polypeptide and a second polypeptide, the first polypeptide includes: (a) at least one variable region of an antibody heavy chain that binds to the human protein Linker of death Programmed Cell 1 (PD-L1); and (b) the human Transforming Growth Factor β Receptor II (TGFpRIl), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΰΡβ), the second polypeptide includes at least one variable region of a chain light of an antibody that binds to PD-L1, and the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen-binding site that binds to PD-L1.
[00220] In certain embodiments of the present invention, containers collectively may include a dose of about 500 mg to about 2400 mg (e.g., about 500 mg to about 2400 mg, about 500 mg to about 2300 mg, about 500 mg to about 2200 mg, about 500 mg to about 2100 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about
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90/127 from 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 2400 mg, about 700 mg to about 2400 mg, about 800 mg to about 2400 mg, about 900 mg to about 2400 mg, about 1000 mg to about 2400 mg, about 1100 mg to about 2400 mg, about 1200 mg to about 2400 mg, about 1300 mg to about 2400 mg, about 1400 mg to about 2400 mg, about 1500 mg to about 2400 mg, about 1600 mg to about 2400 mg, about 1700 mg to about 2400 mg, about 1800 mg to about 2400 mg, about 1900 mg to about 2400 mg, about 2000 mg to about 2400 mg, about 2100 mg to about 2400 mg, about 2200 mg to about 2400 mg, or about 2300 mg to about 2400 mg) of the protein of the present invention (for example, anti-PDL1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO : 3, and a second polypeptide that includes the amino acid sequence of SEQ ID NO: 1)). In certain embodiments, the containers collectively may include 500 to 1800 mg of dose of the protein of the present invention (for example, anti-PD-L1 / TGFP trap). In certain embodiments, the containers collectively may include a 500 mg dose of the protein of the present invention (for example, antiPD-L1 / TGFP Trap). In certain embodiments, the containers collectively may include a 1200 mg dose of the protein of the present invention (for example, anti-PD-L1 / TGFP trap). In certain embodiments, the containers collectively may include a 1800 mg dose of the protein of the present invention (for example,
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91/127 (anti-PD-L1 / TGFP trap). In certain embodiments, the formulation is prepared and packaged as a liquid formulation and stored as about 250 mg / vial to about 1000 mg / vial (for example, about 250 mg / vial to about 1000 mg / vial small, about 250 mg / small bottle to about 900 mg / small bottle, about 250 mg / small bottle to about 800 mg / small bottle, about 250 mg / small bottle to about 700 mg / small bottle, about 250 mg / small bottle to about 600 mg / small bottle, about 250 mg / small bottle to about 500 mg / small bottle, about 250 mg / small bottle to about 400 mg / small bottle, about 250 mg / small bottle to about 300 mg / small bottle, about 300 mg / small bottle to about 1000 mg / small bottle, about 400 mg / small bottle to about 1000 mg / small bottle, about 500 mg / small bottle at about 1000 mg / small bottle, about 600 mg / small bottle about 1000 mg / small bottle, about 700 mg / small bottle about 1000 mg / small bottle, about 800 mg / small bottle about 1000 mg / small bottle, or about 900 mg / small bottle a about 1000 mg / small bottle). For example, in certain embodiments, the formulation is a liquid formulation and stored as about 600 mg / vial, or stored as about 250 mg / vial.
[00221] In certain embodiments, the containers collectively may include a dose of about 1200 mg or about 1800 mg of the protein product with a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the formulation is prepared and packaged as a liquid formulation and stored as about 250 mg / vial
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92/127 small to about 1200 mg / small bottle (for example, about 250 mg / small bottle to about 1200 mg / small bottle, about 250 mg / small bottle to about 1100 mg / small bottle, about 250 mg / small bottle to about 1000 mg / small bottle, about 250 mg / small bottle to about 900 mg / small bottle, about 250 mg / small bottle to about 800 mg / small bottle, about 250 mg / small bottle about 700 mg / small bottle, about 250 mg / small bottle about 600 mg / small bottle, about 250 mg / small bottle about 500 mg / small bottle, about 250 mg / bottle small to about 400 mg / small bottle, about 250 mg / small bottle to about 300 mg / small bottle, about 300 mg / small bottle to about 1200 mg / small bottle, about 400 mg / small bottle to about 1200 mg / small bottle, about 500 mg / small bottle about 1200 mg / small bottle, about 600 mg / small bottle a about 1200 mg / small bottle, about 700 mg / small bottle about 1200 mg / small bottle, about 800 mg / small bottle about 1200 mg / small bottle, about 900 mg / small bottle about 1200 mg / vial, about 1000 mg / vial at about 1200 mg / vial, or about 1100 mg / vial at about 1200 mg / vial) of the protein product with a first polypeptide that includes the sequence of amino acids of SEQ ID NO: 3, and the second polypeptide that includes the amino acid sequence of SEQ ID NO: 1. For example, in certain embodiments, the formulation is a liquid formulation and stored as about 1200 mg / vial, or stored as about 600 mg / vial, or stored as about 250 mg / vial.
[00222] In certain embodiments, the containers collectively can include about 500 mg, about 525 mg, about 550 mg,
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93/127 about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about
1125 mg, about 1150 mg, about 1175 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about
1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about
1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about
1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about
1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about
2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, or about 2400 mg of the protein of the present invention (for example, anti-PD-L1 / TGFp Trap (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the amino acid sequence of
SEQ ID NO: 1)).
[00223] In certain embodiments, the formulation in the containers can be a lyophilized formulation or a liquid formulation.
[00224] In certain modalities, the formulation can be packaged in kits containing an adequate number of small bottles. Medication information can be included, which is in accordance with the approved submission documents. The kit can be shipped in refrigerated shipping containers (2 ^o C. to 8 ^o C.) that are monitored with control devices
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94/127 temperature trolley.
[00225] The formulation can be stored at 2 ^o C. to 8 ^o C. until use. In certain embodiments, the freeze-dried medicine can be reconstituted with 4.5 ml of water for injection and diluted with about 0.9% saline (sodium chloride injection) whereas the liquid formulation can be diluted with about 0.9% saline. The small bottles of the formulations can be sterile and non-pyrogenic, and may not contain bacteriostatic preservatives.
[00226] In certain embodiments, the release device is an injection pen. An injection pen is a device designed to allow a user to self-administer a pre-measured dose of a drug composition subcutaneously or intramuscularly. An injection pen may have a case, inside which is a cartridge. The cartridge may have one or more chambers containing medicamentous compositions or components thereof and is adapted to be attached to a needle assembly. The cartridge can contain either a pre-mixed liquid medicine or a solid medicine and a liquid that are mixed before injection. The case can load a drive assembly with a stored energy source, for example, a compressed spring. The activation of the drive assembly causes a sequence of movements, whereby the needle extends from the injection pen into the user so that the medicated compound is then forced through the needle and into the user. After the dose of medication is released into the injection site, the needle may remain in an extended position. If the injection pen is of a type designed to seal plural components of a drug composition in sealed and separate compartments, a structure may be included that forces the components to
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95/127 mix when the drive assembly is activated.
Protein Production [00227] Cytokine Trap antibody proteins are usually produced recombinantly, using mammalian cells containing a nucleic acid engineered to express the protein. Although an example of a suitable protein production method and cell line is described in Examples 1 and 2 of patent application No. US 20150225483 A1, a wide variety of vectors, cell lines and protein production methods suitable for produce antibody-based biopharmaceuticals and can be used in the synthesis of these antibody proteinsCytokine trap.
Therapeutic Indications [00228] The anti-PD-L1 / TGFp Trap proteins described in the application (for example, including a first polypeptide that includes the amino acid sequence of SEQ ID NO: 3, and a second polypeptide that includes the sequence of amino acids of SEQ ID NO: 1) can be used to treat cancer or reduce tumor growth in a patient. Sample cancers include non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer (for example, pretreated colorectal cancer (CRC)), ovarian cancer, glioblastoma, gastric cancer (for example, Stage IV gastric cancer non-resectable recurrent or pretreated refractory), biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), head or neck adenoma, and squamous carcinoma of the head or neck.
[00229] The cancer or tumor to be treated with an anti-PD-L1 / TGFp Trap can be selected based on the high expression or expression of PD-L1 and TGFp in the tumor, in the correlation of its expression levels with the prognosis or with the progression of the disease
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96/127 ça, and in the preclinical and clinical experience on the tumor's sensitivity to treatments targeting PD-L1 and TGFp. The cancers or tumors referred to include, but are not limited to: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bladder, head and neck, liver, non-small cell lung cancer, advanced non-small cell lung cancer, melanoma, Merkel cell carcinoma, and mesothelioma.
EXAMPLES [00230] The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for purposes of illustrating some aspects and modalities of the present invention, and are not intended to limit in any case the scope of the invention.
EXAMPLE 1: Packaging of the Intravenous Drug Formulation [00231] The anti-PD-L1 / TGFp Trap formulation is prepared as a lyophilized formulation or as a liquid formulation. To prepare the lyophilized formulation, 45 mg of freeze-dried anti-PD-L1 / TGFp Trap is sterilized and stored in a container. Then several similar containers are packaged in a kit to deliver a dose independent of specific body weight to an individual diagnosed with cancer or a tumor. Depending on the dose requirements, the kit contains 12 to 60 small vials. Alternatively, the formulation is prepared and packaged as a liquid formulation and stored as 250 mg / vial at 1000 mg / vial. For example, the formulation is a liquid formulation and stored as 600 mg / vial, or stored as 250 mg / vial.
[00232] The formulation is used to treat cancer or tumor, for example, non-small cell lung cancer, melanoma,
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97/127 pancreatic cancer, colorectal cancer (for example, pretreated colorectal cancer (CRC)), ovarian cancer, glioblastoma, gastric cancer (for example, recurrent or refractory pre-treated refractory Stage IV gastric cancer), cancer biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head or neck, and squamous carcinoma of the head or neck.
[00233] An individual diagnosed with a cancer or similar tumor is administered intravenously with a formulation containing 500 mg to 2000 mg of anti-PD-L1 / TGFp Trap. For example, the subject is administered intravenously with 500 mg of anti-PD-L1 / TGFp Trap or 1200 mg of anti-PD-L1 / TGFp Trap. Intravenous administration is from a saline bag, and administration is once every two weeks. The amount of anti-PD-L1 / TGFβ Trap administered to an individual is independent of the individual's body weight.
EXAMPLE 2: Independent Body Weight Dosage Regimen [00234] In an example embodiment, the 500 mg or 1200 mg body weight independent dose was administered to individuals with non-small cell lung cancer (NSCLC) once every two weeks. Administration was performed intravenously for about an hour (-10 minutes / +20 minutes, that is, 50 minutes to 80 minutes). In order to mitigate potential infusion-related reactions, premedication with an antihistamine and paracetamol (acetaminophen) (for example, 25 to 50 mg of diphenhydramine and 500 to 650 mg of paracetamol [acetaminophen] IV or equivalent oral) approximately 30 to 60 minutes before each dose for the first two infusions. If Grade> 2 infusion related reactions were seen during the first two infusions, premedication was not interrupted. Steroids were not allowed
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98/127 as premedication.
[00235] For individuals who achieved a partial response (PR) or a complete response (CR) with anti-PD-L1 therapy / TGFp trap and then developed disease progression after stopping therapy, 1 re-initiation was allowed course of treatment in the same dose and schedule and duration of treatment up to 12 months. In this example, subjects were> 18 years old and had solid metastatic or locally advanced histologically or cytologically proven tumors, for which there is no effective standard therapy or for which standard therapy was unsuccessful. Preferred individuals had adequate hematological function defined by white blood cell count (WBC)> 3 x 109 / L with absolute neutrophil count (ANC)> 1.5 x 109 / L, lymphocyte count> 0.5 χ 109 / L , platelet count> 120 x 109 / L, and Hgb> 9 g / dL (in the absence of blood transfusion); adequate liver function defined by a total bilirubin level <1.5 χ ULN, an AST level <2.5 χ ULN, and an ALT level <2.5 χ ULN; and adequate renal function defined by an estimated creatinine clearance> 50 mL / min according to the Cockcroft-Gault formula or by measuring the urine collection creatinine clearance for 24 hours. For individuals with liver involvement in their tumor, AST <5.0 x ULN, ALT <5.0 χ ULN, and bilirubin <3.0 were acceptable.
[00236] In other modalities, some individuals had:
Non-small cell lung cancer Stage lllb or IV confirmed histologically or cytologically with relapsing, refractory or progressive disease during or after a single platinum-based chemotherapy line, and no previous treatment with a combination of immunotherapy.
Hepatocellular carcinoma histologically confirmed to be non-resectable or advanced disease not susceptible to healing resection
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99/127 with cancer that progressed after 1 line of therapy with previous sorafenib (at least 14 days of sorafenib at least 400 mg per day) or that was previously considered to be intolerant to sorafenib.
Stage IV, non-small cell lung cancer, or recurrent histologically confirmed without previous systemic anticancer therapy.
Stage IV, or recurrent, non-small cell lung cancer histologically confirmed in patients who received and were unsuccessful with platinum-based chemotherapy as monotherapy and failed with disease progression.
Non-small cell lung cancer in Stage IV, or histologically confirmed recurrent patients who received and were unsuccessful with platinum-based chemotherapy as monotherapy and failed with disease progression and received anti-PD-1 or anti-PD-L1 as monotherapy and failed with disease progression.
Stage III or metastatic non-resectable melanoma (Stage IV).
Pancreatic adenocarcinoma histologically confirmed to be non-resectable, advanced, and / or metastatic, without previous radiation therapy.
Histologically confirmed colon or rectal adenocarcinoma that progressed during or after a second line of systemic treatment including fluoropyrimidine, oxaliplatin, irinotecan and / or bevacizumab.
Triple-negative breast cancer that progressed during or after the first line of chemotherapy.
Epithelial ovarian, fallopian tube, or peritoneal cancer histologically confirmed with non-resectable metastatic disease
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100/127 that is resistant / refractory to platinum in patients previously treated with at least 2 chemotherapy regimens including platinum and taxane agents.
Recurrent or metastatic esophageal adenocarcinoma histologically confirmed, with non-resectable disease (Stage III or IV) in patients who received at least one chemotherapy regimen containing anterior platinum.
Grade IV malignant glioma histologically confirmed previously treated with radiotherapy and temozolomide.
Histologically confirmed or metastatic Head and Neck Squamous Cell Carcinoma (SCCHN) (oral cavity, pharynx, larynx), Stage III / IV, with tumor progression or recurrence within 6 months of the last platinum therapy dose in the adjuvant setting (that is, with radiation after surgery), primary (that is, with radiation), recurrent, or metastatic.
Squamous cell carcinoma, squamous adenocarcinoma, or cervical adenocarcinoma confirmed histologically recurrent or persistent after standard treatment with systemic therapy for advanced disease.
Gastric or gastroesophageal junctional adenocarcinoma Stage IV non-resectable or refractory recurrent histologically or cytologically confirmed for which there is no standard therapy or standard therapy has not been successful.
Esophageal squamous cell cancer confirmed histologically or cytologically for which there is no standard therapy or standard therapy has not been successful.
Biliary tract cancer confirmed histologically or cytologically that has not been successful or is intolerant to a systemic treatment line.
[00237] Selected individuals did not have active tuberculosis or
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101/127 an autoimmune disease that could deteriorate when receiving an immunostimulating agent.
EXAMPLE 3: Efficacy Assessments [00238] The assessment of the tumor response is performed by CT scan or MRI. Baseline sweeps are repeated on subsequent visits. In general, lesions detected at the baseline are followed using the same imaging methodology and preferably the same imaging equipment on subsequent tumor assessment visits. Skin metastases can be used as target lesions according to RECIST 1.1 using calibrator measurements, if they comply with RECIST 1.1 for target lesions.
EXAMPLE 4: Treatment of Patients with Advanced Non-Small Cell Lung Cancer Refractory or Resistant to Previous Treatment with Anti-PD-1 or Anti-PD-L1 Agent [00239] Objective: Stage IV non-small cell lung cancer, or histologically confirmed recurrence in patients who received and were unsuccessful with platinum-based chemotherapy as monotherapy and failed with disease progression and received anti-PD-1 or anti-PD-L1 as monotherapy and failed with disease progression, were selected for treatment with 1200 mg of anti-PD-L1 / TGFp Trap therapy. The rationale for the use of anti-PD-L1 / TGFp Trap in these non-small cell lung cancer sub-cohorts that were unsuccessful with PDx (PDx-ia / 7) was that the simultaneous neutralization of TGF-β , a molecule known to inhibit tumor immune activation, can stimulate clinical response in patients who have not been successful in responding to PDx therapy alone.
[00240] Summary: Patients who had advanced non-small cell lung cancer and were previously treated with anti-PD-1 or anti-PD-L1 agent (“PDx therapy”) and then progressed
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102/127 are of the documented disease, were selected for intravenous administration of 1200 mg of anti-PD-L1 / TGFp Trap. Patients' best overall response (BOR) to previous PDx therapy has been documented. A sub-cohort of patients with progressive disease (PD) after previous PDx therapy was considered to be “primary refractory”, that is, among these patients, disease progression was observed after initiation of PDx therapy without any observed benefit from treatment. Another sub-cohort of patients was characterized as “acquired resistant”, that is, the patients' disease initially responded to previous PDx therapy, but the patients ultimately reverted to the disease progression stage. The acquired resistant patients were characterized with stable disease BOR (SD), partial response (PR) or complete response (CR) to previous PDx therapy before the progression of the subsequent disease.
[00241] Study Design and Results: A total of 83 patients were treated with anti-PD-L1 / TGFp Trap at a dose of 1200 mg every 2 weeks. The median follow-up was 27.3 weeks. The primary endpoint is BOR according to RECIST v1.1, secondary endpoints being safety / tolerability. The baseline characteristics of the patients are listed in the table below. In summary, this was a heavily pre-treated patient population with 74.7% of patients receiving more than 3 previous treatment regimens, and included a range of ages and genders. The response to previous PDx therapy was approximately balanced (primary refractory, 43.4%; acquired resistance, 53%). In addition, all patients had a biopsy within 28 days of starting treatment and a majority (65.1%) was found to have> 1% PDL1 expression in tumor cells based on PD-L1 Dako 73- 10.
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Table 6: Patient characteristics
Characteristics, n (%) N = 83 SexMale 56 (67.5) Feminine 27 (32.5) Age<65 46 (55.4) > 65 36 (43.4) ECOG performance status0 27 (32.5) 1 54 (65.1) 2 1 (1,2) > 3 0 (0.0) Absent 1 (1,2) Tumor PD-L1 expression> 1% 54 (65.1) <1% 21 (25.3) Unknown 8 (9.6) Number of previous anti-cancer drug therapies0 0 (0.0) 1 0 (0.0) 2 21 (25.3) 3 26 (31.3) > 4 36 (43.4) Response to previous anti-PD-1 / PD-L1 therapyPrimary Refractory 36 (43.4) Acquired Resistance 44 (53)
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Characteristics, n (%) N = 83 Absent 3 (3.6)
[00242] From the cut-off date data at the time of the analysis, a total of 2 patients (2.4%) had an answer confirmed by the researcher according to RECIST v1.1. Disease control was reported in 20 patients (24.1%). By independent radiological review, 3 patients (3.6%) had a confirmed partial response. In addition, an additional 1 confirmed PR and 1 unconfirmed PR were reported by the researchers, thus increasing the unconfirmed ORR to 4.8%; 6 patients continued to be treated. Responses occurred in patients with primary refractory disease and in patients with acquired resistance to previous PDx therapy.
[00243] As shown in Figure 13, patients with previously progressive disease (both with primary refractory and acquired resistant disease) achieved important stabilization of the disease. It was observed that patients with disease response and disease stabilization have a range of treatments prior to the start of this study, and even had a range of treatments immediately prior to the start of the test, suggesting clinical activity of anti-PD-L1 / Trap TGFp in a heterogeneous population of patients with exposure to anterior PDx. Responses and disease control were noted both in patients expressing high and low PDL1 regardless of the status of PD-L1 at baseline and also in patients with high or low circulating plasma levels of TGF-βΙ.
[00244] In patients with PDx-fail non-small cell lung cancer, anti-PD-L1 / ΤΟΕβ trap was generally well tolerated by patients with treatment rate-related adverse events (EART) similar to those seen with other anti-PD-1 or anti-PD-L1 monotherapies. Most patients (n = 60, 72.3%)
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105/127 experienced any EART, with a lesser proportion (n = 19, 22.9%) experiencing a grade 3 or higher event. The table representing treatment-related adverse events in> 5% of patients is reported below. Fatigue / asthenia was the most common (36.1%, G3 + 6.0%), followed by pruritus (21.7%, G3 + 2.4%) and decreased appetite (16.9%, G3 + 1.2 %). One patient discontinued the study due to a treatment-related adverse event (G2, plaque eczema). One patient died of pneumonia assessed by the researcher as related to treatment. Noteworthy, skin lesions occurred in 5 patients (6.0%), including keratoacanthoma and squamous cell carcinoma (similar to other TGF-β inhibitory agents) and were well cared for by surgical excision.
[00245] Table 7: Treatment-related adverse events (EARTs)
N = 85 Any Degree Grade> 3 Any EART, n (%) 60 (72.3) 19 (22.9) Anemia 5 (6.0) 1 (1,2) Arthralgia 6 (7.2) 1 (1,2) Decreased appetite 14 (16.9) 1 (1,2) Diarrhea 6 (7.2) 0 (0.0) Dry skin 5 (6.0) 0 (0.0) Epistaxis 8 (9.6) 0 (0.0) Fatigue / Astenia 30 (36.1) 5 (6.0) Itching 18 (21.7) 2 (2.4) Macculopapular rash 6 (7.2) 1 (1,2)
[00246] In summary, it was seen that anti-PD-L1 / ΤΰΕβ Trap is a first class and innovative bifunctional fusion protein designed to simultaneously target 2 immunosuppressive pathways: PD-L1 and
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TGF-β. Inhibition of the TGF-β pathway therefore helps to overcome treatment failure for anti-PD-1 / PD-L1 agents. Treatment with anti-PD-L1 / ΤΰΡβ trap resulted in initial clinical activity in patients with non-small cell lung cancer with strongly pretreated refractory or resistant primary disease acquired from previous treatment with anti-PD-1 or anti therapy -PD-L1.
EXAMPLE 5: Treatment of Patients with Pre-treated Recurrent or Refractory Stage IV Gastric Cancer [00247] Objective: Patients with strongly pre-treated recurrent or refractory Stage IV non-resectable gastric cancer were selected for treatment with 1200 mg anti- PD-L1 / ΤΟΕβ trap and safety and efficacy were evaluated.
[00248] Study Design and Results: A total of 31 patients were treated with anti-PD-L1 / ΤΰΕβ Trap at a dose of 1200 mg every 2 weeks until confirmed progressive disease, unacceptable toxicity, or withdrawal from the study. The cohort consisted of a heavily pre-treated Asian patient population with 67.7% receiving at least 3 previous anti-cancer therapies and 29.3% at least 4 previous anti-cancer therapies.
[00249] The baseline characteristics of the patients are listed in table 8 below.
Table 8: Patient characteristics
Feature
Gender, n (%) Male Female Age, years Median
N = 31 (83.9) (16.1)
Range at 82
Number of anticancer therapies
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Characteristic N = previous 31, n (%) 4 (12.9)
6 (19.4)
12 (38.7)
9 (29.3)> 4
ECOG performance status, n (%)
8 (25.8)
23 (74.2) [00250] From the cutoff date data at the time of the analysis, patients received anti-PD-L1 / TGFp Trap for a median duration of 6.1 (range: 2 to 30) weeks . Among the 31 evaluable patients, 5 patients had a confirmed partial response and 5 patients had stable disease (SD) as their best overall response (BOR) according to RECIST v1.1 as assessed by the researcher. The overall response rate (ORR) was 16.1% and the disease control rate (DCR) was 32.3%.
Table 9: Patient characteristics
N = 31 BOR, n (%)CR 0 PR 5 (16.1) SD 5 (16.1) PD 16 (51.6) HUH 5 (16.1) ORR, n (%)Response rate (CR + PR) 5 (16.1) 95% Cl 5.5 to 33.7 DCR, π (%)Response rate (CR + PR + SD) 10 (32.3) 95% Cl 16.7 to 51.4
[00251] Anti-PD-L1 / TGFp Trap was generally well tolerated by patients with adverse events related to treatment rates (EART) similar to those seen with other anti-Petition monotherapies 870190062642, of 07/04/2019, p. 195/234
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PD-1 I PD-L1. 14 patients (45.2%) experienced treatment-related adverse events. 4 patients (12.9%) experienced grade 3 treatment-related adverse events. There were no grade 4-related adverse events. 1 grade 5 event (received a total of 5 doses) was considered possibly related to treatment, but suspected rupture of pre-existing thoracic aortic aneurysm was mentioned as another probable cause by the researcher.
[00252] Table 10: Treatment-related adverse events (EARTs)
N = 31 Any grade Grade> 3 Any EART, n (%) 14 (45.2) 5 (16.1) Macculopapular rash 6 (19.4) 1 (3.2) Anemia 3 (9.7) 2 (6.5) Rash 3 (9.7) 1 (3.2) Diarrhea 2 (6.5) 1 (3.2) Fatigue 2 (6.5) 0 Infusion related reaction 2 (6.5) 0 Itching 2 (6.5) 0 (sudden death) 1 (3.2) 1 (3.2)
[00253] In summary, it was seen that anti-PD-L1 / TGFp Trap was an innovative first-in-class bifunctional fusion protein designed to simultaneously target 2 immunosuppressive pathways: PD-L1 and TGF-β . Inhibition of the TGF-β pathway may help to overcome treatment failure for anti-PD-1 / PD-L1 agents. Treatment with anti-PD-L1 / ΤΰΕβ trap resulted in initial clinical activity in Asian patients with heavily pretreated gastric cancer.
EXAMPLE 6: Treatment of Heavily Pre-Treated Colorectal Cancer (CRC) Patients
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109/127 [00254] Background and Objective: Colorectal Cancer (CRC) is the second and third most common cancer in women and men worldwide, respectively. Recently, 4 CRC molecular consensus subgroups (CMS) have been described - including poor prognosis, mesenchymal CMS4 group which is characterized by angiogenic, inflammatory, and immunosuppressive qualities. We hypothesized that TGF-β may play a role in mediating this immunosuppressive phenotype, providing a rationale for the use of anti-PD-L1 / TGFp trap in these patients. Anti-PDL1 therapy showed substantial activity for patients with defective incompatibility repair CRC (eg, high microsatellite instability (MSI-H)), however only about 4% of patients with metastatic CRC have MSI-H tumors, and these treatments had minimal activity in patients with proficient incompatibility repair.
[00255] Study Design and Results: A total of 32 patients were treated with anti-PD-L1 / TGFp Trap at a dose of 1200 mg every 2 weeks. The median duration of treatment was 7.1 weeks (range 2 to 38), and from the cutoff date at the time of the analysis, 2 patients remained on active treatment. The primary endpoint is BOR according to RECIST v1.1, secondary endpoints being safety / tolerability. The baseline characteristics of the patients are listed in the table below. In summary, this was a heavily pre-treated patient population with 87.5% of patients receiving more than 3 previous treatment regimens, good general clinical status (PS 0 to 1) and included a range of ages and genders. Approximately 34% of the tumors were KRAS mutated, and a majority of patients (81.3%) had PDL1 expression <1% on tumor cells based on the PD-L1 Dako 73-10 test. The laterality of the tumor was not prospectively collected in the
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110/127 database, but was determined based on the patient's previous cancer surgical history. Using this clinical assessment, 40.6% of the tumors were noted to be on the left, 28.1% were on the right, and 31.3% were unable to be determined based on the available data.
Table 11: Patient characteristics
Feature N = 32 Gender, n (%)MaleFeminine 16 (50.0)16 (50.0) Age, in yearsMedianInterval 6026-81 Number of previous anti-cancer therapies, n (%) 0123> 4 004 (12.5)9 (28.1)19 (59.4) ECOG performance status, n (%)01 11 (34.4)21 (65.6) Mutational state KRAS, n (%)WildMutant 11 (34.4)21 (65.6) Expression of PD-L1 in tumor cells, n (%)> 1%<1% 3 (9.4)26 (81.3)
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Not evaluable 3 (9.4) Primary tumor type, n (%)Colon adenocarcinomaAdenocarcinoma of the rectum 23 (71.9)9 (28.1) Lateral tumor age, n (%) LeftRightNot evaluable 13 (40.6)9 (28.1)10 (31.3)
[00256] From the cut data at the time of the analysis, 1 patient (3.1%) had a confirmed partial response (PR). One additional patient had stable disease, and 27 patients had progressive disease as the best overall response. The response criteria were decided by an independent review committee, and were defined by RECISTv1.1. The patient with a PR had colorectal cancer (CRC) who was proficient in incompatibility repair (ie stable microsatellite), CMS4, mutant KRAS, and PD-L1 + (PD-L1> 1% in tumor cells by IHC). This patient had the highest expression of PD-L1 in tumor cells in our cohort (20%). From 1st. As of November 2017, the patient with a PR remained in the study (12.5 months) with partial response in progress. An additional patient remains on treatment for 13 months with a non-assessable disease due to receiving palliative RT for a target lesion during month 5 of treatment. No new injuries have occurred since that time, and non-target injuries remain stable. The colorectal cancer of this patient was KRAS-mutated; CMS and the micro-satellite status are pending, the status of PD-L1 is unknown.
[00257] In patients with colorectal cancer, anti-PD-L1 / TGFp Trap was generally well tolerated by patients with adverse events related to treatment rates (EART) similar to those seen with other anti-PD-1 / PD monotherapies -L1. Most patients
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112/127 patients (n = 22, 68.8%) experienced any treatment-related adverse event, with a smaller proportion (n = 4, 12.5%) experiencing a grade 3 event. There were no adverse events related to treatment grade 4/5 treatment. The table representing treatment-related adverse events in> 5% of patients is reported below. Anemia, diarrhea, infusion-related reaction and nausea were the most common (all n = 5, 15.6%). There was a single event of grade 3 related anemia (3.1%). Only one treatment-related adverse event led to treatment discontinuation (grade 3 enteritis). The event occurred concurrently with the progression of the disease.
[00258] Table 12: Treatment-related adverse events (EARTs)
N = 32 Any Degree Grade 3 Any EART, n (%) 22 (68.8) 4 (12.5) Anemia 5 (15.6) 1 (3.1) Diarrhea 5 (15.6) 0 Infusion related reaction 5 (15.6) 0 Nausea 5 (15.6) 0 Decreased appetite 4 (12.5) 0 Fatigue 3 (9.4) 1 (3.1) Myalgia 3 (9.4) 0 Pyrexia 3 (9.4) 0 Vomiting 3 (9.4) 0 Abdominal pain 2 (6.3) 0 Acneiform dermatitis 2 (6.3) 0 Indisposition 2 (6.3) 0 Rash 2 (6.3) 0 Macculopapular rash 2 (6.3) 0 Stomatitis 2 (6.3) 0
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Adrenal Insufficiency 1 (3.1) 1 (3.1) Increased blood bilirubin 1 (3.1) 1 (3.1) Enteritis 1 (3.1) 1 (3.1)
[00259] In summary, it was seen that anti-PD-L1 / TGFp Trap was an innovative first class bifunctional fusion protein designed to simultaneously target 2 immunosuppressive pathways, TGFβ and PD-L1. Treatment with anti-PD-L1 / ΤΟΡβ trap resulted in initial clinical activity in patients heavily pretreated with advanced colorectal cancer; 1 patient had a durable PR; 1 patient had DS; and 27 patients had PD as BOR. The patient with an ongoing PR for 8.3 months had CRC which was MSI, CMS4, KRASmutant, and PD-L1 +. A second patient remains well without recurrence at 13 months after an initial progressive disease.
EXAMPLE 7: Establishment of Dose / Effective Dosage Regimen and Exposure in Humans: preliminary dose-response and exposure-response in 2nd Non-Small Cell Lung Cancer. Line (2L NSCLC) after dosing once every 2 weeks (q2w) of anti-PD-L1 / ΤΘΡβ trap [00260] In this study, 80 subjects with advanced / recurrent non-small cell lung cancer after treatment with platinum and not selected for PD-L1, were administered with either 500 mg or 1200 mg of anti-PD-L1 / ΤΟΡβ trap once every 2 weeks (q2w) (n = 40 per cohort) until disease progression , unacceptable toxicity, or withdrawal from the study. Individuals' response and exposure-response were evaluated. From the cut data at the time of the analysis, a total of 17 individuals remained on treatment with a median follow-up of 35.2 (range 1.3 to 47.3) weeks. The overall response rate (ORR) not confirmed by the researcher was 25.0% (ORR at 500 mg, 22.5%; ORR at 1200 mg, 27.5%), with 9 partial responses (PR) seen at 500 mg, and 1
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114/127 complete response (CR) and 10 PRs at 1200 mg. Clinical activity was observed through PD-L1 expression levels with an ORR of 71.4% to 1200 mg noted in patients with> 80% PD-L1 tumor expression (7 patient). The most common treatment-related adverse events (EARTs) were itching (18.8%), maculopapular rash (17.5%), decreased appetite (12.5%), and asthenia (11.3%). Grade> 3 treatment-related adverse events occurred in 20 patients (25%). Treatment-related deaths do not occur. [00261] For the exposure-response assessment, the population pharmacokinetic model was used to predict first cycle exposures based on the dosage and covariate information of these 80 patients. Specifically, AUG and Cminima were predicted after a single dose for each individual using Bayes empirical estimates of population pharmacokinetic parameters (see Tables 2 and 3). Predicted exposure data were combined for 500 mg q2w and 1200 mg q2w cohorts to calculate a response rate for each quartile of predicted exposure, as shown in Table 4 and Table 5.
SEQUENCES SEQ ID NO: 1
light chain peptide sequence lambda anti-PD-L1 secreted QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
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SEQ ID NO: 2
chain peptide sequence H secreted anti-PDL1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 3
peptide sequence of the H chain secreted anti-TGF PDL1 I Trap (PDL anti-N.T .: 1 / ΤΰΕβ Trap) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPetição 870 190 062 642 of 4.7.2019, p. 203/234
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PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO: 4
The DNA sequence of the translation initiation codon of the light chain of the translation stop codon lambda anti-PD-L1 (the leader sequence preceding the VL is the peptide activating signal type plasminogen urokinase) atqaqqqccctqctqqctaqactqctqctqtqcqtqctqqtcqtqtccqacaqcaaqqqcCAGTCCGCCCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCCTGGCCAGTCCATCACCATCAGCTGCACCGGCACCTCCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGTCCAACCGGCCCTCCGGCGTGTCCAACAGATTCTCCGGCTCCAAGTCCGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCAGAGGACGAGGCCGACTACTACTGCTCCTCCTACACCTCCTCCAGCACCAGAGTGTTCGGCACCGGCACAAAAGTGACCGTGCTGggccagcccaag g ccaacccaaccg tg acactg ttccccccatcctccg g g AACTG cag ctg g ccaacaaggccaccctggtctgcctgatctcag atttctatccag gcgccgtgaccgtggcctggaaggctgatggctccccagtgaaggccggcgtggaaaccaccaagccctccaagcagtccaacaacaaatacgccgcctcctcctacctgtccctgacccccgagcagtggaagtcccaccggtcctacag CCAG tcacacacg g g gg ctccaccg TGG TCG aaaag accg cccccaccgagtgctcaTGA
SEQ ID NO: 5
DNA sequence of the translation initiation codon to the translation stop codon (mVK SP leader: underlined lowercase letters; VH: uppercase letters; lgG1m3 with K to A mutation: lowercase letters; (G4S) x4-G (SEQ ID NO: 11) interlinker: bold capital letters; TGFpRIl: lower case letters underlined in bold; two stop codons: upper case letters underlined in bold) atqqaaacaqacaccctqctqctqtqqqtqctqctqctqtqqqtqcccqqctccacaq
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118/127 tqqccqtqtqqaqqaaqaacqacqaaaacatcaccctcqaqaccqtqtqccatqaccccaaqctqccctaccacqacttcatcctqqaaqacqccqcctcccccaaqtqcatcatqaaqqaqaaqaaqaaqcccqqcqaqaccttcttcatqtqcaqctqcaqcaqcqacqaqtqcaatqacaacatcatctttaqcqaqqaqtacaacaccaqcaaccccqacTGATAA
SEQ ID NO: 6
AntiPD-LI (mut) I secreted light chain polypeptide sequence TGFp trap (N.T .: anti-PD-L1 (mut) / ΤϋΡβ Trap), with A31G, D52E, R99Y mutations
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 7
Sequence of polypeptide secreted heavy chain of anti-PDL1 (mut) / TGF trap (N.T .: anti-PDL1 (mut) / ΤϋΡβ Trap) EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDPetição 870 190 062 642 of 7.4.2019, p. 206/234
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MIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO: 8
Human TGFpRII Isoform A Precursor Polypeptide (NCBI RefSeq Accession No: NP_001020018)
MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK
SEQ ID NO: 9
Human TGFpRII Isoform B Precursor Polypeptide (NCBI RefSeq Accession No: NP_003233
MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLPetição 870190062642, of 07.04.2019, p. 207/234
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TAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK
SEQ ID NO: 10
An Extracellular Domain Polypeptide of the Human TGFpRII Isoform B
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
SEQ ID NO: 11
Linker (Gly4Ser) 4Gly
GGGGSGGGGSGGGGSGGGGSG
SEQ ID NO: 12
Polypeptide sequence of the secreted heavy chain variable region of the anti-PD-L1 antibody MPDL3289A
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS
SEQ ID NO: 13
Polypeptide sequence of the light chain variable region secreted from the anti-PD-L1 antibody MPDL3289A
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR
SEQ ID NO: 14
Polypeptide sequence of the secretory heavy chain variable region
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121/127 of the anti-PD-L1 antibody YW243.55S70 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRWWGG
INCORPORATION BY REFERENCE [00262] The entire disclosure of each of the patent documents and scientific articles referred to here, in this patent application, is incorporated by reference for all purposes.
EQUIVALENTS [00263] The invention can be incorporated in other specific forms without departing from the spirit or the essential characteristics of it. Therefore, the foregoing modalities should be considered in all illustrative aspects rather than limiting the invention described here, in this patent application. Various structural elements of the different modalities and various stages of the method disclosed can be used in various combinations and permutations, and all the variants mentioned should be considered forms of the invention. Therefore, the scope of the invention is indicated by the appended claims, rather than the preceding description, and all modifications that come within the meaning and equivalence range of the claims are intended to be encompassed therein.
[00264] The numbered embodiments of the present invention are listed below:
1. a method of treating cancer or inhibiting tumor growth in an individual who needs it, the method in which it comprises administering to the individual a dose of at least 500 mg of a protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: (a) at least one variable region of an antibody heavy chain that binds to the protein
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122/127 Programmed Cell Death Ligand 1 human PD (L-L1); and (b) the Receptor II of the Human Transforming Growth Factor β (ΤΟΡβΗΙΙ), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΟΡβ), in which the second polypeptide comprises at least one variable region of a light chain of an antibody that binds to PD-L1, and where the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds PD-L1.
A method according to claim 1, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
Method according to claim 1 or 2, wherein the dose is from 500 mg to 2400 mg.
Method according to claim 1 or 2, wherein the dose is 1200 mg to 1800 mg.
Method according to any one of claims 1 to 4, wherein the dose is 1200 mg.
A method according to claim 1 or 2, wherein the dose is 1800 mg.
Method according to any one of claims 1 to 6, wherein the dose is administered once every two weeks or once every three weeks.
A method according to any one of claims 1 to 7, wherein the protein is administered by intravenous administration.
A method according to claim 8, wherein the intravenous administration is carried out with a filled pouch, filled pen, or filled syringe comprising a formulation comprising the protein.
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A method according to claim 9, wherein the bag is connected to a channel comprising a tube and / or a needle.
11. Method according to any one of claims 1 to 10, in which the cancer or tumor is selected from the group consisting of: non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer, ovarian cancer, glioblastoma, gastric cancer, biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head or neck, and squamous carcinoma of the head or neck.
12. Method according to any one of claims 1 to 10, in which the cancer or tumor is selected from the group consisting of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloma, lymphoma , leukemia, thyroid, endometrial, uterine, vesical, neuroendocrine, head and neck, liver, nasopharynx, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, cell skin cancer basal cells, squamous cell skin cancer, protuberant dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.
A method according to any one of claims 1 to 10, wherein the tumor is an advanced solid tumor.
A method according to claim 13, wherein the tumor is refractory and / or resistant to previous treatment.
15. Intravenous drug delivery formulation comprising 500 mg to 2400 mg of a protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises: (a) at least one variable region of an antibody heavy chain that binds to the Programmed Cell Death Link human protein 1 (PD-L1); and (b) the human Transforming Growth Factor β Receptor II
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124/127 (TGFpRIl), or a fragment thereof, capable of binding to Transforming Growth Factor β (ΤΰΕβ), in which the second polypeptide comprises at least one variable region of an antibody light chain that binds to PD -L1, and where the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen-binding site that binds PD-L1.
An intravenous drug delivery formulation according to claim 15, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
An intravenous drug delivery formulation according to claim 15 or 16, wherein it comprises 1200 mg of the protein.
An intravenous drug delivery formulation according to claim 15 or claim 16, comprising 1200 mg to 2400 mg of the protein.
An intravenous drug delivery formulation according to claim 15 or 16, comprising 1800 mg of the protein.
An intravenous drug delivery formulation according to any one of claims 15 to 19, wherein the formulation is contained in a bag, a pen, or a syringe.
21. An intravenous drug delivery formulation according to claim 20, wherein the pouch is connected to a channel comprising a tube and / or a needle.
22. An intravenous drug delivery formulation according to any one of claims 15 to 21, wherein the formulation is a lyophilized formulation or a liquid formulation.
23. Drug delivery device, comprising a formulation comprising 500 mg to 2400 mg of a protein comprising a first polypeptide and a second polypeptide,
Petition 870190062642, of 07/04/2019, p. 212/234
125/127 wherein the first polypeptide comprises: (a) at least one variable region of an antibody heavy chain that binds to the Programmed Cell Death Link 1 human protein (PD-L1); and (b) the Receptor II of the Human Transforming Growth Factor β (ΤΟΡβΡΙΙ), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΟΡβ), in which the second polypeptide comprises at least one variable region of a light chain of an antibody that binds to PD-L1, and where the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds PD-L1.
24. A drug delivery device according to claim 23, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
25. A drug delivery device according to claim 23 or 24, comprising 1200 mg of the protein.
26. A drug delivery device according to claim 23 or claim 24, comprising 1200 mg to 2400 mg of the protein.
27. A drug delivery device according to claim 23 or 24, comprising 1800 mg of the protein.
28. A drug delivery device according to any one of claims 23 to 27, wherein the device is a bag, pen, or syringe.
A drug delivery device according to claim 28, wherein the bag is connected to a channel comprising a tube and / or a needle.
30. Kit comprising one or more containers collectively comprising a formulation comprising 500 mg to 2400 mg of a protein comprising a first polypeptide and a second
Petition 870190062642, of 07/04/2019, p. 213/234
126/127 polypeptide, wherein the first polypeptide comprises: (a) at least one variable region of an antibody heavy chain that binds to the Programmed Cell Death Link 1 human protein (PD-L1); and (b) the Receptor II of the Human Transforming Growth Factor β (ΤΟΡβΗΙΙ), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΟΡβ), in which the second polypeptide comprises at least one variable region of a light chain of an antibody that binds to PD-L1, and where the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds PD-L1.
31. The kit of claim 30, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
32. The kit of claim 30 or 31, wherein the containers collectively comprise 1200 mg of the protein.
33. The kit of claim 30 or 31, wherein the containers collectively comprise 1200 to 2400 mg of the protein.
34. The kit of claim 30 or 31, wherein the containers collectively comprise 1800 mg of the protein.
35. A kit according to any one of claims 30 to 34, wherein the formulation is a lyophilized formulation or a liquid formulation.
36. An intravenous drug delivery formulation according to any one of claims 15 to 22, a drug delivery device according to any one of claims 23 to 29, or a kit according to any one of claims 30 to 35, wherein is for use in the treatment of cancer or in inhibiting tumo growth
Petition 870190062642, of 07/04/2019, p. 214/234
127/127 ral in an individual who needs it.
37. Intravenous drug delivery formulation, drug delivery device, or kit according to claim 36, wherein the cancer or tumor is selected from the group consisting of: non-small cell lung cancer, melanoma, pancreatic cancer , colorectal cancer, ovarian cancer, glioblastoma, gastric cancer, biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head or neck, and squamous carcinoma of the head or neck.
38. Intravenous drug delivery formulation, drug delivery device, or kit according to claim 36, wherein the cancer or tumor is selected from the group consisting of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, vesical, neuroendocrine, head and neck, liver, nasopharynx, testicular, small cell lung cancer, cell lung cancer not small, melanoma, basal cell skin cancer, squamous cell skin cancer, protruding dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.
39. Intravenous drug delivery formulation, drug delivery device, or kit according to claim 36, wherein the tumor is an advanced solid tumor.
40. Intravenous drug delivery formulation, drug delivery device, or kit according to claim 36, wherein the tumor is refractory to previous treatment.
41. Intravenous drug delivery formulation, drug delivery device, or kit according to any of claims 36 to 40, wherein the formulation is administered to the individual once every two weeks.

权利要求:
Claims (25)
[1]
1. Use of a protein comprising a first polypeptide and a second polypeptide, characterized by the fact that it is for the manufacture of a drug to treat cancer or inhibit tumor growth in an individual who needs it, in which the said treatment comprises administration to the individual a dose of at least 500 mg of the protein, where the first polypeptide comprises: (a) at least one variable region of an antibody heavy chain that binds to the Programmed Cell Death Link 1 human protein (PD- L1); and (b) the Receptor II of the human Transforming Growth Factor β (ΤΟΡβΡΙΙ), or a fragment thereof, capable of binding to the Transforming Growth Factor β (ΤΟΡβ), in which the second polypeptide comprises at least one variable region of a light chain of an antibody that binds PD-L1, and where the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds PD-L1.
[2]
2. Use according to claim 1, characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
[3]
3. Use according to claim 1 or 2, characterized by the fact that the dose is from 500 mg to 2400 mg.
[4]
4. Use according to claim 1 or 2, characterized in that the dose is 1200 mg to 1800 mg.
[5]
Use according to any one of claims 1 to 4, characterized in that the dose is 1200 mg.
[6]
6. Use according to claim 1 or 2, characterized
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2/4 due to the fact that the dose is 2400 mg.
[7]
7. Use according to any one of claims 1 to 6, characterized in that the dose is administered once every two weeks or once every three weeks.
[8]
8. Use, according to claim 7, characterized by the fact that the dose is administered to the individual once every two weeks.
[9]
Use according to any one of claims 1 to 8, characterized in that the drug comprising the protein is administered by intravenous administration.
[10]
10. Use according to claim 9, characterized by the fact that intravenous administration is carried out with a filled pouch, filled pen, or filled syringe comprising a formulation comprising the protein.
[11]
11. Use according to claim 10, characterized by the fact that the bag is connected to a channel comprising a tube and / or a needle.
[12]
12. Use according to any one of claims 1 to 11, characterized by the fact that the cancer or tumor is selected from the group consisting of: non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer, cancer of ovary, glioblastoma, gastric cancer, biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head or neck, and squamous carcinoma of the head or neck.
[13]
13. Use according to any one of claims 1 to 11, characterized by the fact that the cancer or tumor is selected from the group consisting of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical , myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, vesical, neuroendocrine, head
Petition 870190062642, of 07/04/2019, p. 227/234
3/4 and neck, liver, nasopharynx, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, protuberant dermatofibrosarcoma, carcinoma of Merkel cells, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.
[14]
14. Use according to any one of claims 1 to 11, characterized in that the tumor is an advanced solid tumor.
[15]
15. Use, according to claim 14, characterized by the fact that the tumor is refractory and / or resistant to previous treatment.
[16]
16. Intravenous drug delivery formulation, characterized in that it comprises 500 mg to 2400 mg of a protein, as defined in claim 1 or 2.
[17]
17. Intravenous drug delivery formulation according to claim 16, characterized in that it comprises 1200 mg of the protein.
[18]
18. Intravenous drug delivery formulation according to claim 16, characterized in that it comprises 1200 mg to 2400 mg of the protein.
[19]
19. Intravenous drug delivery formulation according to claim 16, characterized in that it comprises 2400 mg of the protein.
[20]
20. Intravenous drug delivery formulation according to any one of claims 16 to 19, characterized in that it is contained in a bag, a pen, or a syringe.
[21]
21. Intravenous drug delivery formulation according to claim 20, characterized in that the pouch is connected to a channel comprising a tube and / or a needle.
Petition 870190062642, of 07/04/2019, p. 228/234
4/4
[22]
22. Intravenous drug delivery formulation according to any one of claims 16 to 21, characterized in that the formulation is a lyophilized formulation or a liquid formulation.
[23]
23. Drug delivery device, characterized in that it comprises a formulation, comprising a protein, as defined in any one of claims 1 to 22.
[24]
24. Kit, characterized by the fact that it comprises one or more containers collectively comprising a formulation, comprising a protein, as defined in any one of claims 16 to 22.
[25]
25. Invention, characterized by any of its embodiments or categories of claim encompassed by the material initially disclosed in the patent application or in its examples presented here.

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法律状态:
2021-10-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762443698P| true| 2017-01-07|2017-01-07|

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US62/443,698|2017-01-07|

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US201762581978P| true| 2017-11-06|2017-11-06|

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PCT/US2018/012604|WO2018129331A1|2017-01-07|2018-01-05|Dosing regimens and dosage forms for targeted tgf-b inhibition|

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