HK40012523A

HK40012523A - DOSING REGIMENS AND DOSAGE FORMS FOR TARGETED TGF-β INHIBITION

Info

Publication number: HK40012523A
Application number: HK62020002493.8A
Authority: HK
Inventors: I‧度赛特; S‧厄尔巴瓦博; Y‧乌戈梅斯特; A‧坎德瓦
Original assignee: 默克专利有限公司
Priority date: 2017-01-07
Filing date: 2018-01-05
Publication date: 2020-07-24

Description

Dosing regimens and dosage forms for targeted TGF- β inhibition

Cross reference to related applications

This application claims the benefit and priority of U.S. application No. 62/443,698 filed on 7/1/2017 and U.S. application No. 62/581,978 filed on 6/11/2017, each of which is incorporated herein by reference in its entirety for all purposes.

Technical Field

The present disclosure relates generally to weight-independent (BW-independent) dosing regimens and dosage forms for bifunctional proteins targeting the human protein programmed death ligand 1(PD-L1) and transforming growth factor β (TGF β).

Background

Programmed death 1(PD-1)/PD-L1 axis is an important mechanism for tumor immune evasion. Effector T cells that sense antigens chronically exhibit a depletion phenotype marked by PD-1 expression, a state closely associated with tumor cells by upregulation of PD-L1. In addition, in the tumor microenvironment, bone marrow cells, macrophages, parenchymal cells and T cells upregulate PD-L1. Blocking this axis restores effector function in these T cells.

U.S. patent application publication No. US20150225483a1, incorporated herein by reference, describes a bifunctional fusion protein that combines an anti-programmed death ligand 1(PD-L1) antibody with a soluble extracellular domain of tumor growth factor type II β receptor (TGF β RII) as a TGF β neutralizing "Trap" (in particular, the protein is a heterotetramer consisting of two immunoglobulin light chains against PD-L1, and two heavy chains of a heavy chain against PD-L1 comprising an extracellular domain genetically fused to human TGF β RII by a flexible glycine-serine linker (see figure 1.) the anti-PD-L1/TGF β Trap molecule is designed to target two major mechanisms of immunosuppression in the tumor microenvironment. U.S. patent application publication No. US20150225483a1 describes the administration of the Trap molecule at a dose based on the patient's body weight.

Disclosure of Invention

The present disclosure provides improved dosing regimens for administering bifunctional proteins targeting PD-L1 and TGF β in particular, weight-independent (BW-independent) dosing regimens and related dosage forms involving administration of at least 500mg of bifunctional protein at various dosing frequencies are useful as anti-tumor and anti-cancer therapies.

The bifunctional proteins of the present disclosure (anti-PD-L1/TGF β trap molecules) include first and second polypeptides the first polypeptide includes (a) a variable region of a heavy chain of an antibody that binds at least to human protein programmed death ligand 1(PD-L1), and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment (e.g., a soluble fragment) thereof that is capable of binding transforming growth factor β (TGF β). the second polypeptide includes at least a variable region of a light chain of an antibody that binds PD-L1, wherein the heavy chain of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site (e.g., any antibody or antibody fragment described herein) that binds to PD-L1. because the bifunctional proteins of the present disclosure bind to two targets, (1) PD-L1, which is primarily membrane bound, and (2) β, which is soluble in blood and interstitial BW-independent dosing regimens require dosages that not only effectively inhibit TGF-L1, but also TGF-L β at the tumor site.

In one aspect, the disclosure provides for the treatment of cancer or inhibition of tumors, such as non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer (e.g., pre-treated colorectal cancer (CRC)), ovarian cancer, glioblastoma, gastric cancer (e.g., pre-treated recurrent or refractory unresectable IV gastric cancer), biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head and neck, and squamous carcinoma of the head and neck.

The invention also features the use of a bifunctional protein as described above for the treatment of cancer or for inhibiting tumor growth. The cancer or tumor may be selected from: colorectal cancer (e.g., pre-treated colorectal cancer (CRC)), breast cancer, ovarian cancer, pancreatic cancer, gastric cancer (e.g., pre-treated recurrent or refractory unresectable stage IV gastric cancer), prostate cancer, renal cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. The use may also include administration of radiation or administration of a chemotherapeutic agent, biologic agent or vaccine.

The present disclosure also features a method of promoting local consumption of TGF β, the method comprising administering the above-described protein, wherein the protein binds TGF β in solution, binds PD-L1 on the surface of a cell, and carries the bound TGF β into the cell (e.g., a cancer cell).

The disclosure also features a method of inhibiting phosphorylation of SMAD3 in a cell (e.g., a cancer cell or an immune cell), the method comprising contacting the cell in a tumor microenvironment with a protein as described above.

The disclosure also features a method of inhibiting tumor growth or treating cancer. The method comprises exposing the tumor to a protein as described above. The method may further comprise: exposing the tumor to radiation or exposure to a chemotherapeutic agent, biologic, or vaccine. In certain embodiments, the cancer or tumor is selected from: colorectal cancer (e.g., pre-treated colorectal cancer (CRC)), breast cancer, ovarian cancer, pancreatic cancer, gastric cancer (e.g., pre-treated recurrent or refractory unresectable stage IV gastric cancer), prostate cancer, renal cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

"TGF β RII" or "TGF β receptor II" refers to a polypeptide having a wild-type human type 2 TGF β receptor isoform A sequence (e.g., the amino acid sequence of NCBI reference sequence (RefSeq) accession number NP-001020018 (SEQ ID NO:8)), or a wild-type human type 2 TGF β receptor isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq accession number NP-003233 (SEQ ID NO:9)) or a polypeptide having a sequence that is substantially identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9 TGF β TGF RII can retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99% of the binding activity of wild-type sequence β TGF β RII polypeptide has NO signal sequence.

"TGF β RII fragment capable of binding TGF β" refers to any portion of NCBI RefSeq accession number NP 001020018(SEQ ID NO:8) or NCBI RefSeq accession number NP 003233(SEQ ID NO:9) or a sequence substantially identical to SEQ ID NO:8 or SEQ ID NO:9, a fragment at least 20 (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, or 200) amino acids in length and retaining at least a portion of the TGF β binding activity of a wild-type receptor or a wild-type fragment thereof (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99%). typically, these fragments are soluble fragments.

By "substantially identical" is meant that the polypeptide exhibits at least 50%, preferably 60%, 70%, 75% or 80%, more preferably 85%, 90% or 95%, and most preferably 99% amino acid sequence identity to the reference amino acid sequence. The length of the comparison sequences is generally at least 10 amino acids, preferably at least 15 contiguous amino acids, more preferably at least 20, 25, 50, 75, 90, 100, 150, 200, 250, 300 or 350 contiguous amino acids, and most preferably the full-length amino acid sequence.

"patient" means a human or non-human animal (e.g., a mammal). "patient," "subject," "patient in need thereof," and "subject in need thereof" are used interchangeably in this disclosure and refer to a living organism that is suffering from or susceptible to a disease or condition that can be treated by administration using the methods and compositions provided in this disclosure.

The terms "treat," "treating," and other grammatical equivalents as used in this disclosure include alleviating, ameliorating, or preventing a disease, disorder, or symptom, preventing other symptoms, ameliorating, or preventing an underlying metabolic cause of a symptom, inhibiting a disease or disorder, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, causing regression of a disease or disorder, alleviating a disorder caused by a disease or disorder, or stopping a symptom of a disease or disorder, and are intended to include preventing. The term also includes achieving a therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, although the patient may still be suffering from the underlying disorder.

"cancer" refers to a population of cells that abnormally proliferate. As used herein, the term "cancer" refers to all types of cancer, tumors, malignant or benign tumors found in mammals, including leukemias, carcinomas and sarcomas. Exemplary cancers include breast cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer, glioblastoma. Other examples include brain cancer, lung cancer, non-small cell lung cancer, melanoma, sarcoma, prostate cancer, cervical cancer, stomach cancer, head and neck cancer, uterine cancer, mesothelioma, metastatic bone cancer, medulloblastoma, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, essential thrombocytosis, primary macrophage blood disease, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, and tumors of the endocrine and exocrine pancreas.

Throughout the description and claims of this specification, the word "comprise" and other forms of the word, such as "comprises" and "comprising", mean including but not limited to, and are not intended to exclude, for example, other components.

By "co-administration" is meant that the compositions described herein are administered concurrently with, immediately before, or after the administration of additional therapy. The proteins and compositions of the present disclosure may be administered alone, or may be co-administered to a patient with a second, third or fourth therapeutic agent. Co-administration is meant to include simultaneous or sequential administration of the protein or composition (more than one therapeutic agent), either alone or in combination.

The terms "a" and "an" are not meant to be limiting. In certain embodiments, the terms "a" and "an" may refer to the plural. As used throughout this document, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a plurality of such compositions, as well as a single composition.

A "reconstituted" formulation is one prepared by dissolving a lyophilized formulation in an aqueous carrier such that the bifunctional molecule is dissolved in the reconstituted formulation. The reconstituted formulation is suitable for intravenous administration (IV) to a patient in need thereof.

The term "about" refers to any minimal change in the concentration or amount of an agent that does not alter the efficacy of the agent in the preparation of the agent and in the treatment of a disease or disorder. In embodiments, the term "about" may include ± 15% of a specified numerical value or data point.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that a number of values are disclosed in this disclosure, and that each value is also disclosed as "about" that particular value, in addition to the value itself. It should also be understood that throughout this application, data is provided in a number of different formats and represents endpoints and starting points and ranges for any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, equal to 10 and 15 and between 10 and 15 are considered disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic preparations typically have about 250 to 350mOsmol/KgH₂Osmotic pressure of O. The term "hypertonic" is used to describe formulations with an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using vapor pressure or freezing type osmometers.

The term "buffer" refers to one or more components that are capable of protecting a solution from pH changes when added to an aqueous solution, when added to an acid or base, or when diluted with a solvent. In addition to phosphate buffer, glycinate, carbonate, citrate buffer, etc. may also be used, in which case sodium, potassium or ammonium ions may be used as counterions.

An "acid" is a substance that generates hydrogen ions in an aqueous solution. "pharmaceutically acceptable acids" include inorganic and organic acids which are non-toxic in the concentrations and manner in which they are formulated.

"base" is a substance that generates hydroxide ions in an aqueous solution. "pharmaceutically acceptable bases" include inorganic and organic bases which are non-toxic in the concentrations and manner in which they are formulated.

A "lyoprotectant" is a molecule that, when combined with a protein of interest, prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage.

"preservatives" are agents that reduce the action of bacteria and may optionally be added to the formulations herein. The addition of a preservative may, for example, facilitate the production of a multi-use (multi-dose) formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, where the alkyl group is a long chain compound), 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.

A "surfactant" is a surface active molecule containing a hydrophobic moiety (e.g., an alkyl chain) and a hydrophilic moiety (e.g., a carboxyl group and a carboxylate group). Surfactants may be added to the formulations of the present invention. Surfactants suitable for use in the formulations of the present invention include, but are not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and derivatives; a triton; sodium lauryl sulfate; sodium octyl glucoside; dodecyl-, myristoyl-, linoleyl-or stearyl-sulfobutadiene; dodecyl-, myristoyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-or cetyl-Betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoylpropyl-or isostearamidopropylbetaines (e.g. lauramidopropyl); myristoylamidopropyl-, palmitoylpropyl-or isostearamidopropyl-dimethylamine; sodium methyl cocoyl, or disodium methyl oleyl-taurate; and MONAQUAT^TMSeries (Mona Industries, Inc.), Patterson, N.J.), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.).

Other embodiments and details of the present disclosure will be apparent hereinafter.

Description of the figures figure 1 is a schematic representation of an anti-PD-L1/TGF β trap molecule comprising a (Gly) through (Gly) molecule₄Ser)₄An anti-PD-L1 antibody with a Gly (SEQ ID NO:11) linker fused to the two extracellular domains (ECDs) of the TGF β type II receptor.

Figure 2 shows a two-step ELISA graph demonstrating that an anti-PD-L1/TGF β trap binds both PD-L1 and TGF β simultaneously.

Figure 3 is a graph showing that anti-PD-L1/TGF β trap induces a significant increase in IL-2 levels.

FIG. 4A is a graph showing in vivo consumption of TGF β 1 in response to anti-PD-L1/TGF β trapsIsotype controls, and three different doses, as shown in the legend figure 4B is a graph showing TGF β 2 in vivo consumption in response to anti-PD-L1/TGF β wells figure 4C is a graph showing TGF β 3 in vivo consumption in response to anti-PD-L1/TGF β wells figure 4D is a graph showing naive, isotype controls, and three different doses, as shown in the legend figure 4D is a graph showing occupancy of anti-PD-L1/TGF β wells to PD-L1 supporting a receptor binding model in the EMT-6 tumor system.

FIG. 5 is a graph showing the anti-tumor efficacy of anti-PD-L1/TGF β trap control (anti-PD-L1 (mut)/TGF β) in the detroit 562 xenograft model.

Fig. 6A is a scatter diagram showing the relationship between clearance (clearance) and body weight. This line represents the regression line, indicating the relationship between CL and BW. Fig. 6B is a scatter diagram showing the relationship between the distribution volume (V) and the body weight. The line represents the regression line, indicating the relationship between V and BW.

FIG. 7A is C for the entire population for a fixed (1200mg) versus mg/kg-based administration (17.65mg/kg) in a simulated population of 68kg median body weight_avgBox plot of the distribution. Figure 7B is a box plot of the exposure AUC distribution in the simulated population of 68kg median body weight for the whole population at a fixed (1200mg) versus mg/kg based dosing (17.65 mg/kg). FIG. 7C is the C of the entire population for a fixed (1200mg) versus mg/kg-based administration (17.65mg/kg) in a simulated population of 68kg median body weight_{Low valley}Box plot of the distribution. FIG. 7D is C of the entire population for a fixed (1200mg) versus mg/kg-based administration (17.65mg/kg) in a simulated population of 68kg median body weight_maxBox plot of the distribution.

FIG. 7E is C of the entire population for a fixed (500mg) vs. mg/kg based administration (7.35mg/kg) in a simulated population of 68kg median body weight_avgBox plot of the distribution. Figure 7F is a box plot of the exposure AUC distribution in the simulated population of 68kg median body weight for the whole population at a fixed (500mg) versus mg/kg based dosing (7.35 mg/kg). FIG. 7G is C of the entire population for a fixed (500mg) vs. mg/kg based administration (7.35mg/kg) in a simulated population of 68kg median body weight_{Low valley}Box plot of the distribution. FIG. 7H is C of the entire population for a fixed (500mg) vs. mg/kg based administration (7.35mg/kg) in a simulated population of 68kg median body weight_maxBox plot of the distribution.

FIG. 8A is C between quartiles of body weight for a fixed (1200mg) versus mg/kg based dosing (17.65mg/kg) in a simulated population of 68kg median body weight_avgBox plot of the distribution. Drawing (A)8B is a boxplot of the exposure (AUC) distribution between quartiles of body weight for a fixed (1200mg) versus mg/kg-based dosing (17.65mg/kg) in a simulated population of 68kg median body weight. FIG. 8C is C between body weight quartiles for fixed (1200mg) versus mg/kg based dosing (17.65mg/kg) in a simulated population of 68kg median body weight_{Low valley}Box plot of the distribution. FIG. 8D is C between body weight quartiles for fixed (1200mg) versus mg/kg based dosing (17.65mg/kg) in a simulated population of 68kg median body weight_maxBox plot of the distribution.

FIG. 8E is C between quartiles of body weight for a fixed (500mg) versus mg/kg based dosing (7.35mg/kg) in a simulated population of 68kg median body weight_avgBox plot of the distribution. Figure 8F is a box plot of exposure (AUC) distribution between quartiles of body weight for a fixed (500mg) versus mg/kg-based dosing (7.35mg/kg) in a simulated population of 68kg median body weight. FIG. 8G is C between quartiles of body weight for a fixed (500mg) versus mg/kg based dosing (7.35mg/kg) in a simulated population of 68kg median body weight_{Low valley}Box plot of the distribution. FIG. 8H is C between quartiles of body weight for a fixed (500mg) versus mg/kg based dosing (7.35mg/kg) in a simulated population of 68kg median body weight_maxBox plot of the distribution.

Fig. 9A is a goodness-of-fit scatter plot of the PK-potency model, showing predicted tumor volume versus observed tumor volume. Figure 9B is a goodness-of-fit scatter plot of the PK-potency model showing the condition weighted residual (GWRES) versus time after dose.

10A-10C are graphs showing predicted PK and PD-L1 receptor occupancy ("RO") of anti-PD-L1/TGF β trap molecules in doses and regimens related to tumor regression in mice, FIG. 10A is a graph showing predicted plasma concentrations versus time, FIG. 10B is a graph showing predicted PD-L1 RO versus time in PBMCs, and FIG. 10C is a graph showing predicted PD-L1 RO versus time in tumors.

11A-11C are graphs showing predicted PK and PD-L1 receptor occupancy ("RO") of anti-PD-L1/TGF β trap molecules in doses and regimens associated with tumor arrest in mice, FIG. 11A is a graph showing predicted plasma concentrations versus time, FIG. 11B is a graph showing predicted PD-L1 RO versus time in PBMCs, and FIG. 11C is a graph showing predicted PD-L1 RO versus time in tumors.

FIGS. 12A-12B are boxplots of simulated exposure distributions for the entire population for various dosing regimens in a simulated population of 68kg median body weight (FIGS. 12A: C)_AverageAnd, FIG. 12B: c_{Low valley})。

Figure 13 is a spider graph showing that patients previously with progressive disease (with primary refractory and acquired drug resistant disease) achieved significant disease stability, noting that patients with disease response and stable disease had a series of prior treatments prior to the initiation of the study, even immediately prior to the start of the trial, indicating clinical activity against the PD-L1/TGF β trap in a heterogeneous population of patients with prior PDx exposure (filled triangles: subjects stopped treatment (off-treatment); filled diamonds: first appearance of new lesions).

FIG. 14 shows a histogram of the efficacy of anti-PD-L1/TGF β trap molecules in patients treated with anti-PD-1/PD-L1 the efficacy of anti-PD-L1/TGF β trap molecules was observed in some of the anti-PD-1/PD-L1 population in patients identified as refractory (black bars) and drug-resistant (white bars) (the percent change in the sum of diameters is approximately zero (0) or negative values indicate efficacy).

Detailed Description

Weight-independent dosing regimen

Two studies investigated the safety, tolerability and pharmacokinetics of the molecule and included the assessment of PD-L1 target occupancy on peripheral blood mononuclear cells obtained from the blood of treated patients and the measurement of TGF β, TGF β 2 and TGF β concentrations, these assessments were based on data from a total of 350 subjects (dose escalation groups of 1, 3, 10 and 20mg/kg in solid tumors, and expansion groups of 3mg/kg, 10mg/kg, 500mg and 1200mg in selected tumor types), the median treatment duration was approximately 28 days by the date of expiration of the data at the time of analysis.

PK/efficacy model (mouse model)

Experiments were also performed to determine the efficacy of anti-PD-L1/TGF β trap molecules in tumor models the PK/efficacy models were established using the efficacy results of EMT-6 xenografts the PK models established in mice were used to mimic anti-PD-L1/TGF β trap plasma exposure for efficacy experimental settings the estimated parameters are reported in table 1 the estimated KC50 value is 55.3 μ g/ml this value represents the mean plasma concentration that can achieve 50% of the maximum anti-tumor activity of anti-PD-L1/TGF β trap molecules.

The basic diagnostic map of the model shows no model misidentification. The model prediction was able to capture the tumor volume distribution (fig. 9A). The conditionally weighted residuals are typically distributed with 0 means and 1 variance without trends (fig. 9B). Tumor Growth Inhibition (TGI) was then simulated using a PK/efficacy model with human predicted concentration-time curves at different doses.

TABLE 1 mouse PK/efficacy model parameters for anti-PD-L1/TGF β trap molecules in EMT-6 xenograft mice

Parameter(s)	Estimating	Std	CV％	％IIV
					K_g(h^-1)	0.068	0.0005	0.82	40
K_tr(h^-1)	0.055	0.0024	4.4	76
					KC₅₀(ng/mL)	55324.6	522.3	4.4	232
K_max	2	0.09	1	93
					Base line (mm)³)	88.3	0.87	1	47

Using efficacy experiments based on PD-L1 occupied response analysis (in a mouse model), the response in mice has been analyzed and sorted by tumor regression or tumor arrest, and PK and PD-L1 Receptor Occupancy (RO) has been predicted based on an integrated PK/RO model, this method demonstrates that tumor regression requires anti-PD-L1/TGF β trap molecule plasma concentrations of 40 to 100 μ g/mL associated with PD-L1 RO above 95% in tumors (fig. 9A-9B), anti-PD-L1/TGF β trap molecule plasma concentrations of 10 to 40 μ g/mL associated with PD-L1 RO above 95% in the periphery to achieve tumor arrest (fig. 10A-10C).

Response analysis and predicted PK/RO in mice results in FIGS. 11A-11C, which summarize the PK/RO/efficacy of anti-PD-L1/TGF β trap molecules in mice 95% PD-L1 RO was achieved at plasma concentrations of 40 μ g/mL, with an expected/estimated TGI of only about 65%.

Based on the population PK model described below, a flat dose of at least 500mg (flatdose) is required to be administered every two weeks to maintain an average concentration of about 100 μ g/mL, while a flat dose of about 1200mg is required to be administered every two weeks to maintain a C of about 100 μ g/mL_{Low valley}In certain embodiments, from about 1200mg to about 3000mg (e.g., about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, etc.) of a protein product of the present disclosure (e.g., anti-PD-L1/TGF β trap) is administered to a subject.

In embodiments, about 1200mg to about 3000mg (e.g., about 1200mg, about 1300mg, about 1400mg, about 1500mg, about 1600mg, about 1700mg, about 1800mg, about 1900mg, about 2000mg, about 2100mg, about 2200mg, about 2300mg, about 2400mg, etc.) of a polypeptide having a sequence comprising SEQ ID NO:3 and a first polypeptide comprising the amino acid sequence of SEQ ID NO:1 to a subject.

In certain embodiments, about 1200mg of a polypeptide having an amino acid sequence comprising SEQ ID NO:3 and a first polypeptide comprising the amino acid sequence of SEQ ID NO:1 is administered to the subject biweekly. In certain embodiments, about 1800mg of a polypeptide having an amino acid sequence comprising SEQ ID NO:3 and a first polypeptide comprising the amino acid sequence of SEQ id no:1 is administered to the subject once every three weeks.

Pharmacokinetic (PK) analysis sampling in humans

Serum samples for Pharmacokinetic (PK) data analysis were collected at the following time points before the start of the first dose and after the first dose: day 1 immediately after infusion and 4 hours after infusion began; day 2 at least 24 hours after the end of the infusion on day 1; and on days 8 and 15. Samples were collected on days 15, 29, and 43 prior to dosing, at the end of the infusion and at selected subsequent dosing occasions from 2 to 8 hours after the end of the infusion. For subsequent time points on days 57, 71, and 85, pre-dose samples were collected or scheduled to be collected, followed by PK sampling every 6 weeks for up to 12 weeks, followed by PK sampling every 12 weeks. During the amplification phase, sparse PK sampling was performed.

Establishing a weight-independent dosing regimen through clinical and preclinical data, we have created a new, weight-independent dosing regimen for administering anti-PD-L1/TGF β trap molecules to reduce the variability of exposure, reduce dosing errors, reduce the time required for dose preparation, reduce drug waste compared to mg/kg dosing, thereby promoting favorable therapeutic results.

The PK data from the above "PK analysis sampling in humans" are used to generate a population PK model and to perform a simulation of a possible dosing regimen, a total Covariate model, as an Alternative to the method, is used as a statistical Significance for the inference about Covariate Effects, which is called a total approximation model (Full approach model) and described in gastonguy, m., a total Covariate model for an intervention about Covariate Effects, and a statistical Significance for 42 cases Studies (Full Covariate Models as an Alternative to the method, a statistical Significance for an intervention about Covariate Effects, a Review of the Methods and 42 cases Studies), (2011), page 20, 2229, is applied to the population model data obtained from the simulation to obtain parameters with a linear elimination of the two-chamber PK model, CL, V1 and V2, a combined weight and weight adjustment and a ratio of the combined weight and a further dose of the combined weight and the calculated as a cumulative mean of the plasma profile of the subjects, as a cumulative mean dose per dose of the two cycles of the plasma profile of the plasma exposure, a total exposure model, a total dose of the plasma profile, a plasma.

Use the followingMethod for simulation: using the final PK model variance-covariance matrix, N200 sets of parameter estimates were extracted from the multivariate normal distribution of parameter estimates. For each parameter estimate, 200 IIV estimates were extracted from the $ OMEGA multivariate normal distribution, resulting in a total of 40000(200 × 200) subjects. The original dataset (N-380) was resampled for replacement to generate 40000 sets of matched covariates and steady state exposure metrics (AUC, C) were generated for each dosing regimen_avg，C_{Low valley}And C_max)。

Simulations show that the variability of exposure is slightly higher over a wide BW range for BW-based dosing ranges compared to fixed doses. FIGS. 7A and 7B show examples of exposure profiles for a median body weight of 68kg, 17.65mg/kg and 1200mg flat doses or 7.35mg/kg and 500mg flat doses, respectively. The simulations also show the opposite trend of the exposure distribution in the patient population for weight quartiles: low weight patients had higher exposure with a fixed dose, while high weight patients had higher exposure with a BW adjusted dose.

FIGS. 8A and 8B show examples of weight interquartile exposure profiles for median body weights of 68kg, 17.65mg/kg and 1200mg flat doses or 7.35mg/kg and 500mg flat doses, respectively.

Establishing effective dose/dose regimens and exposures in humans initial dose response and exposure response of second-line non-small cell lung cancer (2L NSCLC) following once every 2 weeks (q2w) administration of anti-PD-L1/TGF β traps

In one aspect, dose response and exposure-response assessment is based on data from 80 subjects, in the treatment of 2L NSCLC, subjects were administered 500mg or 1200mg once every 2 weeks anti-PD-L1/TGF β wells (q2w) (n ═ 40 per group) to assess subject dose response and exposure response until data cutoff at analysis, a total of 17 subjects were still receiving treatment with a median follow-up time of 35.2 (range, 1.3-47.3) weeks, investigators assessed an unacknowledged Overall Response Rate (ORR) of 25.0% (500mg ORR, 22.5%; 1200mg ORR 27.5%), with 9 Partial Responses (PR) at 500mg, 1 Complete Response (CR) at 1200mg and 10 PR. observed clinical activity at PD-L1 expression level, with a loss of attention to patients (7 patients) with 80% PD-L1 tumor expression (7 patients) and a loss of attention to the incidence of 3.5% (% tre) of 3.5%) and the most common incidence of signs of 3.5% craving (20%) of 3.5%) and 9% of the treatment.

For exposure-response assessment, the first cycle exposure was predicted based on dose and covariate information from these 80 patients using the population PK model described above. In particular, AUC and C after a single dose were predicted for each subject using empirical bayesian estimates of population PK parameters (tables 2 and 3)_{Low valley}。

Table 2: predicted AUC_0-336h,sdGroup summary of

AUC_0-336h,sdAUC, as predicted using the population PK model, over 0-336 hours after a single dose.

Table 3: predicted C_{Valley, sd}Group summary of

C_{Valley, sd}After a single dose C_{Low valley}As predicted using the population PK model.

The predicted exposure data for the 500mg q2w and 1200mg q2w groups were combined to calculate the response rate of the predicted exposure per quartile as shown in tables 4 and 5 below. These preliminary data indicate that 1200mg q2w may provide a more favorable efficacy profile compared to 500mg q2 w. Furthermore, these data indicate that the exposure range achieved using the 1200mg q2w dosing regimen correlates with response in 2L NSCLC (1.1 per RECIST v), and that this exposure range can be used to design an alternative dosing regimen @ (figure 12) as shown in the examples below.

TABLE 4 AUC in 2L NSCLC subjects treated once every 2 weeks with 500mg or 1200mg anti-PD-L1/TGF β traps_0-336h,sdObserved response rate of

TABLE 5C in 2L NSCLC subjects treated once every 2 weeks with 500mg or 1200mg anti-PD-L1/TGF β wells_{Valley, sd}Observed response rate (n-40 per dose group).

Establishing dosing regimens with various dosing frequencies

Data regimens with various dosing frequencies have been created to allow for less frequent dosing and/or to allow for the use of coordinated dosing regimens with concomitant medications. In particular, the preliminary population PK modeling and simulation methods described above have been used to simulate exposure to various dosing regimens and compare the regimens based on the exposure.

Based on these simulations, for a typical subject, a flat dose of at least 500mg administered every two weeks is required to maintain an average concentration of about 100 μ g/mL, while a flat dose of about 1200mg administered every two weeks is required to maintain a C of about 100 μ g/mL_{Low valley}。

Based on C_avgThe 1200mg once every two weeks corresponds to 1800mg once every three weeks (FIG. 12A), while for C_{Low valley}1200mg every two weeks corresponds to 2800mg every three weeks (FIG. 12B). And for C_avgFor example, 500mg once every two weeks is equivalent to 750mg once every three weeks; for C_{Low valley}In other words, 500mg once every two weeks is equivalent to 1167mg once every three weeks.

TGF β as cancer target

The present disclosure enables the local reduction of TGF β in tumor microenvironments by capturing TGF β with soluble cytokine receptor (TGF β RII) linked to an antibody moiety that targets a cell immune checkpoint receptor on the outer surface of certain tumor cells or immune cells an example of an antibody moiety of the present invention is such as anti-PD-L1 directed to an immune checkpoint protein, which is sometimes also referred to herein as an "antibody-cytokine trap" is indeed effective because the anti-receptor antibody is physically linked to the cytokine trap.

Indeed, as described below, treatment with anti-PD-L1/TGF β trap induces a synergistic anti-tumor effect due to simultaneous blockade of the interaction between PD-L1 on tumor cells and PD-1 on immune cells and neutralization of TGF β in the tumor microenvironment, without being bound by theory, this may be due to a synergistic effect obtained by simultaneous blockade of two major immune escape mechanisms, and consumption of TGF β by a single molecular entity in the tumor microenvironment, the clearance being achieved by (1) autocrine/paracrine of TGF β in the tumor microenvironment being bound by TGF β trap and (3) disruption of bound TGF β by endocytosis mediated by the PD-L1 receptor of TGF β RII, and fusion to the C-terminus of TGF β to Fc (a crystalline fragment of IgG) being several times stronger than the C-terminus of TGF 24 RII- β RII-Fc that places TGF 23 RII Fc to the N-terminus.

TGF β has been once questioned as a target for Cancer immunotherapy because its dual action is more developmentally and context dependent than the molecular dual personality of Cancer (Jeklyl and Hyde) (Bierie et al, Nat Rev cancer.2006; 6: 506-20). like some other cytokines, TGF β activity is developmentally and context dependent. indeed, TGF β acts both as a tumor promoting factor and a tumor suppressor to influence tumor development, progression and metastasis. the potential mechanism of this dual action of TGF β remains unclear (Yang et al, Trends Immunol.2010; 31: 220-.

There are three isoforms of ligand, TGF β,2 and 3, all homodimers, there are also three TGF β 0 receptors (TGF β R) called type I, type II and type III TGF β 2R (L Lopez-Casillas et al, J Cell biol.1994; 124: 557-68). TGF β RI is a signaling chain and does not bind ligands.TGF β 4RII binds with high affinity to ligands TGF β and 3, and not TGF β 62. TGF β RII/TGF β complex recruits TGF 588 RII β RI to form a signaling complex (Won et al, Cancer Res.1999; 59: 1273-7). TGF β RIII is a positive regulator to which TGF β binds its signaling receptor and binds with high affinity to all 3 isoforms 68692. TGF RIII binds to Cell surface 3527 RIII, TGF RIII binds to β, TGF RIII binds and then binds to β RIII.

Mice that have knocked out three different TGF- β isoforms have different phenotypes, suggesting that they have many non-compensatory functions (Bujak et al, Cardiovasc res.2007; 74: 184-95). TGF 3501 deleted mice have hematopoietic and angiogenic deficiencies, TGF β deleted mice show defects in pulmonary development and palate formation, TGF β 2 deleted mice show various developmental abnormalities, most notably various cardiac malformations (Bartram et al, circulation.2001; 103: 2745-52; Yamagishi et al, Anat rec.2012; 295: 257-67). furthermore, TGF 362 also plays an important role in the repair of myocardial injury after ischemia and reperfusion injury in adult hearts, TGF cell secretion β maintains spontaneous secretion rates as a cardiac autocrine, TGF cell secretion rates are important, TGF cell secretion rates are β -4835, TGF kinase inhibitors are observed with the same rat toxicity, but TGF kinase inhibitors are used to treat heart diseases including heart attack, kidney disease, heart disease, kidney disease, heart disease, kidney.

The natural form of TGF β RIII is, however, 280-330kD glycosaminoglycan (GAG) -glycoprotein, an extracellular domain of 762 amino acid residues, a protein that is very complex for biotherapeutic development.GAG-depleted soluble TGF β RIII can be produced in insect cells and shown to be an effective TGF β neutralizing agent (Vilchis-Landers et al, Biochem J.355:215,2001). two separate binding domains of TGF β RIII (endoglin) related and uromodulin related) can each be expressed independently, but their affinities are 20 to 100 times lower than those of soluble TGF β RIII, and the neutralizing activity is also much lower (Mendoza et al, biochemistry 2009.2009: β 8, 55. RII) as the only extracellular domain of amino acid 136, in the receptor trap methodResidues, which can be produced as 25-35kD glycoproteins it has also been shown that recombinant soluble TGF β RII has a K of 200pM_DBinding TGF β 1, which is very similar to the KD for full-length TGF β RII 50pM on cells (Lin et al, J Biol chem.1995; 270: 2747-54.) soluble TGF β 0RII-Fc, tested as an anti-cancer agent, appeared to be able to inhibit the growth of established murine malignant mesothelioma in tumor models (Suzuki et al, clin. cancer res.2004; 10: 5907-18). since TGF β 1RII does not bind TGF β 2, TGF β RII binds TGF β 1 and 3 with lower affinity than TGF β RII, so a fusion Protein of the endoglin domain of TGF β RIII with TGF β RII ectodomain was produced in bacteria that appeared to inhibit signaling of TGF β 1 and 2 in cell experiments more effectively than TGF β RII or RIII (Verona et al, Protein degs Des. en. 21: 463-73).

Another approach to neutralizing all three isoforms of TGF β ligand is to screen for broadly neutralizing anti-TGF β antibodies or anti-receptor antibodies that block receptor binding to TGF β,2 and 3 GC1008 is a human antibody specific for all TGF β 1 isoforms, phase I/II studies that have been carried into patients with advanced malignant melanoma or renal cell carcinoma (Morris et al, J Clin Oncol 2008; 26: 9028 (conference Abstract)), although this treatment was found to be safe and well tolerated, only limited clinical effects were observed, and therefore it was difficult to explain the importance of anti-TGF β treatment without further characterization of immune effects (Flavelll et al, Nat RevImmunol.2010; 10:554-67) also TGF β 3 isoform specific antibodies into clinical trials.TGF β -specific melissa (TGF-. beta.) that was used to prevent scar formation after glaucoma over surgery; TGF β) 2-specific melissa (TGF-. beta.) was found to be highly effective in phase 2 clinical trials; TGF-. beta.7, 160. TGF-. beta.8, no more effective in anti-TGF-. beta.: anti-TGF-. beta. (human sperm) antibody targeting TGF-. beta.: No. 7, 2-7, No. 5. No. 7, No. 5. the anti-5. TGF-5. the anti-TGF-5-inflammatory disease, No. 5. the anti-TGF-III-beta. the anti-TGF-beta. the anti-TGF-.

The antibody TGF β trap of the present disclosure is a bifunctional protein comprising at least part of human TGF β 0 receptor II (TGF β 1RII) capable of binding TGF β in certain embodiments, a TGF β 2 trap polypeptide is a soluble portion of human TGF β 24 receptor type 2 isoform A (SEQ ID NO:8) capable of binding TGF β in certain embodiments, a TGF β trap polypeptide comprises at least amino acids 73-184 of SEQ ID NO:8 in certain embodiments, a TGF 7 trap polypeptide comprises amino acids 24-184 of SEQ ID NO:8 in certain embodiments, a TGF β trap polypeptide is a soluble portion of human TGF 2 receptor type β receptor type B (SEQ ID NO:9) capable of binding TGF β in certain embodiments, a TGF β trap polypeptide comprises at least amino acids 48-159 of SEQ ID NO:9 in certain embodiments, a TGF β trap polypeptide comprises amino acids 24-159 of SEQ ID NO:9 in certain embodiments, a TGF β trap polypeptide comprises amino acids 24-159 of SEQ ID NO:9 in certain embodiments.

Mechanism of action

Targeting T cells with therapeutic antibodies to inhibit checkpoints to de-inhibit (dis-inhibition) is an area of intense research (reviewed in pardol, Nat Rev cancer.2012; 12: 253-264). In one of the approaches, the antibody moiety or antigen binding fragment thereof targets T cells on T cells to inhibit checkpoint receptor proteins, such as: CTLA-4, PD-1, BTLA, LAG-3, TIM-3 or LAIR 1. In another approach, the antibody moiety targets counter-receptors (counter-receptors) on antigen presenting cells and tumor cells (which also select some of these counter-receptors for their own immune escape), such as: PD-L1(B7-H1), B7-DC, HVEM, TIM-4, B7-H3 or B7-H4.

To this end, applicants tested the anti-tumor effect of TGF β wells in combination with antibodies targeting multiple T cell inhibitory checkpoint receptor proteins (e.g., anti-PD-1, anti-PD-L1, anti-TIM-3, and anti-LAG 3).

The programmed death 1(PD-1)/PD-L1 axis is an important mechanism for tumor immune escape, long-term induction of antigen effector T cells exhibit depletion phenotype expressed by PD-1 as a marker, this is a state closely related to tumor cells by upregulating PD-L1 additionally, in the tumor microenvironment, bone marrow cells, macrophages, parenchymal cells and T cells upregulate PD-L1 blocking this axis restores effector functions in these T cells, anti-PD-L1/TGF β trap also binds TGF β (1, 2 and 3 isoforms), which is an inhibitory cytokine produced by cells including apoptotic neutrophils, myeloid-derived suppressor cells, T cells and tumors in the tumor microenvironment. inhibition of TGF 26 by soluble TGF β RII reduces malignant mesothelioma in a manner related to increased anti-tumor effects of CD8+ T cells.

TGF β has a growth inhibitory effect on normal epithelial cells, acts as a modulator of epithelial cell homeostasis, and acts as a tumor suppressor during early carcinogenesis the growth inhibitory effect of TGF β on tumors is lost through mutations in one or more TGF β pathway signaling components or through oncogenic reprogramming as tumors progress to malignancy, tumors continue to produce high levels of TGF β after losing sensitivity to TGF β inhibition, which is then used to promote tumor growth TGF β cytokines are overexpressed in various cancer types, associated with the staging of tumors TGF 86535 is produced by many types of cells in the tumor microenvironment, including the tumor cells themselves, immature bone marrow cells, regulatory T cells and stromal fibroblasts, which together produce large TGF 6 s in the extracellular matrix, TGF β signaling promotes tumor progression by promoting metastasis, stimulating angiogenesis and inhibiting innate and adaptive anti-tumor immunity strategies.

In view of the emerging blue map of the PD-1/PD-L1 class, where the response is significant but has space to increase the size of the effect, it is hypothesized that a co-targeted complementary immunomodulatory step will improve the tumor response.a similar TGF- β targeting agent, frasolimumab (fresolimumab), a monoclonal antibody targeting TGF β,2 and 3, shows preliminary evidence of tumor response in phase I trials in subjects with melanoma.

In certain embodiments, the present disclosure provides experiments demonstrating that the TGF β RII portion of the anti-PD-L1/TGF β well (well control "anti-PDL-1 (mut)/TGF β well") elicits anti-tumor activity, for example, the anti-PDL-1 (mut)/TGF β well causes a dose-dependent decrease in tumor volume when administered at 25 μ g, 76 μ g, or 228 μ g following subcutaneous implantation in the detroit 562 human pharyngeal cancer model (fig. 5).

In certain embodiments, the present disclosure provides experiments demonstrating that the proteins of the present disclosure bind to both PD-L1 and TGF β (fig. 2).

In certain embodiments, the present disclosure provides an experiment demonstrating that a protein of the present disclosure (e.g., an anti-PD-L1/TGF β trap) inhibits PD-L1 and TGF β -dependent signaling in vitro in certain embodiments, the present disclosure provides an experiment demonstrating that a protein of the present disclosure enhances T cell effector function in vitro by blocking PD-L1-mediated immunosuppression, as measured by an IL-2 induction assay following superantigen stimulation (fig. 3) at about 100ng/ml, a protein of the present disclosure induces a significant increase in IL-2 levels in vitro (fig. 3).

In certain embodiments, the disclosure provides experiments demonstrating that proteins of the disclosure (e.g., anti-PD-L1/TGF β trap) cause depletion of TGF β in the blood in vivo treatment of EMT-6 breast cancer cells implanted in situ in JH mice with 55 μ g, or 164 μ g, or 492 μ g of the proteins of the disclosure results in efficient and specific elimination of TGF β 1 (fig. 4A), TGF β 2 (fig. 4B), and TGF β 3 (fig. 4C) additionally, the disclosure provides experiments demonstrating that the proteins of the disclosure occupy the PD-L1 target, supporting the concept that the proteins of the disclosure fit the receptor binding model in the EMT-6 tumor system (fig. 4D).

In certain embodiments, the present disclosure provides experiments demonstrating that the proteins of the present disclosure bind PD-L1 and TGF β efficiently, specifically, and simultaneously, with potent antitumor activity, inhibit tumor growth and metastasis, and prolonged survival and confer long-term protective antitumor immunity in various mouse models.

In addition to monitoring the pharmacokinetics of the anti-PD-L1/TGF β trap molecule in the first human phase I dose escalation study, the mechanism of action, particularly on TGF β cytokines, was also studied.

Patients are administered intravenously anti-PD-L1/TGF β trap molecules at 5 dose levels of about 0.3, about 1, about 3, about 10, or about 20mg/kg once every two weeks, with PK analysis from the samples up to day 85. blood levels of TGF β -3 and proinflammatory cytokines are measured at these time points with additional time points of D8 by flow cytometry from patient blood collected at day 2 (D2), D15, and D43 before dosing.

The results show that anti-PD-L1/TGF β trap molecule PK exposure increases between 3 and 20mg/kg in an approximately dose proportional manner in the first cycle, with no significant accumulation within the first 85 days of treatment.during the entire dosing interval, approximately 80% PD-L1 target occupancy is maintained at 3mg/kg-20 mg/kg.a small fraction (1.7 times over D2) but significant IFN γ induction (p 0.001, n 19) is also maintained at 0.3-20 mg/kg. TGF β, TGF β 2 and TGF β 3 levels in blood are reduced by a minimum of 99%, 92% and 91%, respectively, at lower doses of 0.3mg/kg, TGF β 1-3 levels are depleted at D2 and D8, but not at D15. furthermore, there is a strong correlation between the drug levels and TGF 23 levels and thus a higher PK capture level is achieved at full TGF 3 mg/kg.368651.

anti-PD-L1 antibody

The present disclosure may 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, lot 329701). The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody. Antibody fragments include Fab, F (ab') 2, scFv and Fv fragments, as described in more detail below.

Exemplary antibodies can be found in PCT publication WO 2013/079174. These antibodies may comprise a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(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-H1 sequence is IKLGTVTGVX₆Y(SEQ ID NO:23)；

Wherein: x₁Is K, R, T, Q, G, A, W, M, I or S; x₂Is V, R, K, L, M or I; x₃Is H, T, N, Q, A, V, Y, W, F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D.

In one embodiment, X₁Is M, I or S; x₂Is R, K, L, M or I; x₃Is F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D.

In another embodiment X₁Is M, I or S; x₂Is L, M or I; x₃Is F or M; x₄Is I; x₅Is S or T; x₆Is D.

In another embodiment, X₁Is S; x₂Is I; x₃Is M; x₄Is I; x₅Is T; x₆Is D.

In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence located between HVRs, as shown below: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR 4).

In another aspect, the framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

In another aspect, at least one of the framework 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). In another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2, and HVR-L3, wherein:

(a) the HVR-L1 sequence is TGTX₇X₈DVGX₉YNYVS(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)；

Wherein: x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is I, N or S; x₁₂Is D, H or N; x₁₃Is F or Y; x₁₄Is N or S; x₁₅Is R, T or S; x₁₆Is G or S; x₁₇Is I or T.

In another embodiment, X₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is N or S; x₁₂Is N; x₁₃Is F or Y; x₁₄Is S; x₁₅Is S; x₁₆Is G or S; x₁₇Is T.

In another embodiment, X₇Is S; x₈Is S; x₉Is G; x₁₀Is D; x₁₁Is S; x₁₂Is N; x₁₃Is Y; x₁₄Is S; x₁₅Is S; x₁₆Is S; x₁₇Is T.

In another aspect, the light chain further comprises a variable region light chain framework sequence located between the HVRs, as shown below: (LC-FR1MHVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

In another aspect, the light chain framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

In another aspect, the light chain framework sequence is a lambda light chain sequence.

In another aspect, at least one of the framework 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).

In another embodiment, the present disclosure provides an anti-PD-L1 antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, and wherein: (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 IKLGTVTGVX₆Y (SEQ ID NO:23), and; (b) the light chain includes HVR-L1, HVR-L2, and HVR-L3, and wherein: (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); wherein: x₁Is K, R, T, Q, G, A, W, M, I, or S; x₂Is V, R, K, L, M, or I; x₃Is H, T, N, Q, A, V, Y, W, F, or M; x₄Is F or I; x₅Is S or T; x₆Is E or D; x₇Is N or S; x₈Is a combination of T, R,or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is I, N, or S; x₁₂Is D, H, or N; x₁₃Is F or Y; x₁₄Is N or S; x₁₅Is R, T, or S; x₁₆Is G or S; x₁₇Is I or T.

In one embodiment, X₁Is M, I or S; x₂Is R, K, L, M or I; x₃Is F or M; x₄Is F or I; x₅Is S or T; x₆Is E or D; x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is N or S; x₁₂Is N; x₁₃Is F or Y; x₁₄Is S; x₁₅Is S; x₁₆Is G or S; x₁₇Is T.

In another embodiment, X₁Is M, I or S; x₂Is L, M or I; x₃Is F or M; x₄Is I; x₅Is S or T; x₆Is D; x₇Is N or S; x₈Is T, R or S; x₉Is A or G; x₁₀Is E or D; x₁₁Is N or S; x₁₂Is N; x₁₃Is F or Y; x₁₄Is S; x₁₅Is S; x₁₆Is G or S; x₁₇Is T.

In another embodiment, X₁Is S; x₂Is I; x₃Is M; x₄Is I; x₅Is T; x₆Is D; x₇Is S; x₈Is S; x₉Is G; x₁₀Is D; x₁₁Is S; x₁₂Is N; x₁₃Is Y; x₁₄Is S; x₁₅Is S; x₁₆Is S; x₁₇Is T.

In another aspect, the heavy chain variable region comprises one or more framework sequences located between HVRs as shown below: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and said light chain variable region comprises one or more framework sequences located between HVRs as shown below: (LC-FR1MHVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

In another aspect, the framework sequence is derived from a human consensus framework sequence or a human germline sequence.

In another aspect, one or more of the heavy chain framework sequences are 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).

In another aspect, one or more of the light chain framework sequences are 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).

In another aspect, the heavy chain variable region polypeptide, antibody or antibody fragment further comprises at least C_H1 domain.

In a more specific aspect, the heavy chain variable region polypeptide, antibody or antibody fragment further comprises C_H1、C_H2 and C_H3 domain.

In another aspect, the variable region light chain, antibody or antibody fragment further comprises C_LA domain.

In another aspect, the antibody isComprising C_H1、C_H2、C_H3 and C_LA domain.

In another more specific aspect, the antibody further comprises a human or murine constant region.

In another aspect, the human constant region is selected from the group consisting of: IgG1, IgG2, IgG2, IgG3, IgG 4.

In a more specific aspect, the human or murine constant region is lgG 1.

In another embodiment, the disclosure features an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, which have at least 80% overall sequence identity to SYIMM (SEQ ID NO:35), SIYPSGGITFYADTVKG (SEQ ID NO:36), and IKLGTVTTVDY (SEQ ID NO:37), respectively, and

(b) the light chain includes HVR-L1, HVR-L2, and HVR-L3, which have at least 80% overall sequence identity to TGTSSDVGGYNYVS (SEQ ID NO:38), DVSNRPS (SEQ ID NO:39), and SSYTSSSTRV (SEQ ID NO:40), respectively.

In a particular 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%.

In another embodiment, features of the disclosure include an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, which have at least 80% overall sequence identity to MYMMM (SEQ ID NO:41), SIYPSGGITFYADSVKG (SEQ ID NO:42), and IKLGTVTTVDY (SEQ ID NO:37), respectively, and

(b) the light chain includes HVR-L1, HVR-L2, and HVR-L3, which have at least 80% overall sequence identity with TGTSSDVGAYNYVS (SEQ ID NO:43), DVSNRPS (SEQ ID NO:39), and SSYTSSSTRV (SEQ ID NO:40), respectively.

In another aspect, at least those amino acids highlighted by underlining as shown below remain unchanged compared to the sequences of HVR-H1, HVR-H2, and HVR-H3 in the antibodies or antibody fragments of the present disclosure:

(a) in HVR-H1SYIMIn M (SEQ ID NO:35),

(b) in HVR-H2SIYPSGGITFYADTVKG(SEQ ID NO:36) in the sequence,

(c) in HVR-H3IKLGTVTTVDY(SEQ ID NO: 37);

and wherein at least those amino acids highlighted by underlining as shown below remain unchanged compared to the sequences of HVR-L1, HVR-L2, and HVR-L3:

(a)HVR-L1 TGTSSDVGGYNYVS(SEQ ID NO:38)

(b)HVR-L2 DVSNRPS(SEQ ID NO:39)

(c)HVR-L3 SSYTSSSTRV(SEQ ID NO:40)。

in another aspect, the heavy chain variable region comprises one or more framework sequences located between HVRs as shown below: (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4), and said light chain variable region comprises one or more framework sequences located between HVRs as shown below: (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4).

In another aspect, the framework sequence is derived from a human germline sequence.

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).

In another aspect, the light chain framework sequence is derived from a lambda light chain sequence.

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).

In certain embodiments, features of the present disclosure include an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of seq id no:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMVWRQAPGKGLEWVSSIYPSGGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS (SEQ ID NO:44), and

(b) the light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 45). In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In certain embodiments, the present disclosure provides an anti-PD-L1 antibody comprising heavy and light chain variable region sequences, wherein:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEVWSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWG QGTLVTVSS (SEQ ID NO:46), and

QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 47). In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In another embodiment, the antibody binds human, mouse, or cynomolgus PD-L1. In a particular aspect, the antibody is capable of blocking the interaction between human, mouse or cynomolgus PD-L1 and the corresponding human, mouse or cynomolgus PD-1 receptor.

In another embodiment, the antibody is at 5x10^-9M or lower K_DPreferably at 2x10^-9M or lower K_DEven more preferably at 1x10^-9M or lower K_DBinds to human PD-L1.

In another embodiment, the disclosure relates to an anti-PD-L1 antibody or antigen-binding fragment thereof that binds to a functional epitope comprising residues Y56 and D61 of human PD-L1.

In a specific aspect, the functional epitope further comprises E58, E60, Q66, R113, and M115 of human PD-L1.

In a more specific aspect, the antibody binds to a conformational epitope comprising residues 54-66 and 112-122 of human PD-L1.

In certain embodiments, the disclosure relates to an anti-PD-L1 antibody or antigen-binding fragment thereof that cross-competes for binding to PD-L1 with an antibody of the disclosure described herein.

In certain embodiments, the disclosure features proteins and polypeptides comprising any of the above anti-PD-L1 antibodies, in combination with at least one pharmaceutically acceptable carrier.

In certain embodiments, the disclosure features an isolated nucleic acid encoding a polypeptide, or a light chain or heavy chain variable region sequence, of an anti-PD-L1 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the present disclosure provides an isolated nucleic acid encoding a light chain or heavy chain variable region sequence of an anti-PD-L1 antibody, wherein:

(a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3 sequences that have 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 HVR-L1, HVR-L2, and HVR-L3 sequences that have at least 80% sequence identity to TGTSSDVGGYNYVS (SEQ ID NO:38), DVSNRPS (SEQ ID NO:39), and SSYTSSSTRV (SEQ ID NO:40), respectively.

In another aspect, the nucleic acid sequence of the heavy chain is:

(SEQ ID NO:48)

and the nucleic acid sequence of the light chain is:

(SEQ ID NO:49).

other exemplary anti-PD-L1 antibodies that can be used in an anti-PD-L1/TGF β trap can be found in U.S. patent application publication No. US 2010/0203056 in one of the embodiments of the disclosure, the antibody moiety is yw243.55s70 in another embodiment of the disclosure, the antibody moiety is MPDL 3289A.

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:12), and

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:13)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:14), and

other exemplary anti-PD-L1 antibodies that may be used in anti-PD-L1/TGF β traps may be found in U.S. patent publication No. US 7,943,743.

In one embodiment of the disclosure, the anti-PD-L1 antibody is MDX-1105.

In certain embodiments, the anti-PD-L1 antibody is MEDI-4736.

Constant region

The proteins and peptides of the present disclosure may comprise a constant region of an immunoglobulin, 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, such as IgG1, IgG2, IgG3, IgG4, or other species. In certain embodiments, the constant region comprises a CH2 domain. In other certain embodiments, the constant region comprises CH2 and CH3 binding domains or comprises the hinge-CH 2-CH 3. Alternatively, the constant region may comprise all or part of the hinge region, the CH2 domain, and/or the CH3 domain.

In one embodiment, the constant region comprises a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region may comprise a mutation that eliminates a glycosylation site in the IgG heavy chain constant region. In some embodiments, the constant region comprises a mutation, deletion, or insertion at an amino acid position corresponding to Leu234, Leu235, Gly236, Gly237, Asn297, or Pro331 of IgG1 (amino acids numbered according to EU nomenclature). In a specific embodiment, the constant region contains a mutation at the amino acid position corresponding to Asn297 of IgG 1. In another embodiment, the constant region comprises a mutation, deletion or insertion of an amino acid position corresponding to Leu281, Leu282, Gly283, Gly284, Asn344 or Pro378 of IgG 1.

In some embodiments, the constant region comprises a CH2 domain derived from a human IgG2 or IgG4 heavy chain. Preferably, the CH2 domain comprises a mutation that eliminates the glycosylation site in the CH2 domain. In one embodiment, the mutation alters an asparagine within the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence within the CH2 domain of the IgG2 or IgG4 heavy chain. Preferably, the mutation changes asparagine to glutamine. Alternatively, the mutation alters both phenylalanine and asparagine within the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence. In one embodiment, the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence is substituted with a Gln-Ala-Gln-Ser (SEQ ID NO:16) amino acid sequence. The asparagine within the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence corresponds to Asn297 of IgG 1.

In another embodiment, the constant region comprises a CH2 domain and at least a portion of a hinge region. The hinge region may be derived from an immunoglobulin heavy chain such as IgG1, IgG2, IgG3, IgG4, or other species. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other species. More preferably, the hinge region is derived from the heavy chain of human IgG 1. In one embodiment, the cysteine in the IgG1 hinge region Pro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO:17) amino acid sequence is altered. In certain embodiments, the Pro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO:17) amino acid sequence is replaced by a Pro-Lys-Ser-Ser-Asp-Lys (SEQ ID NO:18) amino acid sequence. In certain embodiments, the constant region comprises 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, and the hinge region is derived from an altered human IgG1 heavy chain.

Amino acid changes near the junction of the Fc portion and the non-Fc portion significantly increase the serum half-life of the Fc fusion protein (PCT publication WO 01/58957, the disclosure of which is incorporated herein by reference). Thus, the linking region of a protein or polypeptide of the present disclosure may contain alterations relative to the immunoglobulin heavy chain and erythropoietin native sequences, preferably within about 10 amino acids from the point of attachment. These amino acid changes result in increased hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which a C-terminal lysine residue is substituted. Preferably, the C-terminal lysine of the IgG sequence is replaced with a non-lysine amino acid (e.g., alanine or leucine) to further increase serum half-life. In another embodiment, the constant region is derived from an IgG sequence, wherein the Leu-Ser-Leu-Ser (SEQ ID NO:19) amino acid sequence near the C-terminus of the constant region has alterations that eliminate potential conjugative T cell epitopes. For example, in one embodiment, the Leu-Ser-Leu-Ser (SEQ ID NO:19) amino acid sequence is substituted with an Ala-Thr-Ala-Thr (SEQ ID NO:20) amino acid sequence. In other embodiments, amino acids within the Leu-Ser-Leu-Ser (SEQ ID NO:19) segment are replaced with other amino acids such as glycine or proline. Methods for making amino acid substitutions in the Leu-Ser-Leu-Ser (SEQ ID NO:19) segment near the C-terminus of IgG1, IgG2, IgG3, IgG4, or other immunoglobulin molecules are described in detail in U.S. patent publication No. 20030166877, the disclosure of which is incorporated herein by reference.

Suitable hinge regions of the present disclosure may be derived from IgG1, IgG2, IgG3, IgG4, and other immunoglobulin classes. The IgG1 hinge region has three cysteines, two of which are involved in the disulfide bonds between the two heavy chains of immunoglobulins. These same cysteines allow efficient and consistent disulfide bond formation between the Fc portions. Thus, the hinge region of the present disclosure is derived from IgG1, e.g., human IgG 1. In a preferred embodiment, the first cysteine in the hinge region of human IgG1 is mutated to another amino acid, preferably serine. The hinge region of the IgG2 isotype has four disulfide bonds, which tend to contribute to oligomerization and possibly incorrect disulfide bonds during secretion of the recombinant system. Suitable hinge regions may be derived from the IgG2 hinge, where the first two cysteines are each mutated to other amino acids. The hinge region of IgG4 is known to be less effective in forming interchain disulfide bonds. However, suitable hinge regions of the present disclosure may be derived from the IgG4 hinge region, preferably containing mutations that enhance the correct disulfide bond formation between heavy chain derived portions (Angal S et al, (1993) mol.Immunol.,30: 105-8).

According to the present disclosure, the constant region may comprise CH2 and/or CH3 domains and a hinge region, i.e., a hybrid (hybrid) constant region, derived from different antibody isotypes. For example, in one embodiment, the constant region comprises a CH2 and/or CH3 domain derived from IgG2 or IgG4 and a mutated hinge region derived from IgG 1. Alternatively, mutant hinge regions derived from other IgG subclasses may be employed in the hybrid constant region. For example, a mutated version of the IgG4 hinge can be used to effect disulfide bond bonding between two heavy chains. Mutant hinges may also be derived from the IgG2 hinge, in which the first two cysteines are each mutated to other amino acids. The assembly of hybrid constant regions can be found in U.S. patent publication 20030044423, the disclosure of which is incorporated herein by reference.

According to the present disclosure, the constant region may comprise one or more of the mutations described herein. The combination of mutations in the Fc portion has additive or synergistic effects on extending serum half-life and increasing potency of the bifunctional molecule in vivo. Thus, in one exemplary embodiment, the constant region may comprise (i) a region derived from an IgG sequence in which the Leu-Ser-Leu-Ser (SEQ ID NO:19) amino acid sequence is replaced with an Ala-Thr-Ala-Thr (SEQ ID NO:20) amino acid sequence; (ii) a C-terminal alanine residue instead of lysine; (iii) CH2 domains and hinge regions derived from different antibody isotypes, such as an IgG2CH2 domain and an altered IgG1 hinge region; and (iv) a mutation that eliminates the glycosylation site within the IgG 2-derived CH2 domain, such as the Gln-Ala-Gln-Ser (SEQ ID NO:16) amino acid sequence within the IgG 2-derived CH2 domain rather than the Gln-Phe-Asn-Ser (SEQ ID NO:15) amino acid sequence.

Antibody fragments

The proteins and polypeptides of the present disclosure may also include antigen-binding fragments of antibodies. Exemplary antibody fragments include scFv, Fv, Fab, F (ab')₂And single domain VHH fragments, such as those from camelids.

Single-chain antibody fragments, also known asSingle chain antibodies (scFv), are recombinant polypeptides that typically bind to an antigen or receptor; these fragments comprise at least one antibody variable light chain sequence (V) linked with or without one or more interconnecting linkers_L) Fragment and at least one antibody variable heavy chain amino acid sequence (V)_H) And (3) fragment. Such linkers may be short flexible peptides selected to ensure correct three-dimensional folding after association of the VL and VH domains, thereby retaining the target molecule binding specificity of the whole antibody from which the single chain antibody fragment is derived. In general, V_LOr V_HThe carboxy terminus of the sequence is covalently linked to complementary V through such a peptide linker_LAnd V_HThe amino acid terminus of the sequence. Single chain antibody fragments may be generated by molecular cloning, antibody phage display or similar techniques. These proteins can be produced in eukaryotic cells as well as prokaryotic cells, including bacteria.

Single chain antibody fragments comprise amino acid sequences having at least one of the variable regions or CDRs of intact antibodies described herein, but lacking all or part of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute an integral part of the complete antibody structure. Thus, single chain antibody fragments may overcome some of the problems associated with the use of antibodies comprising part or all of the constant region. For example, single chain antibody fragments tend to be free of undesired interactions or other undesired biological activities between biomolecules and heavy chain constant regions. Furthermore, single chain antibody fragments are much smaller than intact antibodies and therefore can have higher capillary permeability than intact antibodies, which enables the single chain antibody fragments to more efficiently address and bind to the target antigen binding site. Also, antibody fragments can be produced in prokaryotic cells on a relatively large scale, facilitating their production. Furthermore, the relatively small size of single chain antibody fragments makes them less likely to elicit an immune response in a recipient than intact antibodies.

Antibody fragments having the same or comparable binding characteristics as the intact antibody may also be present. Such fragments may contain one or two Fab fragments or F (ab')₂And (3) fragment. The antibody fragment may comprise the entirety of the intact antibodyFragments that contain six CDRs, but less than all of these regions, e.g., three, four, or five CDRs, are also functional.

Pharmaceutical composition

The disclosure also features pharmaceutical compositions comprising a therapeutically effective amount of a protein described herein. The compositions can be formulated to be suitable for use in a variety of drug delivery systems. The compositions may also contain one or more physiologically acceptable excipients or carriers to make suitable formulations. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, 17 th edition, Mark Publishing Company (Mack Publishing Company), 1985, of Iston, Pa. For reviews on drug delivery methods see, for example, Langer (Science249: 1527) -1533, 1990).

In one aspect, the present disclosure provides an intravenous drug delivery formulation comprising 500mg-2000mg of a protein comprising a first polypeptide comprising (a) a heavy chain variable region of an antibody that binds to at least human protein programmed death ligand 1(PD-L1), and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment thereof capable of binding transforming growth factor β (TGF β), and a second polypeptide comprising at least a light chain variable region of an antibody 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 PD-L1.

In certain embodiments, a protein product of the present disclosure comprises a first polypeptide comprising a polypeptide of SEQ ID NO:3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

In certain embodiments of the disclosure, an intravenous drug delivery formulation may comprise about 500mg to about 2400mg (e.g., about 500mg to about 2300mg, about 500mg to about 2200mg, about 500mg to about 3000mg, about 500mg to about 2000mg, about 500mg to about 3000mg, about 1200mg, about 3000mg, about 1200mg, about 3000mg, about 1200mg, about 2000 to about 3000mg, about 1, about 3000mg, about 200mg, about 1, about 3000mg, about 200mg, about 3000mg, about 1, about 200mg, about 3000mg, about 3000mg, about 1, about 1, about 3000, about 3000mg, about 3000mg, about 3000, about.

In certain embodiments, the intravenous drug delivery formulation may comprise about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, about 1300mg, about 1325mg, about 1350mg, about 1375mg, about 1400mg, about 1425mg, about 1450mg, about 1475mg, about 1500mg, about 1525mg, about 1550mg, about 1575mg, about 1600mg, about 1800mg, about 1650mg, about 1725mg, about 1720 mg, about 1000mg, about 20mg, about 1500mg, about 20mg, about 1525mg, about 20mg, about 1000mg, about 20 mg.

The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen or syringe. In certain embodiments, the bag may be connected to a channel comprising a tube and/or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the formulation may be lyophilized, and about 45mg of the lyophilized formulation may be contained in one vial. In certain embodiments, about 40mg to about 100mg of the lyophilized formulation may be contained in one vial. In certain embodiments, freeze-dried preparations from 12, 27 or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous pharmaceutical preparation. In certain embodiments, the formulation can be a liquid formulation of a protein product having a protein sequence comprising SEQ id no:3, and a first polypeptide comprising the amino acid sequence of SEQ ID NO:1 and stored from about 250 mg/vial to about 2000 mg/vial (e.g., about 250 mg/vial to about 2000 mg/vial, about 250 mg/vial to about 1900 mg/vial, about 250 mg/vial to about 1800 mg/vial, about 250 mg/vial to about 1700 mg/vial, about 250 mg/vial to about 1600 mg/vial, about 250 mg/vial to about 1500 mg/vial, about 250 mg/vial to about 1400 mg/vial, about 250 mg/vial to about 1300 mg/vial, about 250 mg/vial to about 1200 mg/vial, about 250 mg/vial to about 1100 mg/vial, about 250 mg/vial to about 1000 mg/vial, about 250 mg/vial to about 900 mg/vial, about 250 mg/vial to about 800 mg/vial, from about 250 mg/vial to about 700 mg/vial, from about 250 mg/vial to about 600 mg/vial, from about 250 mg/vial to about 500 mg/vial, from about 250 mg/vial to about 400 mg/vial, from about 250 mg/vial to about 300 mg/vial, from about 300 mg/vial to about 2000 mg/vial, from about 400 mg/vial to about 2000 mg/vial, from about 500 mg/vial to about 2000 mg/vial, from about 600 mg/vial to about 2000 mg/vial, from about 700 mg/vial to about 2000 mg/vial, from about 800 mg/vial to about 2000 mg/vial, from about 900 mg/vial to about 2000 mg/vial, from about 1000 mg/vial to about 2000 mg/vial, from about 1100 mg/vial to about 2000 mg/vial, from about 1200 mg/vial to about 2000 mg/vial, from about 1300 mg/vial to about 2000 mg/vial, from about 1400 mg/hr vial to about 2000 mg/vial, from about 1500 mg/vial to about 2000 mg/vial, from about 1600 mg/vial to about 2000 mg/vial, from about 1700 mg/vial to about 2000 mg/vial, from about 1800 mg/vial to about 2000 mg/vial, or from about 1900 mg/vial to about 2000 mg/vial). In certain embodiments, the formulation may be a liquid formulation and stored at about 600 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 1200 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 1800 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 250 mg/vial.

The present disclosure provides liquid aqueous pharmaceutical formulations comprising a therapeutically effective amount of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap) in a buffered solution forming the formulation.

These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged as is ("use as-is") type product or lyophilized, the lyophilized formulation being combined with a sterile aqueous carrier prior to administration. The pH of the formulation is generally between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, e.g.between 7 and 7.5. The resulting composition in solid form may be packaged as a plurality of single dose units, each unit dose unit containing a fixed amount of one or more of the agents described above. The composition in solid form can also be packaged in containers to obtain flexible amounts.

In certain embodiments, the present disclosure provides formulations having extended shelf life, comprising a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)), with mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

In certain embodiments, an aqueous formulation comprising a protein of the present disclosure in a pH buffered solution is prepared (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). the pH of the buffer of the present disclosure can be from about 4 to about 8, e.g., from about 4 to about 8, from about 4.5 to about 8, from about 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 5.5 to about 7.5, from about 6 to about 6.5, from about 4 to about 7, from about 4.5 to about 7, from about 5 to about 7.5, from about 6.5 to about 6.5, from about 4 to about 7.5, from about 5 to about 6.5, from about 5 to about 5.5, from about 5 to about 6.5, from about 5 to about 5, as an example, or as an alternative, a pH buffering agent that can be included within the range of the pH buffered sodium acetate buffer of the aforementioned organic buffer, a pH buffer that is also included as an upper limit, a buffer of the range of the pH buffer of the organic buffer of the pH-L865, or a buffer of the invention.

In certain embodiments, the formulation includes a buffer system comprising citrate and phosphate to maintain the pH in the range of about 4 to about 8. In certain embodiments, the pH range may be from about 4.5 to about 6.0, or from about pH4.8 to about 5.5, or in the pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3mg/ml citric acid (e.g., 1.305mg/ml), about 0.3mg/ml sodium citrate (e.g., 0.305mg/ml), about 1.5mg/ml dibasic sodium phosphate dihydrate (e.g., 1.53mg/ml), about 0.9mg/ml monobasic sodium phosphate dihydrate (e.g., 0.86), and about 6.2mg/ml sodium chloride (e.g., 6.165 mg/ml). In certain embodiments, the buffer system comprises about 1-1.5mg/ml citric acid, about 0.25 to about 0.5mg/ml sodium citrate, about 1.25 to about 1.75mg/ml dibasic sodium phosphate dihydrate, about 0.7 to about 1.1mg/ml monobasic sodium phosphate dihydrate, and 6.0 to 6.4mg/ml sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

Polyols that act as conditioning agents and can stabilize antibodies may also be included in the formulation. The polyol is added to the formulation in an amount that can vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also vary relative to the molecular weight of the polyol. For example, a lower amount of monosaccharide (e.g., mannitol) may be added as compared to a disaccharide (e.g., trehalose). In certain embodiments, the polyol that can be used as a tonicity agent in the formulation is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg/ml. In certain embodiments, the mannitol concentration may be about 7.5 to about 15 mg/ml. In certain embodiments, the mannitol concentration may be about 10 to about 14 mg/ml. In certain embodiments, the mannitol concentration may be about 12 mg/ml. In certain embodiments, the polyol sorbitol may be included in the formulation.

Detergents or surfactants may also be added to the formulation. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and/or minimizes particle formation 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 the detergent polysorbate 80 or tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, encyclopedia of excipients (Lexikon der Hilfsstuffe), edition published by Editio Cantor Verlag Aulendorf, 4 th edition, 1996). In certain embodiments, the formulation may contain from about 0.1mg/mL to about 10mg/mL, or from about 0.5mg/mL to about 5mg/mL of polysorbate 80. In certain embodiments, polysorbate 80 may be added to the formulation at about 0.1%.

Freeze-dried preparation

Lyophilized formulations of the present disclosure include an anti-PD-L1/TGF β trap molecule and a lyoprotectant.

The amount of sucrose or maltose that can be used to stabilize the lyophilized pharmaceutical product can be at least 1: 2 to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose may be 1: 2 to 1: 5.

in certain embodiments, the pH of the formulation may be set by the addition of a pharmaceutically acceptable acid and/or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

Prior to lyophilization, the pH of a solution containing a protein of the present disclosure may be adjusted between about 6 to about 8. In certain embodiments, the pH of the lyophilized drug product can range from about 7 to about 8.

In certain embodiments, the salt or buffer component may be added in an amount of about 10mM to about 200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) having "base forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions may be used as counterions.

In certain embodiments, a "filler" may be added. A "bulking agent" is a compound that can increase the quality of the lyophilized mixture and aid in the physical structure of the lyophilized cake (e.g., aid in producing a substantially uniform lyophilized cake that retains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulation of the present invention may contain such a bulking agent.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multi-dose) formulation.

In certain embodiments, the lyophilized pharmaceutical product can be comprised of an aqueous carrier. Aqueous carriers of interest herein are pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) and can be used to prepare liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, ringer's solution, or dextrose solution.

In certain embodiments, the lyophilized pharmaceutical products of the present disclosure are reconstituted with sterile water for injection, USP (swfi) or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder dissolves into solution.

In certain embodiments, the lyophilized protein product of the present disclosure comprises about 4.5mL of water for injection and is diluted with 0.9% saline solution (sodium chloride solution).

Liquid preparation

In embodiments, the protein product of the present disclosure is formulated as a liquid formulation, the liquid formulation may be present at a concentration of 10mg/mL in a USP/Ph Eur type I50R vial, which is closed with a rubber stopper and sealed with an aluminum crimp stopper the stopper may be made of an elastomer conforming to USP and Ph Eur in certain embodiments, the vial may be filled with about 61.2mL of the protein product solution to allow for an extractable volume of 60mL, in certain embodiments, the liquid formulation may be diluted with a 0.9% saline solution, in certain embodiments, the vial may contain about 61.2mL of about 20mg/mL to about 50mg/mL (e.g., about 20 mg/2200 mL, about 25mg/mL, about 30mg/mL, about 35mg/mL, about 40mg/mL, about 45mg/mL or about 50mg/mL) of the protein product (e.g., anti-PD-L1/β (e.g., a first, comprising an amino acid sequence of SEQ ID: 3, and about 3000, about 3000mg, about 1200mg to 1200mg, about 3000mg of a polypeptide, about 3000mg to about 3000mg, about 3000mg to 3000, about 3000mg, about 3000mg to about 3000mg, about 3000mg of the polypeptide, about 3000.

In certain embodiments, a vial may contain about 61.2mL of about 20mg/mL to about 50mg/mL (e.g., about 20mg/mL, about 25mg/mL, about 30mg/mL, about 35mg/mL, about 40mg/mL, about 45mg/mL, or about 50mg/mL) of a protein product solution (e.g., a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1) to allow an extractable volume of 60mL for delivery of about 1200mg to about 3000mg (e.g., about 1200mg to about 3000mg, about 1200mg to about 2900mg, about 1200mg to about 2800mg, about 1200mg to about 2700mg, about 1200mg to about 2600mg, about 1200mg to about 2500mg, about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, from about 1200mg to about 2000mg, from about 1200mg to about 1900mg, from about 1200mg to about 1800mg, from about 1200mg to about 1700mg, from about 1200mg to about 1600mg, from about 1200mg to about 1500mg, from about 1200mg to about 1400mg, from about 1200mg to about 1300mg, from about 1300mg to about 3000mg, from about 1400mg to about 3000mg, from about 1500mg to about 3000mg, from about 1600mg to about 3000mg, from about 1700mg to about 3000mg, from about 1800mg to about 3000mg, from about 1900mg to about 3000mg, from about 2000mg to about 3000mg, from about 2100mg to about 3000mg, from about 2200mg to about 3000mg, from about 2300mg to about 3000mg, from about 2500mg to about 3000mg, from about 2600mg to about 3000mg, from about 2900mg to about 3000mg, from about 1200mg, from about 1300mg, from about 1600mg, about 1500mg, from about 3000mg to about 3000mg, from about 2100mg, from about 3000mg to about 3000mg, from about 3000mg about 3000, about 2700mg, about 2800mg, about 2900mg, or about 3000mg) of the protein product to a subject.

In certain embodiments, the liquid formulations of the present invention may be prepared as a 10mg/mL concentration solution in combination with a stabilizing level of sugar. In certain embodiments, the liquid formulation may be prepared in an aqueous vehicle. In certain embodiments, the stabilizer may be added in an amount no greater than that which would result in an undesirable viscosity or would not be suitable for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may further comprise one or more of a buffer, a surfactant, and a preservative.

In certain embodiments, the pH of the liquid formulation may be set by the addition of 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.

In addition to aggregation, deamidation is a common product variant of peptides and proteins, which can occur during fermentation, harvest/cell clarification, purification, drug/drug product storage and during sample analysis. Deamidation is the loss of NH from proteins that form succinimide intermediates that can undergo hydrolysis₃. The succinimide intermediate resulted in a 17u mass reduction of the parent peptide. Subsequent hydrolysis resulted in an 18u mass increase. Due to instability under aqueous conditions, it is difficult to isolate the succinimide intermediate. Thus, deamidation can generally be detected as a 1u mass increase. Deamidation of asparagine produces aspartic acid or isoaspartic acid. 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 the deamidation rate. Gly and Ser after Asn in the protein sequence lead to higher sensitivity to deamidation.

In certain embodiments, the liquid formulations of the present invention may be stored under conditions of pH and humidity to prevent deamination of the protein product.

The aqueous vehicles contemplated herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, ringer's solution, or dextrose solution.

In certain cases, Intravenous (IV) formulations may be the preferred route of administration, for example when a patient receives all drugs via the IV route in a hospital after transplantation. In certain embodiments, the liquid formulation is diluted with a 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted pharmaceutical product for injection is isotonic and suitable for administration by intravenous infusion.

Methods of treating cancer or inhibiting tumor growth

In one aspect, the present disclosure provides a method of treating cancer or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject a dose of at least 500mg of a protein comprising a first polypeptide comprising (a) a variable region of a heavy chain of an antibody that binds at least to human protein programmed death ligand 1(PD-L1) and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment thereof that is capable of binding transforming growth factor β (TGF β), a second polypeptide comprising at least a variable region of a light chain of an antibody 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 PD-L1.

In certain embodiments, the methods of the present disclosure for treating cancer or inhibiting tumor growth comprise administering to a subject a protein comprising two peptides, wherein a first polypeptide comprises the amino acid sequence of SEQ ID NO. 3 and a second polypeptide comprises the amino acid sequence of SEQ ID NO. 1.

In certain embodiments, a method of the disclosure for treating cancer or inhibiting tumor growth comprises administering to a subject a dose of about 1200mg to about 3000mg (e.g., about 1200mg to about 3000mg, about 1200mg to about 2900mg, about 1200mg to about 2800mg, about 1200mg to about 2700mg, about 1200mg to about 2600mg, about 1200mg to about 2500mg, about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, about 1200mg to about 2000mg, about 1200mg to about 1900mg, about 1200mg to about 1800mg, about 1200mg to about 1700mg, about 1200mg to about 1600mg, about 1200mg to about 1500mg, about 1200mg to about 1400mg, about 1200mg to about 1300mg, about 1300mg to about 1300mg, about 1400mg to about 1400mg, about 1400mg to about 3000mg, about 1500mg to about 3000mg, about 3000mg of a polypeptide sequence of a polypeptide in a TGF or about 1 or about 200 a polypeptide sequence of a polypeptide sequence in a TGF protein in a first or about 200 or about 20 or 3000, wherein a TGF protein sequence of a first or about 20 or about 9 or 3000, wherein a first or about polypeptide sequence of a first or about 3000, such as a polypeptide sequence of a polypeptide having a polypeptide sequence in a first or a polypeptide sequence of a first or a polypeptide sequence of a first or second embodiment of a first or a polypeptide of a first or second embodiment of a polypeptide of about 3000 of a first or.

In certain embodiments, the dose may be about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, about 1300mg, about 1325mg, about 1350mg, about 1375mg, about 1400mg, about 1425mg, about 1450mg, about 1475mg, about 1500mg, about 1525mg, about 1550mg, about 1575mg, about 1600mg, about 1800mg, about 1650mg, about 1725mg, about 1720 mg, about 170 mg, about 175mg, about 2100mg, about 2200mg, about 2300mg, or about 2400 mg.

In certain embodiments, the dose may be administered once every two weeks. In certain embodiments, the protein may be administered intravenously, for example, with a pre-filled bag, a pre-filled pen, or a pre-filled syringe. In certain embodiments, the protein is administered intravenously from a 250ml saline bag, and the intravenous infusion may last about 1 hour (e.g., 50 to 80 minutes). In certain embodiments, the bag is connected to a channel comprising a tube and/or a needle.

In certain embodiments, the method treats cancer or inhibits tumor growth, e.g., the cancer or tumor comprises: non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, glioblastoma, gastric cancer, biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), head and neck adenomas, head and neck squamous cell carcinoma, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma, merkel cell carcinoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. In certain embodiments, the method treats a cancer in a pre-treated patient, such as pre-treated non-small cell lung cancer, pre-treated melanoma, pre-treated pancreatic cancer, pre-treated colorectal cancer, pre-treated ovarian cancer, pre-treated breast cancer, pre-treated glioblastoma, pre-treated recurrent or refractory unresectable stage IV gastric cancer, pre-treated biliary tract cancer, pre-treated esophageal cancer (squamous cell carcinoma or adenocarcinoma), pre-treated head and neck adenoma, pre-treated head and neck squamous cell carcinoma, pre-treated prostate cancer, pre-treated renal cancer, pre-treated cervical cancer, pre-treated myeloma, pre-treated lymphoma, pre-treated leukemia, pre-treated thyroid cancer, pre-treated endometrial cancer, pre-treated uterine cancer, pre-treated bladder cancer, pre-treated neuroendocrine cancer, pre-treated liver cancer, pre-treated nasopharyngeal carcinoma, pre-treated testicular carcinoma, pre-treated small cell lung carcinoma, pre-treated basal cell skin carcinoma, pre-treated squamous cell skin carcinoma, pre-treated dermatofibrosarcoma, pre-treated merkel cell carcinoma, pre-treated glioma, pre-treated sarcoma, pre-treated mesothelioma, and pre-treated myelodysplastic syndrome.

In certain embodiments, patients with advanced NSCLC and previously treated with anti-PD-1 or anti-PD-L1 agents ("PDx therapy") and then having recorded disease progression are treated by intravenous administration of about 1200mg of anti-PD-L1/TGF β trap, recording optimal overall response (BOR) of patients to previous PDx therapy, in certain embodiments, patients with Progressive Disease (PD) after previous PDx therapy are therefore considered to be primarily refractory (i.e., in these patients, disease progression is observed after PDx therapy begins without any observed therapeutic benefit) by intravenous administration of about 1200mg to about 2400mg (e.g., about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 1200mg, about 1200mg to about 2400mg, about 1200mg to about 1200mg, about 1200mg to about 2000mg, about 1200mg to about 1200mg of anti-PD-1 to about 500mg, about 500mg of anti-PD-L389, about 1200mg of anti-PD-1, about 1200mg of anti-PD-2400, about 1200mg of anti-PD-500 mg of anti-PD-1, about 500mg, about 1200mg of anti-PD-2400, about 1200mg, about 500mg of anti-PD-2400, about 1200mg of anti-PD-2000, about 500-1200 mg of anti-PD-180, about 500-1200 mg of anti-180, about 500-1200 mg of anti-180, about 500-180, about 200, about 500-1200 mg of anti-PD alone, about 500-200, and about 500-1200 mg of anti-200, about 500-1200 mg of anti-180, about 500-2400, about 500-1200 mg of anti-180, about 500-1200 mg of anti-PD alone, for anti-1200 mg of anti-180, for a clinical anti-180, and about 500-1200 mg of anti-1200, about 500-1200, for a clinical treatment, for a clinical course of anti-1200, about 200-1200 mg of anti-1200, about 200, about.

In certain embodiments, patients with advanced NSCLC who have refractory, recurrent or progressive disease at or after single-line platinum-based chemotherapy are treated by intravenous administration of about 1200mg to about 2400mg (e.g., about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, about 1200mg to about 2000mg, about 1200mg to about 1900mg, about 1200mg to about 1800mg, about 1200mg to about 1700mg, about 1200mg to about 1600mg, about 1200mg to about 1400mg, about 1300mg to about 2400mg, about 1400mg to about 2400mg, about 1500mg to about 2400mg, about 1600mg to about 2400mg, about 1700mg to about 2400mg, about 1800mg to about 2400mg, about 1900mg to about 2400mg, about 2000mg to about 2000mg, about 2100mg to about 2200mg, about 1600mg to about 2400mg, about 500mg to about 500mg, about 500mg of TGF-500 mg, or about 500mg of a single-line platinum-based chemotherapy, or about 500mg of a polypeptide sequence, including about 500mg, 2, 500mg to 500mg of a single-line platinum-based chemotherapy, or about 500mg of a single-2 or about 500mg of a single-line platinum-based chemotherapy, including a single-based chemotherapy or about 500-1 to about 500-2.

In certain embodiments, a patient having a heavily pretreated relapsed or refractory unresectable IV gastric cancer has been treated with intravenous therapy by intravenous administration of about 1200mg to about 2400mg (e.g., about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, about 1200mg to about 2000mg, about 1200mg to about 1900mg, about 1200mg to about 1800mg, about 1200mg to about 1700mg, about 1200mg to about 1600mg, about 1200mg to about 1500mg, about 1200mg to about 1400mg, about 1200mg to about 1300mg, about 1300mg to about 2400mg, about 1400mg to about 2400mg, about 1500mg to about 2400mg, about 1600mg to about 2400mg, about 1700mg to about 2400mg, about 2400mg to about 2400mg, about 1900mg to about 2400mg, about 2000mg to about 2400mg, about 2100mg to about 2100mg, about 2000 to about 2400mg, about 2100mg to about 2300mg, or about 3556 mg of a heavily pretreated gastric cancer in a patient having had been treated with intravenous administration of anti-TGF IV therapy in at least one previous cycles of a previous mode of anti-TGF IV, a previous anti-2 therapy, in certain embodiments, a previous anti-TGF-phst embodiment, a patient having been treated with intravenous administration of a previous mode of a previous anti-phst 2 therapy, a previous anti-TGF-phst embodiment, a previous anti-TGF-phb therapy in at least one dose, a previous embodiment, a previous treatment mode of about 1200mg to treat patient, a previous embodiment.

In certain embodiments, a patient having pre-treated colorectal cancer (CRC) receives anti-TGF-3, anti-TGF-35, anti-cancer therapy by intravenous administration of about 1200mg to about 2400mg (e.g., about 1200mg to about 2400mg, about 1200mg to about 2300mg, about 1200mg to about 2200mg, about 1200mg to about 2100mg, about 1200mg to about 2000mg, about 1200mg to about 1900mg, about 1200mg to about 1800mg, about 1200mg to about 1700mg, about 1200mg to about 1600mg, about 1200mg to about 1500mg, about 1400mg to about 2400mg, about 1500mg to about 2400mg, about 1600mg to about 2400mg, about 1700mg, about 1800mg to about 2400mg, about 1900mg to about 2400mg, about 2000mg to about 2000mg, about 2100mg to about 2400mg, about 2400mg to about 2400mg, about 1900mg to about 3556 mg) of anti-TGF-3, per week of a pre-TGF-2400, pre-3, anti-TGF-35, and anti-TGF-3, and anti-35.

Delivery device

In one aspect, the present disclosure provides a drug delivery device comprising a formulation comprising about 500mg to about 3000mg of a protein comprising a first polypeptide comprising (a) a heavy chain variable region of an antibody that binds at least to human protein programmed death ligand 1(PD-L1), and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment thereof capable of binding transforming growth factor β (TGF β), and a second polypeptide comprising at least a light chain variable region of an antibody that binds PD-L1, and the heavy chain variable region of the first polypeptide and the light chain of the second polypeptide, when combined, form an antigen binding site that binds PD-L1.

In some embodiments, the device may be a bag, pen or syringe. In certain embodiments, the bag may be connected to a channel comprising a tube and/or a needle.

In certain embodiments of the disclosure, a drug delivery device may comprise about 500mg to about 3000mg (e.g., about 500mg to about 3000mg, about 500mg to about 2900mg, about 500mg to about 2800mg, about 500mg to about 2700mg, about 500mg to about 2600mg, about 500mg to about 2500mg, about 500mg to about 2400mg, about 500mg to about 2300mg, about 500mg to about 2200mg, about 500mg to about 2100mg, about 500mg to about 2000mg, about 500mg to about 1900mg, about 500mg to about 1800mg, about 500mg to about 1700mg, about 500mg to about 1600mg, about 500mg to about 1500mg, about 500mg to about 1300mg, about 500mg to about 1200mg, about 500mg to about 1100mg, about 500mg to about 1000mg, about 500mg to about 900mg, about 500mg to about 800mg, about 500mg to about 3000mg, about 500mg to about 3000mg, about 3000mg of a second amino acid sequence of a polypeptide in a first embodiment of the present invention, a TGF-3000-polypeptide-containing sequence of this invention, a TGF-3000-1-3000-1-3000-1-containing TGF-3000-polypeptide-1-containing first-containing polypeptide-containing sequence of this invention-containing first-polypeptide-containing first-containing polypeptide-3000-containing first-polypeptide-containing sequence of this invention-polypeptide-3000-containing-polypeptide-1-3000-1-polypeptide-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-1-3000-.

In certain embodiments, the drug delivery device may comprise a dose of about 1200mg of a protein of the present disclosure (e.g., anti-PD-L1/TGF β trap (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1)). in certain embodiments, the drug delivery device may comprise about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 5mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1300mg, about 1325mg, about 1075mg, about 1000 mg.

Reagent kitIn one aspect, the present disclosure provides a kit comprising one or more containers collectively comprising from about 500mg to about 2400mg (e.g., from about 500mg to about 2400mg, from about 500mg to about 2300mg, from about 500mg to about 2200mg, from about 500mg to about 2100mg, from about 500mg to about 2000mg, from about 500mg to about 1900mg, from about 500mg to about 1800mg, from about 500mg to about 1700mg, from about 500mg to about 1600mg, from about 500mg to about 1500mg, from about 500mg to about 1400mg, from about 500mg to about 1300mg, from about 500mg to about 1200mg, from about 500mg to about 1100mg, from about 500mg to about 1000mg, from about 500mg to about 900mg, from about 500mg to about 800mg, from about 500mg to about 700mg, from about 500mg to about 600mg, from about 600mg to about 2400mg, from about 700mg to about 2400mg, from about 800mg to about 900mg, from about 900mg to about 2400mg, from about 500mg to about 1000mg, from about 2400mg to about 2400mg, about 1400mg to about 2400mg, about 1500mg to about 2400mg, about 1600mg to about 2400mg, about 1700mg to about 2400mg, about 1800mg to about 2400mg, about 1900mg to about 2400mg, about 2000mg to about 2400mg, about 2100mg to about 2400mg, about 2200mg to about 2400mg, or about 2300mg to about 2400mg) of a formulation of a protein comprising a first polypeptide and a second polypeptide, the first polypeptide comprising: (a) a variable region of a heavy chain of an antibody that binds at least to human protein programmed death ligand 1 (PD-L1); and (b) capable of binding to a transformantHuman transforming growth factor β receptor II (TGF β RII) of length factor β (TGF β), or a fragment thereof, the second polypeptide comprising at least the variable region of a light chain of an antibody 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 PD-L1.

In certain embodiments of the disclosure, the containers collectively include from about 500mg to about 2400mg (e.g., from about 500mg to about 2300mg, from about 500mg to about 2200mg, from about 500mg to about 2100mg, from about 500mg to about 2000mg, from about 500mg to about 1900mg, from about 500mg to about 1800mg, from about 500mg to about 1700mg, from about 500mg to about 1600mg, from about 500mg to about 1500mg, from about 500mg to about 1400mg, from about 500mg to about 1200mg, from about 500mg to about 1100mg, from about 500mg to about 1000mg, from about 500mg to about 900mg, from about 500mg to about 800mg, from about 500mg to about 700mg, from about 500mg to about 2400mg, from about 500mg to about 500mg, from about 500mg to about 1100mg, from about 500mg to about 1000mg, from about 500mg to about 500mg, from about 500mg to about 500mg of the disclosed herein and in a vial, or in certain embodiments of the disclosure, as a vial, and in a vial, as a vial, or as a vial, as a TGF vial, as disclosed and as a TGF/500 mg or as a TGF/500 mg or as well as a TGF/500 mg/800 mg/500 mg/800 mg, as well as disclosed, as well as disclosed, as well as disclosed in certain embodiments of a single vial, or as well as disclosed in certain embodiments of a TGF/500 mg or as disclosed in certain embodiments of a TGF/500 mg.

In certain embodiments, the container collectively may comprise a dose of about 1200mg or about 1800mg of a protein product having a first polypeptide and a second polypeptide, wherein the first polypeptide comprises SEQ ID NO:3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the formulations are prepared and packaged as liquid formulations and are administered in a dosage range of from about 250 mg/vial to about 1200 mg/vial (e.g., from about 250 mg/vial to about 1200 mg/vial, from about 250 mg/vial to about 1100 mg/vial, from about 250 mg/vial to about 1000 mg/vial, from about 250 mg/vial to about 900 mg/vial, from about 250 mg/vial to about 800 mg/vial, from about 250 mg/vial to about 700 mg/vial, from about 250 mg/vial to about 600 mg/vial, from about 250 mg/vial to about 500 mg/vial, from about 250 mg/vial to about 400 mg/vial, from about 250 mg/vial to about 300 mg/vial, from about 300 mg/vial to about 1200 mg/vial, from about 400 mg/vial to about 1200 mg/vial, about 500 mg/vial to about 1200 mg/vial, about 600 mg/vial to about 1200 mg/vial, about 700 mg/vial to about 1200 mg/vial, about 800 mg/vial to about 1200 mg/vial, about 900 mg/vial to about 1200 mg/vial, about 1000 mg/vial to about 1200 mg/vial, or about 1100 mg/vial to about 1200 mg/vial) of a protein product having a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 1. For example, in certain embodiments, the formulation is a liquid formulation and is stored at about 1200 mg/vial, or at about 600 mg/vial, or at about 250 mg/vial.

In certain embodiments, the container may collectively comprise about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, about 1000mg, about 1025mg, about 1050mg, about 1075mg, about 1100mg, about 1125mg, about 1150mg, about 1175mg, about 1200mg, about 1225mg, about 1250mg, about 1275mg, about 1300mg, about 1325mg, about 1350mg, about 1375mg, about 1400mg, about 1425mg, about 1450mg, about 1475mg, about 1500mg, about 1525mg, about 1550mg, about 1575mg, about 1600mg, about 1800mg, about 1650mg, about 1725mg, about 1720 mg, about 1000mg, about 200mg, about 1000mg, about 75mg, about 1000mg, about 20mg, about 1000mg, about 20mg, about 1000 mg.

In certain embodiments, the formulation in the container may be a lyophilized formulation or a liquid formulation.

In certain embodiments, the formulations may be packaged in kits containing an appropriate number of vials. Information about the drug may be included that is in compliance with the approved filing document. The kit can be shipped in a shipping cooling container (2 ℃ to 8 ℃), which is monitored with a temperature control device.

The formulation may be stored at 2 ℃ to 8 ℃ until use. In certain embodiments, the lyophilized drug product may be reconstituted with 4.5mL of water for injection and diluted with about 0.9% saline solution (sodium chloride injection), while the liquid formulation may be diluted with about 0.9% saline solution. The vial of formulation may be sterile and pyrogen-free, and may be free of bacteriostatic preservatives.

In some embodiments, the delivery 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 pharmaceutical composition subcutaneously or intramuscularly. The injection pen may have a housing inside which is a cartridge. The cartridge may have one or more chambers containing the pharmaceutical composition or components thereof and is adapted to be attached to a needle assembly. The cartridge may contain a premixed liquid medicament or solid medicament and a liquid that is mixed prior to injection. The housing may carry an actuating assembly with a stored energy source, such as a compression spring. Activation of the actuation assembly causes a series of movements whereby the needle extends from the injection pen into the user such that the pharmaceutical compound is then forced through the needle and into the user. The needle may be held in the extended position after delivery of the dose of medicament to the injection site. If the injector pen is of a type designed to carry multiple components of a pharmaceutical composition in separate sealed compartments, a structure may be included that forces the components to mix when the actuation assembly is activated.

Protein production

Antibody-cytokine trap proteins are typically produced by recombinant techniques using mammalian cells containing nucleic acids engineered to express the protein. Although one example of a suitable cell line and protein production method is described in examples 1 and 2 of US20150225483a1, a number of suitable vectors, cell lines and protein production methods exist for the production of antibody-based biopharmaceuticals and may be used to synthesize the antibody-cytokine trap proteins herein.

Treatment indications

Exemplary cancers include non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer (e.g., pre-treated colorectal cancer (CRC)), ovarian cancer, glioblastoma, gastric cancer (e.g., pre-treated recurrent or refractory unresectable stage IV gastric cancer), biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head and neck, and squamous carcinoma of the head and neck.

Cancers or tumors to be treated with the anti-PD-L1/TGF β trap may be selected based on expression or elevated expression of PD-L1 and TGF β in the tumor, correlation of their expression levels with prognosis or disease progression, and preclinical and clinical experience with respect to the sensitivity of the tumor to treatment targeting PD-L1 and TGF β.

Examples

The foregoing is a general description of the invention that will be more readily understood by reference to the following examples, which are intended as illustrations of certain aspects and embodiments of the invention, and are not intended to limit the scope of the invention in any way.

Example 1: packaging for intravenous pharmaceutical formulations

The kit comprises 12-60 vials depending on the dosage requirements, or the formulation is prepared and packaged as a liquid formulation and stored at 250 mg/vial to 1000 mg/vial, for example, the formulation is a liquid formulation and stored at 600 mg/vial, or at 250 mg/vial.

The formulations are useful for treating cancer or tumors, such as non-small cell lung cancer, melanoma, pancreatic cancer, colorectal cancer (e.g., pre-treated colorectal cancer (CRC)), ovarian cancer, glioblastoma, gastric cancer (e.g., pre-treated recurrent or refractory unresectable stage IV gastric cancer), biliary tract cancer, esophageal cancer (squamous cell carcinoma or adenocarcinoma), adenoma of the head and neck, and squamous carcinoma of the head and neck.

For example, 500mg of anti-PD-L1/TGF β trap or 1200mg of anti-PD-L1/TGF β trap is administered intravenously to a subject.

Example 2: BW-independent dosing regimen

In an exemplary embodiment, a subject with non-small cell lung cancer (NSCLC) is administered a BW independent dose of 500mg or 1200mg once every two weeks. Intravenous administration is for about 1 hour (-10 min/+ 20 min, i.e. 50 min to 80 min). To alleviate potential infusion-related reactions, a predose of antihistamine and paracetamol (acetaminophen) (e.g., 25-50mg diphenhydramine and 500-650mg paracetamol [ acetaminophen ] intravenous or oral equivalent) is administered about 30 to 60 minutes prior to each dose for the first 2 infusions. If a grade 2 infusion response is seen during the first two infusions, pre-administration is not stopped. Steroids are not allowed as a pre-medication.

For subjects who achieve PR or CR in anti-PD-L1/TGF β well therapy and then develop disease progression after cessation of therapy, 1 reinitiation of the therapy session with the same dose and schedule and duration of treatment for up to 12 months is allowed⁹/L, Absolute Neutrophil Count (ANC) ≥ 1.5X 10⁹L, lymphocyte count ≥ 0.5X 10⁹L, platelet count is not less than 120X 10⁹/L，Hgb≥9gAdequate blood function as determined by/dL (no transfusion); sufficient liver function determined by total bilirubin level less than or equal to 1.5 × ULN, AST level less than or equal to 2.5 × ULN, ALT level less than or equal to 2.5 × ULN; and creatinine clearance measurement according to the Cockcroft-Gault equation or 24 hour urine collection from the estimated creatinine clearance>Sufficient kidney function as determined at 50 mL/min. For subjects with liver involvement in tumors, AST ≦ 5.0 × ULN, ALT ≦ 5.0 × ULN, and bilirubin ≦ 3.0 are acceptable.

In other embodiments, some objects have:

-histologically or cytologically confirmed stage IIIb or IV NSCLC with recurrent, refractory or progressive disease at or after single-line platinum chemotherapy and no previous combination immunotherapy treatment.

Histologically confirmed hepatocellular carcinoma, which is a late-stage disease that is unresectable or not suitable for radical resection, with a cancer that progressed after 1-line prior sorafenib treatment (at least 14 days of sorafenib, at least 400mg per day) or was previously considered sorafenib intolerant.

Histologically confirmed stage IV, or recurrent NSCLC, without prior systemic anti-cancer therapy.

-histologically confirmed stage IV or recurrent NSCLC in patients who received a monotherapy form of platinum-based chemotherapy but failed, and failed disease progression.

Histologically confirmed stage IV or recurrent NSCLC in patients who failed to receive a monotherapy form of platinum-based chemotherapy and failed disease progression, and who had received a monotherapy form of anti-PD-1 or anti-PD-L1 and failed disease progression.

Stage III or metastatic (stage IV) melanoma, which cannot be resected.

Histologically confirmed pancreatic cancer, which is unresectable, advanced and/or metastatic, previously untreated with radiation.

-histologically confirmed colon or rectal adenocarcinoma that progressed during or after a second line systemic treatment comprising fluoropyrimidine, oxaliplatin, irinotecan and/or bevacizumab.

Triple negative breast cancer that progresses during or after the first line of chemotherapy.

Histologically confirmed epithelial ovarian, fallopian tube or peritoneal cancer with unresectable metastatic disease that was platinum resistant/refractory in patients previously treated with at least 2 chemotherapeutic regimens including platinum and taxanes.

Histologically confirmed recurrent or metastatic esophageal adenocarcinoma with unresectable (stage III or IV) disease in patients who received at least one prior platinum-containing chemotherapy regimen.

Histologically confirmed grade IV glioblastoma previously treated with radiotherapy and temozolomide.

Histologically confirmed recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) (oral cavity, pharynx, larynx), stage III/IV, tumor progression or recurrence (i.e. post-operative radiotherapy), primary (i.e. radiotherapy), recurrent or metastatic within 6 months after the last dose of platinum treatment.

Histologically confirmed recurrent or persistent squamous cell carcinoma, adenosquamous carcinoma or cervical adenocarcinoma, following standard of care treatment for systemic treatment of advanced diseases.

Histologically or cytologically confirmed recurrent or refractory unresectable stage IV gastric or gastroesophageal junctional adenocarcinomas for which there is no standard therapy or standard therapy failure.

-histologically or cytologically confirmed esophageal squamous cell carcinoma for which no standard therapy or standard therapy failed.

Histologically or cytologically confirmed biliary tract cancer, which has failed or is intolerant to first-line systemic treatment.

The selected subject is free of autoimmune disease or active tuberculosis that may worsen when receiving an immunostimulant.

Example 3: evaluation of efficacy

Tumor response assessment was performed by CT scan or MRI. The scans performed at the baseline are repeated in subsequent visits. Typically, the same imaging method and preferably the same imaging device is used to track the lesion detected at baseline at the subsequent tumor assessment visit. According to RECIST 1.1 measured using calipers, skin metastases can be used as target lesions if they meet RECIST 1.1 for the target lesion.

Example 4: treatment of advanced NSCLC patients who are resistant or refractory to prior treatment with anti-PD-1 or anti-PD-L1 agents

The objective was to select histologically confirmed stage IV in patients who received platinum-based chemotherapy as monotherapy and failed disease progression, and had received anti-PD-1 or anti-PD-L1 as monotherapy and failed disease progression, or relapsing NSCLC were treated with 1200mg anti-PD-L1/TGF β trap therapy the rationale for using anti-PD-L1/TGF β trap in these NSCLC PDx-failure subgroups was to simultaneously neutralize TGF- β, a molecule known to inhibit tumor immune activation, possibly stimulating clinical response in patients who did not respond to PDx treatment alone.

Summary patients with advanced NSCLC and previously treated with anti-PD-1 or anti-PD-L1 agents ("PDx therapy") and subsequently with documented disease progression were selected for treatment with an intravenous administration of approximately 1200mg of an anti-PD-L1/TGF β trap.

Study design and results a total of 83 patients received anti-PD-L1/TGF β well therapy at a dose of 1200mg per 2 weeks median follow-up time of 27.3 weeks the primary endpoint was BOR according to RECIST v1.1 and the secondary endpoint was safety/tolerance the baseline characteristics of the patients are listed in the following table taken together this is a population of patients that had undergone extensive pretreatment where 74.7% of the patients received more than 3 previous treatment regimens and included many different ages and sexes the response to previous PDx treatment was roughly balanced (primary refractory, 43.4%; acquired resistance, 53%) in addition all patients were biopsied within 28 days after treatment initiation and most (65.1%) patients had > 1% tumor cell PD-L1 expression as determined by Dako 73-10 PD-L1.

Table 6: patient characteristics

By the date of data expiration at the time of analysis, a total of 2 patients (2.4%) had investigator-confirmed responses according to RECIST v 1.1. Disease control was reported in 20 patients (24.1%). 3 patients (3.6%) had confirmed partial responses by independent radiology examinations. In addition, the investigators reported 1 additional confirmed PR and 1 unacknowledged PR, thus increasing the unacknowledged ORR to 4.8%; treatment was continued for 6 patients. Responses occur in patients with primary refractory disease and in patients with acquired resistance to prior anti-PDx treatment.

As shown in FIG. 13, patients with previously progressive disease (with primary refractory and acquired drug resistant disease) achieved significant disease stability it was noted that patients with disease response and disease stability had undergone a series of treatments in previous treatments prior to initiation of the study, even received a series of treatments immediately prior to initiation of the trial, indicating clinical activity against the PD-L1/TGF β trap in a heterogeneous population of patients with prior PDx exposure, that response and disease control was noted in high and low PD-L1 expressing patients (regardless of the PD-L1 state at the beginning of the trial), and that this was also noted in patients with high or low circulating TGF- β 1 plasma levels.

Patients with similar incidence of treatment-related adverse events (TRAE) to other anti-PD-1 or anti-PD-L1 monotherapies in NSCLC PDx-failure patients were generally well-tolerated anti-PD-L1/TGF β wells most patients (n 60, 72.3%) experienced any TRAE, a smaller proportion (n 19, 22.9%) experienced grade 3 or higher events the table below reports indicating TRAEs in > 5% of patients, fatigue/weakness was most common (36.1%, G3+ 6.0%), followed by pruritus (21.7%, G3+ 2.4%) and loss of appetite (16.9%, G3+ 1.2%), one patient was stopped the study due to treatment-related AEs (G2, plaque eczema), one patient died of pneumonia, assessed by the investigator as treatment-related, 5 patients (6.0%) had cutaneous lesions, including dermatoma and TGF-64) were well-controlled by other squamous cell surgery, and were similarly protected by other keratotic procedures.

Table 7: treatment-related adverse events (TRAE)

N＝85	Any ofRank of	Grade ≥ 3
			Any TRAE, n (%)	60(72.3)	19(22.9)
Anemia (anemia)	5(6.0)	1(1.2)
			Arthralgia pain	6(7.2)	1(1.2)
Loss of appetite	14(16.9)	1(1.2)
			Diarrhea (diarrhea)	6(7.2)	0(0.0)
Dry skin	5(6.0)	0(0.0)
			Nose bleeding	8(9.6)	0(0.0)
Fatigue/weakness	30(36.1)	5(6.0)
			Itching (pruritus)	18(21.7)	2(2.4)
Maculopapular rash	6(7.2)	1(1.2)

In summary, anti-PD-L1/TGF β trap was found to be an innovative first-class bifunctional fusion protein aimed at targeting 2 immunosuppressive pathways simultaneously, PD-L1 and TGF- β. therefore, inhibition of the TGF- β pathway helps to overcome treatment failures of anti-PD-1/PD-L1 agents treatment with anti-PD-L1/TGF β trap treatment results in initial clinical activity in NSCLC patients with severe pre-treatment of primary refractory or acquired-resistant disease to prior treatment with anti-PD-1 or anti-PD-L1.

Example 5: treatment of pre-treated relapsed or refractory stage IV gastric cancer patients

Aims to select patients with recurrent or refractory unresectable gastric cancer in stage IV after severe pretreatment, treat the patients with 1200mg of anti-PD-L1/TGF β trap therapy and evaluate the safety and the curative effect.

Study design and results A total of 31 patients received anti-PD-L1/TGF β trap treatment at a dose of 1200mg every 2 weeks until progressive disease is confirmed, unacceptable toxicity or trial exit the group includes a population of Asian patients who have undergone extensive pretreatment, 67.7% of which received at least 3 prior anti-cancer treatments and 29.3% of which received at least 4 anti-cancer treatments.

The baseline characteristics of the patients are listed in table 8 below.

Table 8: patient characteristics

Median duration of patients receiving anti-PD-L1/TGF β wells was 6.1 weeks (range: 2-30 weeks) by date of data expiration at the time of analysis, of 31 evaluable patients, 5 patients had confirmed partial response, 5 patients had Stable Disease (SD) as their BOR according to RECIST v1.1, as assessed by the investigator, Overall Response Rate (ORR) was 16.1%, and Disease Control Rate (DCR) was 32.3%.

Table 9: patient characteristics

Patients with a similar rate of treatment-related adverse events (TRAE) as other anti-PD-1/PD-L1 monotherapies were generally well-tolerated anti-PD-L1/TGF β trap 14 patients (45.2%) experienced treatment-related adverse events 4 patients (12.9%) experienced grade 3 TRAE, failure to develop treatment-related grade 4 ae.1 grade 5 events (5 doses total) were considered likely to be treatment-related, but the investigator suspected whether the preexisting rupture of the thoracic aortic aneurysm was classified as other possible cause.

Table 10: treatment-related adverse events (TRAE)

In summary, the anti-PD-L1/TGF β trap was found to be an innovative first-class bifunctional fusion protein aimed at targeting 2 immunosuppressive pathways simultaneously, PD-L1 and TGF- β inhibition of the TGF- β pathway could help overcome the therapeutic failure of anti-PD-1/PD-L1 agents treatment with the anti-PD-L1/TGF β trap resulted in the initial clinical activity of Asian patients with heavily pretreated gastric cancer.

Example 6: treatment of largely pretreated colorectal cancer (CRC) patients

Recently, a Consensus Molecular Subgroup (CMS) of 4 CRCs has been described-including the poor prognosis, the mesenchymal CMS4 group, characterized by angiogenic, inflammatory and immunosuppressive properties.given that TGF- β may play a role in mediating this immunosuppressive phenotype, the rationale for using anti-PD-L1/TGF β traps in these patients is provided.

Study design and results a total of 32 patients received anti-PD-L1/TGF β well therapy at a dose of 1200mg every 2 weeks with a median duration of treatment of 7.1 weeks (range: 2-38) and 2 patients were still actively treated by the date of data cut-off at the time of analysis, the primary endpoint was BOR according to RECIST v1.1, the secondary endpoint was safety/tolerance, the baseline characteristics of the patients are listed in the table below, taken together, this is a population of greatly pre-treated patients, 74.7% of whom received more than 3 prior treatment regimens, overall good clinical status (PS 0-1) and including many different ages and sexes, approximately 34% of which were KRAS mutated based on Dako 73-10PD-L1, and most patients (81.3%) had a PD-L1 expression of < 1% on tumor cells, no prospective collection of tumor-side (tumor-side) in the database, but 3.3% of which were assessed on the left side of the tumor based on prior surgical history, 3.3% of which was not considered as estimated based on prior surgical history, 3.6% of cancer.

Table 11: patient characteristics

By the date of data expiration at the time of analysis, 1 patient (3.1%) had a confirmed Partial Response (PR). The other patient had stable disease, with progressive disease in 27 patients being the best overall response. The response criteria were adjudged by the independent examination committee and defined according to recistv 1.1. Patients with PR have CRC with precise mismatch repair (i.e. microsatellite stability), CMS4, KRAS mutant and PD-L1+ (PD-L1 > 1% in tumor cells by IHC). This patient had the highest tumor cell PD-L1 expression (20%) in our group. By 11/1/2017, patients with PR were still under study (12.5 months) and continued to respond partially. Since the remission RT on the target lesion was received during the 5 th month of treatment, additional patients were still under treatment for the disease that could not be assessed at 13 months. No new lesions appeared since then, non-target lesions remained stable. The CRC of the patient is KRAS mutated; CMS and microsatellite status were investigated, and PD-L1 status was unknown.

Among CRC patients, patients with a similar rate of treatment-related adverse events (TRAEs) as other anti-PD-1/PD-L1 monotherapies were generally well-tolerated anti-PD-L1/TGF β traps most patients (n-22, 68.8%) experienced any TRAE, a smaller proportion (n-4, 12.5%) experienced grade 3 events no grade 4/5 trae.table indicating TRAEs in > 5% of patients is reported in the table below anemia, diarrhea, infusion-related responses and nausea are the most common (all n-5, 15.6%), there is one grade 3 related anemic event (3.1%) table below.

Table 12: treatment-related adverse events (TRAE)

In summary, anti-PD-L1/TGF β trap was found to be an innovative first-class bifunctional fusion protein aimed at targeting 2 immunosuppressive pathways simultaneously, TGF- β and PD-L1 treatment with anti-PD-L1/TGF β trap resulted in initial clinical activity in pre-treated patients with advanced CRC, 1 patient with persistent PR, 1 patient with SD, and 27 patients with PD BOR. PR for 8.3 months had CRC of MSI, CMS4, KRAS mutant and PD-L1 +. the second patient remained good and non-relapsing 13 months after the initial progressive disease.

Example 7 establishment of effective dose/dosing regimens and Exposure in humans preliminary dose response and Exposure response to second-line non-Small cell Lung cancer (2L NSCLC) following anti-PD-L1/TGF β trap once every 2 weeks (q2w)

In this study, 80 subjects with advanced/recurrent NSCLC, post-platinum treatment and non-selection for PD-L1 were given 500mg or 1200mg of anti-PD-L1/TGF β trap (n ═ 40 per group) once every 2 weeks (q2w) until disease progression, unacceptable toxicity or trial withdrawal. subjects were evaluated for dose response and exposure response until the data was cut off at the time of analysis, a total of 17 subjects were still receiving treatment with a median follow-up time of 35.2 (range, 1.3-47.3) weeks. investigators evaluated for an unproven Overall Response Rate (ORR) of 25.0% (500mg ORR, 22.5%; 1200mg ORR 27.5%), with 9 Partial Responses (PR) at 500mg and 1 Complete Response (CR) and 10 PR. at 1200mg with observed clinical activity at PD-L1 expression level, with a decline of treatment of 3.7% or more of PD-1 (7) and 10 of the patients with 20% of evidence of signs of craving (20%) and a decline of the incidence of wheal (TRAE) of the most common treatment incidence of the disease (20% of 3.5%) and 4% of the patients with 80% of anorexia.

For exposure-response assessment, the first cycle exposure was predicted based on dose and covariate information from these 80 patients using a population PK model. In particular, AUC and C after a single dose were predicted for each subject using empirical bayesian estimates of population PK parameters (see tables 2 and 3)_{Low valley}. The predicted exposure data for the 500mg q2w and 1200mg q2w groups were combined to calculate the response rate of the predicted exposure per quartile as shown in tables 4 and 5.

Sequence of

SEQ ID NO:1

Secreted anti-PD-L1 lambda light chain peptide sequence

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:2

Secreted peptide sequence against the H chain of PDL1

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:3

Secreted anti-PDL 1/TGF β trap H chain peptide sequences

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:4

DNA sequence of anti-PD-L1 lambda light chain from translation initiation codon to translation termination codon (leader sequence before VL is signal peptide from urokinase plasminogen activator)

atgagggccctgctggctagactgctgctgtgcgtgctggtcgtgtccgacagcaagggcCAGTCCGCCCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCCTGGCCAGTCCATCACCATCAGCTGCACCGGCACCTCCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGTCCAACCGGCCCTCCGGCGTGTCCAACAGATTCTCCGGCTCCAAGTCCGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCAGAGGACGAGGCCGACTACTACTGCTCCTCCTACACCTCCTCCAGCACCAGAGTGTTCGGCACCGGCACAAAAGTGACCGTGCTGggccagcccaaggccaacccaaccgtgacactgttccccccatcctccgaggaactgcaggccaacaaggccaccctggtctgcctgatctcagatttctatccaggcgccgtgaccgtggcctggaaggctgatggctccccagtgaaggccggcgtggaaaccaccaagccctccaagcagtccaacaacaaatacgccgcctcctcctacctgtccctgacccccgagcagtggaagtcccaccggtcctacagctgccaggtcacacacgagggctccaccgtggaaaagaccgtcgcccccaccgagtgctcaTGA

SEQ ID NO:5

DNA sequence from translation initiation codon to translation termination codon (mVK SP leader: lower case underlined; VH: upper case; IgG1m3 containing the K to A mutation: lower case; (G4S) x4-G (SEQ ID NO:11) linker: bold upper case; TGF β RII: bold underlined lower case; two termination codons: bold underlined upper case)

atggaaacagacaccctgctgctgtgggtgctgctgctgtgggtgcccggctccacaggcGAGGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACATCATGATGTGGGTGCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGTCCTCCATCTACCCCTCCGGCGGCATCACCTTCTACGCCGACACCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGATCAAGCTGGGCACCGTGACCACCGTGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtgctGGCGGCGGAGGAAGCGGAGGAGGTGGCAGCGGTGGCGGTGGCTCCGGCGGAGGTGGCTCCGGAatccctccccacgtgcagaagtccgtgaacaacgacatg atcgtgaccgacaacaacggcgccgtgaagttccctcagctgtgcaagttctgcgacgtgaggttcagcacctgcg acaaccagaagtcctgcatgagcaactgcagcatcacaagcatctgcgagaagccccaggaggtgtgtgtggccgt gtggaggaagaacgacgaaaacatcaccctcgagaccgtgtgccatgaccccaagctgccctaccacgacttcatc ctggaagacgccgcctcccccaagtgcatcatgaaggagaagaagaagcccggcgagaccttcttcatgtgcagct gcagcagcgacgagtgcaatgacaacatcatctttagcgaggagtacaacaccagcaaccccgacTGATAA

SEQ ID NO:6

Polypeptide sequence of secreted anti-lambda light chain of PD-L1(mut)/TGF β trap with mutations A31G, D52E, R99Y

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:7

Polypeptide sequence of secreted anti-PD-L1 (mut)/TGF β trap heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:8

Human TGF β RII isoform A precursor polypeptide (NCBI RefSeq accession No.: NP-001020018)

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

SEQ ID NO:9

Human TGF β RII isoform B precursor polypeptide (NCBI RefSeq accession No.: NP-003233)

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK

SEQ ID NO:10

Human TGF β RII isoform B extracellular domain polypeptides

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID NO:11

(Gly₄Ser)₄Gly linker

GGGGSGGGGSGGGGSGGGGSG

SEQ ID NO:12

Polypeptide sequence of heavy chain variable region of secreted anti-PD-L1 antibody MPDL3289A

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

SEQ ID NO:13

Polypeptide sequence of light chain variable region of secreted anti-PD-L1 antibody MPDL3289A

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR

SEQ ID NO:14

Polypeptide sequence of YW243.55S70 heavy chain variable region of secreted anti-PD-L1 antibody

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA

Is incorporated by reference

The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes.

Equivalent forms

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The embodiments described above are therefore illustrative in all respects and not restrictive of the invention described herein. The various structural elements and the various method steps described in the different embodiments may be arranged in any combination and all such variations are to be considered in the manner of this disclosure. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Numbered embodiments of the present disclosure are listed below:

1. a method of treating cancer or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject a dose of at least 500mg of a protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises (a) at least the variable region of the heavy chain of an antibody that binds to human protein programmed death ligand 1(PD-L1), and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment thereof capable of binding transforming growth factor β (TGF β),

wherein the second polypeptide comprises at least the variable region of the light chain of an antibody that binds PD-L1, and

wherein 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. The method of claim 1, wherein the first polypeptide comprises SEQ ID NO:3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

3. The method of claim 1 or 2, wherein the dose is 500mg to 2400 mg.

4. The method of claim 1 or 2, wherein the dose is 1200mg to 1800 mg.

5. The method of any one of claims 1-4, wherein the dose is 1200 mg.

6. The method of claim 1 or 2, wherein the dose is 1800 mg.

7. The method of any one of claims 1-6, wherein the dose is administered once every two weeks or once every three weeks.

8. The method of any one of claims 1-7, wherein the protein is administered by intravenous administration.

9. The method of claim 8, wherein the intravenous administration is performed using a pre-filled bag, a pre-filled pen, or a pre-filled syringe comprising a formulation comprising the protein.

10. The method of claim 9, wherein the bag is connected to a channel comprising a tube and/or a needle.

11. The method of any one of claims 1-10, 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), head and neck adenomas, and head and neck squamous cell carcinoma.

12. The method of any one of claims 1-10, wherein the tumor or cancer is selected from the group consisting of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

13. The method of any one of claims 1-10, wherein the tumor is an advanced solid tumor.

14. The method of claim 13, wherein the tumor is refractory and/or resistant to a previous treatment.

15. An intravenous drug delivery formulation comprising 500mg-2400mg of a protein, the protein comprising a first polypeptide and a second polypeptide,

wherein the first polypeptide comprises (a) at least the variable region of the heavy chain of an antibody that binds to human protein programmed death ligand 1(PD-L1), and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment thereof capable of binding transforming growth factor β (TGF β),

16. The intravenous drug delivery formulation of claim 15, wherein the first polypeptide comprises SEQ id no:3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

17. The intravenous drug delivery formulation of claim 15 or 16, comprising 1200mg of the protein.

18. The intravenous drug delivery formulation of claim 15 or 16, comprising 1200mg to 2400mg of the protein.

19. The intravenous drug delivery formulation of claim 15 or 16, comprising 1800mg of the protein.

20. The intravenous drug delivery formulation of any one of claims 15-19, wherein the formulation is contained in a bag, pen, or syringe.

21. The intravenous drug delivery formulation of claim 20, wherein the bag is connected to a channel comprising a tube and/or a needle.

22. The intravenous drug delivery formulation of any one of claims 15-21, wherein the formulation is a lyophilized formulation or a liquid formulation.

23. A drug delivery device comprising a formulation comprising 500mg to 2400mg of a protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises (a) at least the variable region of the heavy chain of an antibody that binds to human protein programmed death ligand 1(PD-L1), and (b) human transforming growth factor β receptor II (TGF β RII) or a fragment thereof capable of binding transforming growth factor β (TGF β),

24. The drug delivery device of claim 23, wherein the first polypeptide comprises SEQ ID NO:3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

25. The drug delivery device of claim 23 or 24, comprising 1200mg of the protein. 26. The drug delivery device of claim 23 or 24, comprising 1200 to 2400mg of the protein.

27. The drug delivery device of claim 23 or 24, comprising 1800mg of the protein.

28. The drug delivery device of any one of claims 23-27, wherein the device is a bag, a pen, or a syringe.

29. A drug delivery device as in claim 28, wherein the bag is connected to a channel comprising a tube and/or a needle.

30. A kit comprising one or more containers collectively comprising a formulation comprising 500mg to 2400mg of a protein comprising a first polypeptide and a second polypeptide,

31. The kit of claim 30, wherein the first polypeptide comprises 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 1200mg of the protein.

33. The kit of claim 30 or 31, wherein the containers collectively comprise 1200 to 2400mg of the protein.

34. The kit of claim 30 or 31, wherein the containers collectively comprise 1800mg of the protein.

35. The kit of any one of claims 30-34, wherein the formulation is a lyophilized formulation or a liquid formulation.

36. The intravenous drug delivery formulation of any one of claims 15-22, the drug delivery device of any one of claims 23-29, or the kit of any one of claims 30-35, for use in treating cancer or inhibiting tumor growth in a subject in need thereof.

37. The intravenous drug delivery formulation, drug delivery device, or kit of 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), head and neck adenomas, and head and neck squamous cell carcinoma.

38. The intravenous formulation, drug delivery device or kit of 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, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma and myelodysplastic syndrome.

39. The intravenous drug delivery formulation, drug delivery device, or kit of claim 36, wherein the tumor is an advanced solid tumor.

40. The intravenous drug delivery formulation, drug delivery device, or kit of claim 36, wherein the tumor is refractory to a prior treatment.

41. The intravenous drug delivery formulation, drug delivery device, or kit of any one of claims 36-40, wherein the formulation is administered to the subject biweekly.

Sequence listing

<110> Merck patent Co., Ltd (MERCK PATENT GMBH)

<120> dosing regimens and dosage forms for targeted TGF- β inhibition

<130> EMD-005PC

<140>

<141>

<150> 62/443,698

<151> 2017-01-07

<150> 62/581,978

<151> 2017-11-06

<160> 49

<170> PatentIn version 3.5

<210> 1

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 1

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 2

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 2

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 3

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 3

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val

465 470 475 480

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

485 490 495

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

500 505 510

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

515 520 525

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

530 535 540

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

545 550 555 560

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

565 570 575

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

580 585 590

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

595 600 605

<210> 4

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of polynucleotides

<400> 4

atgagggccc tgctggctag actgctgctg tgcgtgctgg tcgtgtccga cagcaagggc 60

cagtccgccc tgacccagcc tgcctccgtg tctggctccc ctggccagtc catcaccatc 120

agctgcaccg gcacctccag cgacgtgggc ggctacaact acgtgtcctg gtatcagcag 180

caccccggca aggcccccaa gctgatgatc tacgacgtgt ccaaccggcc ctccggcgtg 240

tccaacagat tctccggctc caagtccggc aacaccgcct ccctgaccat cagcggactg 300

caggcagagg acgaggccga ctactactgc tcctcctaca cctcctccag caccagagtg 360

ttcggcaccg gcacaaaagt gaccgtgctg ggccagccca aggccaaccc aaccgtgaca 420

ctgttccccc catcctccga ggaactgcag gccaacaagg ccaccctggt ctgcctgatc 480

tcagatttct atccaggcgc cgtgaccgtg gcctggaagg ctgatggctc cccagtgaag 540

gccggcgtgg aaaccaccaa gccctccaag cagtccaaca acaaatacgc cgcctcctcc 600

tacctgtccc tgacccccga gcagtggaag tcccaccggt cctacagctg ccaggtcaca 660

cacgagggct ccaccgtgga aaagaccgtc gcccccaccg agtgctcatg a 711

<210> 5

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of polynucleotides

<400> 5

atggaaacag acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg ctccacaggc 60

gaggtgcagc tgctggaatc cggcggagga ctggtgcagc ctggcggctc cctgagactg 120

tcttgcgccg cctccggctt caccttctcc agctacatca tgatgtgggt gcgacaggcc 180

cctggcaagg gcctggaatg ggtgtcctcc atctacccct ccggcggcat caccttctac 240

gccgacaccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 300

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ccggatcaag 360

ctgggcaccg tgaccaccgt ggactactgg ggccagggca ccctggtgac agtgtcctcc 420

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc cccgggtgct ggcggcggag gaagcggagg aggtggcagc 1440

ggtggcggtg gctccggcgg aggtggctcc ggaatccctc cccacgtgca gaagtccgtg 1500

aacaacgaca tgatcgtgac cgacaacaac ggcgccgtga agttccctca gctgtgcaag 1560

ttctgcgacg tgaggttcag cacctgcgac aaccagaagt cctgcatgag caactgcagc 1620

atcacaagca tctgcgagaa gccccaggag gtgtgtgtgg ccgtgtggag gaagaacgac 1680

gaaaacatca ccctcgagac cgtgtgccat gaccccaagc tgccctacca cgacttcatc 1740

ctggaagacg ccgcctcccc caagtgcatc atgaaggaga agaagaagcc cggcgagacc 1800

ttcttcatgt gcagctgcag cagcgacgag tgcaatgaca acatcatctt tagcgaggag 1860

tacaacacca gcaaccccga ctgataa 1887

<210> 6

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 6

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 7

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 7

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr

20 25 30

Met Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val

465 470 475 480

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

485 490 495

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

500 505 510

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

515 520 525

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

530 535 540

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

545 550 555 560

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

565 570 575

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

580 585 590

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

595 600 605

<210> 8

<211> 592

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 8

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp

20 25 30

Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn

35 40 45

Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr

50 55 60

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

65 70 75 80

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

85 90 95

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

100 105 110

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

115 120 125

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

130 135 140

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

145 150 155 160

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

165 170 175

Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val

180 185 190

Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile

195 200 205

Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser Ser

210 215 220

Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His

225 230 235 240

Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys

245 250 255

Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp

260 265 270

Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu

275 280 285

Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe

290 295 300

Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser

305 310 315 320

Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu

325 330 335

Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala Phe

340 345 350

His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile Ser

355 360 365

Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala

370 375 380

His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile

385 390 395 400

Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu

405 410 415

Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr

420 425 430

Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg

435 440 445

Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val

450 455 460

Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp

465 470 475 480

Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu

485 490 495

Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met

500 505 510

Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe

515 520 525

Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu

530 535 540

Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala

545 550 555 560

Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser

565 570 575

Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr Lys

580 585 590

<210> 9

<211> 567

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 9

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu

145 150 155 160

Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu

165 170 175

Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn

180 185 190

Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys

195 200 205

Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg

210 215 220

Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu

225 230 235 240

Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala

245 250 255

Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu

260 265 270

Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys

275 280 285

Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile

290 295 300

Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln

305 310 315 320

Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr

325 330 335

Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser

340 345 350

Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys

355 360 365

Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn

370 375 380

Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu

385 390 395 400

Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser

405 410 415

Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser

420 425 430

Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr

435 440 445

Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val

450 455 460

Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu

465 470 475 480

His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly

485 490 495

Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met

500 505 510

Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg

515 520 525

Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu

530 535 540

Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp

545 550 555 560

Gly Ser Leu Asn Thr Thr Lys

565

<210> 10

<211> 136

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 11

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 11

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly

20

<210> 12

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 13

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 14

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 15

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 15

Gln Phe Asn Ser

1

<210> 16

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 16

Gln Ala Gln Ser

1

<210> 17

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 17

Pro Lys Ser Cys Asp Lys

1 5

<210> 18

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 18

Pro Lys Ser Ser Asp Lys

1 5

<210> 19

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 19

Leu Ser Leu Ser

1

<210> 20

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 20

Ala Thr Ala Thr

1

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Lys, Arg, Thr, Gln, Gly, Ala, Trp, Met, Ile or Ser

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Val, Arg, Lys, Leu, Met or Ile

<220>

<221> MOD_RES

<222> (5)..(5)

<223> His, Thr, Asn, Gln, Ala, Val, Tyr, Trp, Phe or Met

<400> 21

Xaa Tyr Xaa Met Xaa

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Phe or Ile

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Ser or Thr

<400> 22

Ser Ile Tyr Pro Ser Gly Gly Xaa Thr Phe Tyr Ala Asp Xaa Val Lys

1 5 10 15

Gly

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Glu or Asp

<400> 23

Ile Lys Leu Gly Thr Val Thr Thr Val Xaa Tyr

1 5 10

<210> 24

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 24

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 25

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 25

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 26

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 26

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 27

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 27

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 28

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (4)..(4)

<223> Asn or Ser

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Thr, Arg or Ser

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Ala or Gly

<400> 28

Thr Gly Thr Xaa Xaa Asp Val Gly Xaa Tyr Asn Tyr Val Ser

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Glu or Asp

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Ile, Asn or Ser

<220>

<221> MOD_RES

<222> (4)..(4)

<223> Asp, His or Asn

<400> 29

Xaa Val Xaa Xaa Arg Pro Ser

1 5

<210> 30

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Phe or Tyr

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Asn or Ser

<220>

<221> MOD_RES

<222> (6)..(6)

<223> Arg, Thr or Ser

<220>

<221> MOD_RES

<222> (7)..(7)

<223> Gly or Ser

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Ile or Thr

<400> 30

Ser Ser Xaa Thr Xaa Xaa Xaa Xaa Arg Val

1 5 10

<210> 31

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 31

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys

20

<210> 32

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 32

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

1 5 10 15

<210> 33

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 33

Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

1 5 10 15

Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

20 25 30

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 34

Phe Gly Thr Gly Thr Lys Val Thr Val Leu

1 5 10

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 35

Ser Tyr Ile Met Met

1 5

<210> 36

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 36

Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 37

Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr

1 5 10

<210> 38

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 38

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 39

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 40

Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val

1 5 10

<210> 41

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 41

Met Tyr Met Met Met

1 5

<210> 42

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 42

Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 43

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400> 43

Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr Asn Tyr Val Ser

1 5 10

<210> 44

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 44

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Val Trp Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Trp Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 45

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 45

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 46

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 46

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr

20 25 30

Met Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Val Trp

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 47

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 48

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polynucleotides from human Fab libraries

<400> 48

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 tggtggtgga 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

<210> 49

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<400> 49

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

Claims

1. A method of treating cancer or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject a dose of at least 500mg of a protein comprising a first polypeptide and a second polypeptide,

3. The method of claim 1 or 2, wherein the dose is 500mg to 2400 mg.

4. The method of claim 1 or 2, wherein the dose is 1200mg to 1800 mg.

5. The method of any one of claims 1-4, wherein the dose is 1200 mg.

6. The method of claim 1 or 2, wherein the dose is 1800 mg.

23. A drug delivery device comprising a formulation comprising 500mg to 2400mg of a protein comprising a first polypeptide and a second polypeptide,

25. The drug delivery device of claim 23 or 24, comprising 1200mg of the protein.

26. The drug delivery device of claim 23 or 24, comprising 1200 to 2400mg of the protein.