WO2021196225A1 - Molecular antagonist of gastric inhibitory polypeptide, compositions comprising molecular antagoist of gastric inhibitory polypeptide, and method for treatment of diseases using the same - Google Patents
Molecular antagonist of gastric inhibitory polypeptide, compositions comprising molecular antagoist of gastric inhibitory polypeptide, and method for treatment of diseases using the same Download PDFInfo
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- WO2021196225A1 WO2021196225A1 PCT/CN2020/083352 CN2020083352W WO2021196225A1 WO 2021196225 A1 WO2021196225 A1 WO 2021196225A1 CN 2020083352 W CN2020083352 W CN 2020083352W WO 2021196225 A1 WO2021196225 A1 WO 2021196225A1
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- Disturbances in glucose metabolism or glucose metabolism disorders refer to abnormalities in the structure, function, and concentration of hormones or enzymes that regulate glucose metabolism, or pathophysiological changes in tissues and organs, leading to disturbances in monitored blood glucose.
- Clinically important glucose metabolism disorders are mainly high blood glucose concentrations (hyperglycemia) and low blood glucose concentration (hypoglycemia) , which may be caused by many different reasons.
- Obesity is a medical condition related to glucose metabolism disorders fatty tissue buildup in which excess body fat has accumulated to the extent that it has a negative effect on health. In the United States, obesity is considered to occur when a person's body mass index (BMI) is 30 kg/m 2 or greater. Obesity increases the likelihood of various diseases, including heart disease, type II diabetes, obstructive sleep apnea, some cancers, and osteoarthritis. It is known that following oral glucose administration, serum GIP levels increase 5-to 6-fold, which stimulates insulin release, which promotes glucose uptake. GIP also activates Akt PKB, which promotes the membrane translocation of glucose transporter-4, leading to enhanced adipocyte uptake of glucose, leading to the creation and storage of fat.
- BMI body mass index
- Figure 4 further demonstrates that in cellular level, after binding, the variants can inhibit downstream signals in GIP pathway to a similar extent as the parent molecule.
- HV I31F + HV: K50Q;
- HV K50Q + HV: V108R;
- HV I31F + HV: K50Q + HV: N107A;
- the GIP molecular antagonist is a monoclonal antibody with a light chain variable domain and a heavy variable domain.
- the heavy chain variable domain has an amino acid sequence with at least 95%identity to SEQ ID NO: 7, has a first CDR as represented by SEQ ID NO: 1, a second CDR as represented by SEQ ID NO: 2, and a third CDR as represented by SEQ ID NO: 3, and has one or more mutations selected from the group consisting of HV: I31F, HV: Q45R, HV: K50Q, HV: N107A and HV: V108R.
- the light chain variable domain has at least 95%identity to SEQ ID NO: 9.
- the present disclosure relates to a molecular antagonist of gastric inhibitory polypeptide (GIP) , comprising a heavy chain variable domain having an amino acid sequence with at least 95%identity to SEQ ID NO: 7, wherein said heavy variable domain comprises:
- the present disclosure relates to the GIP molecular antagonist according to the first aspect, wherein the one or more mutations is selected from the following groups:
- HV K50Q + HV: N107A;
- HV N107A + HV: V108R;
- HV I31F + HV: Q45R + HV: K50Q + HV: V108R;
- the parental sequence used in the present disclosure is Protein X, with the full length amino acid sequence of its heavy chain (SEQ ID NO: 8) and the amino acid sequence of its heavy chain variable region (SEQ ID NO: 7) disclosed in Figure 1. a, and the full length amino acid sequence of its kappa light chain (SEQ ID NO: 10) the amino acid sequence of its light chain variable region (SEQ ID NO: 9) disclosed in Figure 1. b.
- the CDRs of Protein X is SEQ ID NO: 1 for HC CDR1, SEQ ID NO: 2 for HC CDR2, SEQ ID NO: 3 for HC CDR3; SEQ ID NO: 4 for LC CDR1, SEQ ID NO: 5 for LC CDR2, and SEQ ID NO: 6 for LC CDR3.
- ProteinX_SP_K. 012 2.51 97.49 ProteinX_SP_K. 013 3.17 96.83 ProteinX_SP_K. 014 7.93 92.07 ProteinX_SP_K. 015 4.08 95.92 ProteinX_SP_K. 016 3.35 96.65 ProteinX_SP_K. 017 5.82 94.18 ProteinX_SP_K. 018 3.90 96.10 ProteinX_SP_K. 019 1.67 98.33 ProteinX_SP_K. 023 3.14 96.86 ProteinX_SP_K. 025 0.98 99.02 ProteinX_SP_K. 026 1.80 98.20 ProteinX_SP_K. 027 2.10 97.90 ProteinX_SP_K. 028 1.55 98.45 ProteinX_SP_K. 029 2.75 97.25 ProteinX_SP_K. 030 1.29 98.71 ProteinX_SP_K. 031 0.94 99.06
- mice On the day of the test, mice were moved to procedure room from 8: 00 am and placed on the bench and these mice were kept on the same bench where the glucose tolerance experiment was conducted so that they can be accustomed to the area to reduce stress during the procedure.
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- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The disclosure relates to molecular antagonist of gastric inhibitory polypeptide (GIP), compositions comprising molecular antagonist of gastric inhibitory polypeptide, and method for treatment of diseases using GIP molecular antagonist.
Description
The present disclosure relates to molecular antagonist of gastric inhibitory polypeptide (GIP) , compositions comprising molecular antagonist of gastric inhibitory polypeptide, and method for treatment of diseases using GIP molecular antagonist.
The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art.
Gastric inhibitory polypeptide (GIP) or gastroinhibitory peptide, also known as the glucose-dependent insulinotropic peptide, is a 42-amino acid peptide secreted from K-cells in the small intestine (duodenum and jejunum) .
GIP has a lot of physiological effects in tissues, including promotion of fat storage in adipocytes, promotion of pancreatic islet β-cell function and glucose-dependent insulin secretion. GIP secretion is induced by food ingestion, and it is a known insulinotropic factor ("incretins") . In addition to its actions on pancreatic beta-cells, GIP exerts a range of secondary extrapancreatic activities, which further augments its antihyperglycaemic properties.
Disturbances in glucose metabolism or glucose metabolism disorders refer to abnormalities in the structure, function, and concentration of hormones or enzymes that regulate glucose metabolism, or pathophysiological changes in tissues and organs, leading to disturbances in monitored blood glucose. Clinically important glucose metabolism disorders are mainly high blood glucose concentrations (hyperglycemia) and low blood glucose concentration (hypoglycemia) , which may be caused by many different reasons.
Diabetes is a group of metabolic diseases characterized by high blood sugar. Hyperglycemia is caused by a defect in insulin secretion or impaired biological effects thereof, or both. The long-term hyperglycemia during diabetes causes chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves.
Obesity is a medical condition related to glucose metabolism disorders fatty tissue buildup in which excess body fat has accumulated to the extent that it has a negative effect on health. In the United States, obesity is considered to occur when a person's body mass index (BMI) is 30 kg/m
2 or greater. Obesity increases the likelihood of various diseases, including heart disease, type II diabetes, obstructive sleep apnea, some cancers, and osteoarthritis. It is known that following oral glucose administration, serum GIP levels increase 5-to 6-fold, which stimulates insulin release, which promotes glucose uptake. GIP also activates Akt PKB, which promotes the membrane translocation of glucose transporter-4, leading to enhanced adipocyte uptake of glucose, leading to the creation and storage of fat. As a result, it was hypothesized that antagonizing GIP and reducing its biological activity would inhibit glucose absorption and thus lead to reduced creation and storage of fat. The action of GIP is considered partly responsible for obesity and it has been reported that obesity is actually suppressed by inhibiting the functions of GIP (Miyawaki K et al., Nat Med. 8 (7) : 738-42, 2002) .
Miyawaki K et al. has further reported that GIP is partly responsible for insulin resistance. When insulin resistance occurs, glucose-absorbing effects mediated by insulin are reduced and the organism secrets overmuch insulin by conpensatory action, consequently causing hyperinsulinemia. Insulin resistance tends to cause metabolic syndrome and type 2 diabetes, so the prevention or amelioration of insulin resistance is also important in terms of the risk reduction of such diseases related to insulin-resistance.
Another disease related to glucose metabolism disorders is the metabolic syndrome, the key factors of which are obesity and insulin resistance. Metabolic syndrome can be diagnosed when three of the following five medical conditions are diagnosed together: increased blood pressure, excessive body fat in the waist, high fasting blood glucose, high serum triglycerides, and low level of high-density cholesterol (HDL) . Metabolic syndrome also increases the risk of diseases including type II diabetes, coronary heart disease, fat metabolism disorders, and potentially some mental illness. Treatment includes lifestyle changes and medications.
As such, these links to obesity, insulin secretion and glucose metabolism disorders drive attention to GIP as a potential therapeutic agent for the treatment of diseases related to glucose metabolism disorders such as obesity, diabetes and metabolic syndrome.
WO 2015/095354 discloses that fat retention in the liver, the omentum, or the subcutaneous tissue was reduced by administering GIP monoclonal antibody to mice. Meanwhile, administration of the monoclonal antibody that binds to GIP significantly improved all of the markers associated with obesity and metabolic syndrome (triglyceride levels are reduced, total cholesterol levels are reduced, ratio of high-density lipoprotein to total cholesterol in the sera are increased, average ratio of low-density lipoprotein to total cholesterol in the sera are reduced, average insulin levels are reduced, average adiponectin levels are reduced, average leptin levels are reduced) .
The present inventor realized that such monoclonal antibodies have unfavorable biophysical attributes including low stability and tendency to aggregate, and thus are not ideal for future clinical stage CMC (chemistry, manufacturing and control) development and commercialization.
SUMMARY
The present disclosure relates to molecular antagonists of gastric inhibitory polypeptide (GIP) with improved developability and manufacturability properties, use thereof, and method for treatment of diseases using the molecular antagonists of gastric inhibitory polypeptide.
The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.
Figure 1a and figure 1b show respectively the heavy chain and kappa light chain amino acid sequences, the numbering methodology as well as different domains of Protein X. Figure 1c shows ABHAND residue types of amino acids.
Figure 2 shows the modelded Fab structure of Protein X and the relative arrangement of key domains.
Figure 3 shows that at in vitro level, the binding affinity of the variants is similar to the parent molecule. Thus the introduced mutation to improve stability and reduce aggregation do not negatively impact the antibody’s biological activity.
Figure 4 further demonstrates that in cellular level, after binding, the variants can inhibit downstream signals in GIP pathway to a similar extent as the parent molecule.
Figure 5a and 5b yet further confirm that in in vivo animal model, the biological activity of a variant is no different than the parent molecule. In a mouse oral glucose challenge test, both antibodies attenuated GIP induced insulin elevation; neither affected blood glucose level.
The singular forms “a, ” “an, ” and “the” include plural referents unless the context clearly dictates otherwise.
As used in the specification and in the claims, the open-ended transitional phrases “comprise (s) , ” “include (s) , ” “having, ” “contain (s) , ” and variants thereof require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. These phrases should also be construed as disclosing the closed-ended phrases “consist of” or “consist essentially of” that permit only the named ingredients/steps and unavoidable impurities, and exclude other ingredients/steps.
Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.
All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 grams to 10 grams” is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values) .
The term “about” can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, “about” also discloses the range defined by the absolute values of the two endpoints, e.g. “about 2 to about 4” also discloses the range “from 2 to 4. ” The term “about” may refer to plus or minus 10%of the indicated number.
The term “mutation” is a change that occurs in a nucleic acid sequence or a polypeptide sequence, either due to mistakes during natural process or as the result of genetic engineering. Mutations in a nucleic acid sequence normally relates to changes in bases such as A, C, G and T. Mutations in a polypeptide sequence normally relates to changes in amino acids such as Asp, Glu and Tyr, etc.
The term “identity” refers to the degree of similarity between a pair of sequences (nucleotide or amino acid) . Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Gaps do not count when assessing identity. Thus, two copies of exactly the same sequence have 100%identity, while sequences that have deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST, are available for determining sequence identity. BLAST nucleotide searches are performed with the NBLAST program, and BLAST protein searches are performed with the BLASTP program, using the default parameters of the respective programs.
Two different sequences may vary from each other without affecting the overall function of the protein encoded by the sequence. In this regard, it is well known in the art that chemically similar amino acids can replace each other, often without change in function. Relevant properties can include acidic/basic, polar/nonpolar, electrical charge, hydrophobicity, and chemical structure. For example, the basic residues Lys and Arg are considered chemically similar and often replace each other, as do the acidic residues Asp and Glu, the hydroxyl residues Ser and Thr, the aromatic residues Tyr, Phe and Trp, and the non-polar residues Ala, Val, Ile, Leu and Met. These substitutions are considered “conserved. ” Similarly, nucleotide codons and acceptable variations are known in the art. For example, the codons ACT, ACC, ACA, and ACG all code for the amino acid threonine, i.e. the third nucleotide can be modified without changing the resulting amino acid. Similarity is measured by dividing the number of similar residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Note that similarity and identity measure different properties.
An antagonist is a molecule that binds to a receptor, however, it does not activate the physiological response induced by the naturally occurring physiological ligand of the receptor. An “molecular antagonist of GIP” according to the present disclosure is a molecule that binds to GIP and interferes with the biological action of GIP.
The term “antibody” is a protein used by the immune system to identify a target antigen. The basic functional unit of an antibody is an immunoglobulin monomer. The monomer is made up of two identical heavy chains and two identical light chains which form a Y-shaped protein. Each light chain is composed of one constant domain and one variable domain. For light chains, the constant domain may also be referred to as the “constant region” , and the variable domain can also be referred to as the “variable region” . Each heavy chain is composed of one variable domain and three or four constant domains. For heavy chains, the constant domains together are referred to as the “constant region” , and the variable domain can also be referred to as the “variable region” . The arms of the Y are called the fragment, antigen-binding (Fab) region, with each arm being called a Fab fragment. Each Fab fragment is composed of one constant domain and one variable domain from a heavy chain, and one constant domain and one variable domain from a light chain. The base of the Y is called the Fc region, and is composed of two or three constant domains from each heavy chain. The variable domains of the heavy and light chains in the Fab region are the part of the antibody that binds to antigen (e.g. GIP in the present disclosure) . More specifically, the complementarity determining regions (CDRs) of the variable domains bind to their antigen (e.g. the GIP) . In the amino acid sequence of each variable domain, there are three CDRs arranged non-consecutively. The term “whole” is used herein to refer to an antibody that contains the Fab region and the Fc region.
In some embodiments, the present disclosure is directed to molecular antagonist of gastric inhibitory polypeptide (GIP) .
In some embodiments, the GIP molecular antagonist comprises a heavy chain variable domain having an amino acid sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%identity to SEQ ID NO: 7. In some embodiments, the GIP molecular antagonist comprises a heavy chain variable domain having a first CDR as represented by SEQ ID NO: 1, a second CDR as represented by SEQ ID NO: 2, and a third CDR as represented by SEQ ID NO: 3. In some embodiments, the GIP molecular antagonist comprises a heavy chain variable domain having one or more mutations selected from the group consisting of HV: I31F, HV: Q45R, HV: K50Q, HV: N107A and HV: V108R, and the numbering of the amino acid sequence is calculated based on the numbering system as shown in Figure 1.
In some embodiments, the one or more mutations in the heavy chain variable domain of the GIP molecular antagonist are selected from the following combinations:
HV: I31F;
HV: Q45R;
HV: K50Q;
HV: N107A;
HV: V108R;
HV: I31F + HV: Q45R;
HV: I31F + HV: K50Q;
HV: I31F + HV: N107A;
HV: I31F + HV: V108R;
HV: Q45R + HV: K50Q;
HV: Q45R + HV: N107A;
HV: Q45R + HV: V108R;
HV: K50Q + HV: N107A;
HV: K50Q + HV: V108R;
HV: N107A + HV: V108R;
HV: I31F + HV: Q45R + HV: K50Q;
HV: I31F + HV: Q45R + HV: N107A;
HV: I31F + HV: Q45R + HV: V108R;
HV: I31F + HV: K50Q + HV: N107A;
HV: I31F + HV: K50Q + HV: V108R;
HV: I31F + HV: N107A + HV: V108R;
HV: Q45R + HV: K50Q + HV: N107A;
HV: Q45R + HV: K50Q + HV: V108R;
HV: Q45R + HV: N107A + HV: V108R;
HV: K50Q + HV: N107A + HV: V108R;
HV: I31F + HV: Q45R + HV: K50Q + HV: N107A;
HV: I31F + HV: Q45R + HV: K50Q + HV: V108R;
HV: I31F + HV: Q45R + HV: N107A + HV: V108R;
HV: I31F + HV: K50Q + HV: N107A + HV: V108R;
HV: Q45R + HV: K50Q + HV: N107A + HV: V108R;
HV: I31F + HV: Q45R + HV: K50Q + HV: N107A + HV: V108R.
In some embodiments, the GIP molecular antagonist has a heavy chain variable domain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and SEQ ID NO: 71.
In some embodiments, the GIP molecular antagonist has a heavy chain with an amino acid sequence as represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.
In some embodiments, the heavy chain variable domain of the GIP molecular antagonist is part of a single-chain variable fragment (scFv) , an F (ab')
2 fragment, a Fab or Fab' fragment, a diabody, a triabody, a tetrabody, or a monoclonal antibody.
In some embodiments, the GIP molecular antagonist further comprises a light chain variable domain comprising a first CDR as represented by SEQ ID NO: 4, a second CDR as represented by SEQ ID NO: 5, and a third CDR as represented by SEQ ID NO: 6. In some embodiments, the light chain variable domain of the GIP molecular antagonist comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%identity to SEQ ID NO: 9. In some embodiments, the light chain of the GIP molecular antagonist comprises the amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%identity to SEQ ID NO: 10.
In some embodiments, the heavy chain variable domain and the light chain variable domain of the GIP molecular antagonist are parts of a single-chain variable fragment (scFv) , an F (ab')
2 fragment, a Fab or Fab' fragment, a diabody, a triabody, a tetrabody, or a monoclonal antibody.
A single-chain variable fragment (scFv) includes a light chain variable domain and a heavy chain variable domain, joined together with a linking group which usually has a length of about 10 to about 25 amino acids (though it does not need to be within this range) . The N-terminus of one variable domain is connected to the C-terminus of the other variable domain. If desired, the scFV can be PEGylated (with polyethylene glycol) to increase its size, as with certolizumab pegol. Two scFvs can be joined together with another linking group to produce a tandem scFv.
If a light chain variable domain and a heavy chain variable domain are joined together with a shorter linking group to form an scFv, the two variable domains cannot fold together, and the scFv will dimerize to form a diabody. Even shorter linking groups can result in the formation of trimers (i.e. a triabody) and tetramers (i.e. a tetrabody) .
A whole monoclonal antibody is formed from two heavy chains and two light chains. Again, each light chain and each heavy chain contains a variable domain. Each light chain is bonded to a heavy chain. The two heavy chains are joined together at a hinge region. If the constant region of the heavy chains are removed below the hinge region, an F (ab’ ) 2 fragment is produced which contains a total of four variable domains. The F (ab’ ) 2 fragment can then be split into two Fab’ fragments. An Fab’ fragment contains sulfhydryl groups from the hinge region. A Fab fragment is formed when the constant region of the heavy chains is removed above the hinge region, and does not sulfhydryl groups from the hinge region. However, all of these fragments contain a light chain variable domain and a heavy chain variable domain.
In some embodiments, the GIP molecular antagonist is a whole monoclonal antibody formed from light chains and heavy chains having the variable regions /domains disclosed above, combined with human constant regions. The constant region of the heavy chain can be any human isotype, including lgA1 , lgA2, IgD, IgE, lgG1 , lgG2, lgG3, lgG4, or IgM. The human constant region of the light chain can be the kappa or lambda isotype. In specific embodiments, the heavy chain constant region is the lgG1 isotype, and the light chain constant region is the kappa isotype.
In some embodiments, the GIP molecular antagonist is a monoclonal antibody with a light chain variable domain and a heavy variable domain. The heavy chain variable domain has an amino acid sequence with at least 95%identity to SEQ ID NO: 7, has a first CDR as represented by SEQ ID NO: 1, a second CDR as represented by SEQ ID NO: 2, and a third CDR as represented by SEQ ID NO: 3, and has one or more mutations selected from the group consisting of HV: I31F, HV: Q45R, HV: K50Q, HV: N107A and HV: V108R. The light chain variable domain has at least 95%identity to SEQ ID NO: 9.
In some embodiments, the GIP molecular antagonist is a whole monoclonal antibody with a light chain variable domain as represented by SEQ ID NO: 9, and a heavy chain variable domain selected from SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 43.
In some embodiments, the GIP molecular antagonist is a whole monoclonal antibody with a light chain as represented by SEQ ID NO: 10, and a heavy chain selected from SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO: 44.
In some embodiments, the present disclosure is directed to DNA sequences encoding molecular antagonist of gastric inhibitory polypeptide (GIP) . In some embodiment, DNA sequences encode for amino acid sequences selected from the group consisting of SEQ ID NOs: 7-72. In some embodiments, the DNA sequences encoding GIP molecular antagonist have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%identity to a DNA sequence selected from the group consisting of SEQ ID NOs: 73-137.
In some embodiments, the present disclosure is directed to a pharmaceutical composition comprising GIP molecular antagonist of the present disclosure.
In some embodiments, the GIP molecular antagonist is used in a composition administered to a person. The composition contains a pharmaceutically effective amount of the GIP molecular antagonist. In some embodiments, the composition contains the GIP molecular antagonist in an amount of from about 0.1 to about 1000 milligrams per milliliter of the composition (w/v) . In some embodiments, the GIP molecular antagonist is particularly monoclonal antibody.
The pharmaceutical compositions containing the GIP molecular antagonist is generally administered by a parenteral (i.e. subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleural, intravesicularly or intrathecally) route, as necessitated by choice of drug and disease. The dose used in a particular formulation or application is determined by the requirements of the particular state of disease and the constraints imposed by the characteristics of capacities of the carrier materials. In some embodiments, the composition is administered intravenously, intraperitoneally, or subcutaneously.
In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier. The carrier acts as a vehicle for delivering the molecular antagonist. Examples of pharmaceutically acceptable carriers include liquid carriers like water, oil, and alcohols, in which the molecular antagonists can be dissolved or suspended.
The pharmaceutical composition may also include excipients. Particular excipients include buffering agents, surfactants, preservative agents, bulking agents, polymers, and stabilizers, which are useful with these molecular antagonists. Buffering agents are used to control the pH of the composition. Surfactants are used to stabilize proteins, inhibit protein aggregation, inhibit protein adsorption to surfaces, and assist in protein refolding. Exemplary surfactants include Tween 80, Tween 20, Brij 35, Triton X-10, Pluronic F127, and sodium dodecyl sulfate. Preservatives are used to prevent microbial growth. Examples of preservatives include benzyl alcohol, m-cresol, and phenol. Bulking agents are used during lyophilization to add bulk. Hydrophilic polymers such as dextran, hydroxyl ethyl starch, polyethylene glycols, and gelatin can be used to stabilize proteins. Polymers with nonpolar moieties such as polyethylene glycol can also be used as surfactants. Protein stabilizers can include polyols, sugars, amino acids, amines, and salts. Suitable sugars include sucrose and trehalose. Amino acids include histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, and mixtures thereof. Proteins like human serum albumin can also competitively adsorb to surfaces and reduce aggregation of the protein-like molecular antagonist. It should be noted that particular molecules can serve multiple purposes. For example, histidine can act as a buffering agent and an antioxidant. Glycine can be used as a buffering agent and as a bulking agent.
The pharmaceutical composition may be in the form of a powder, injection, solution, suspension, or emulsion. It is contemplated that the composition will be delivered by injection. Sometimes, the molecular antagonist of GIP can be lyophilized using standard techniques known to those in this art. The lyophilized antagonist may then be reconstituted with, for example, suitable diluents such as normal saline, sterile water, glacial acetic acid, sodium acetate, combinations thereof and the like.
Dose will depend on a variety of factors, including the therapeutic index of the drugs, disease type, patient age, patient weight, and tolerance. The dose will generally be chosen to achieve serum concentrations from about 1 ng/ml to about 10 mg/ml in the patient. The dose of a particular patient can be determined by the skilled clinician using standard pharmacological approaches in view of the above factors. The response to treatment may be monitored by analysis of blood or body fluid levels of glucose or insulin levels, or by monitoring fat levels in the patient. The skilled clinician will adjust the dose based on the response to treatment revealed by these measurements. A single administration may usually be sufficient to produce a therapeutic effect, but it is contemplated that multiple administrations will be used to assure continued response over a substantial period of time. Because of the protein-like nature of the molecular antagonists disclosed herein, it is believed that the antagonists will have a long half-life in the body, so that the composition will only need to be administered once or twice a month, or possibly once a week. The circulating concentration of the molecular antagonist should be sufficient to neutralize GIP that is generated during and after eating.
In some embodiments, the present disclosure is directed to method of treating diseases related to glucose metabolism disorders such as obesity, diabetes and metabolic syndrome, comprising administering to a person a pharmaceutically effective amount of a GIP molecular antagonist of the present disclosure.
The term "treat" is used to refer to a reduction in progression of the disease, a regression in the disease, and/or a prophylactic usage to reduce the probability of presentation of the disease. This can be measured in several different ways, such as via the improvements of patient conditions, physical parameters and representative in vivo and in vitro indexes of diseases.
Upon administration to patients, GIP molecular antagonist will inhibit binding of GIP to its receptor in enterocytes, diminishing postprandial glucose absorption and nutrient storage associated with an elevated insulin response by attenuating GIP-induced insulin secretion from pancreatic beta cells. Inhibiting binding of GIP to its receptor in adipocytes should also decreased GIP-induced glucose uptake into fat cells and prevent GIP from inhibiting lipolysis. Overall, this results in less efficient nutrient uptake and storage, promoting weight loss. These effects also benefit patients with diseases related to glucose metabolism disorders such as obesity, diseases and metabolic syndrome.
In a first aspect, the present disclosure relates to a molecular antagonist of gastric inhibitory polypeptide (GIP) , comprising a heavy chain variable domain having an amino acid sequence with at least 95%identity to SEQ ID NO: 7, wherein said heavy variable domain comprises:
a first CDR as represented by SEQ ID NO: 1,
a second CDR as represented by SEQ ID NO: 2,
a third CDR as represented by SEQ ID NO: 3, and
one or more mutations selected from the group consisting of HV: I31F, HV: Q45R, HV: K50Q, HV: N107A and HV: V108R, and the numbering of the amino acid sequence is calculated based on the numbering system as shown in Figure 1.
In a second aspect, the present disclosure relates to the GIP molecular antagonist according to the first aspect, wherein the one or more mutations is selected from the following groups:
HV: I31F;
HV: Q45R;
HV: K50Q;
HV: N107A;
HV: V108R;
HV: I31F + HV: Q45R;
HV: I31F + HV: K50Q;
HV: I31F + HV: N107A;
HV: I31F + HV: V108R;
HV: Q45R + HV: K50Q;
HV: Q45R + HV: N107A;
HV: Q45R + HV: V108R;
HV: K50Q + HV: N107A;
HV: K50Q + HV: V108R;
HV: N107A + HV: V108R;
HV: I31F + HV: Q45R + HV: K50Q;
HV: I31F + HV: Q45R + HV: N107A;
HV: I31F + HV: Q45R + HV: V108R;
HV: I31F + HV: K50Q + HV: N107A;
HV: I31F + HV: K50Q + HV: V108R;
HV: I31F + HV: N107A + HV: V108R;
HV: Q45R + HV: K50Q + HV: N107A;
HV: Q45R + HV: K50Q + HV: V108R;
HV: Q45R + HV: N107A + HV: V108R;
HV: K50Q + HV: N107A + HV: V108R;
HV: I31F + HV: Q45R + HV: K50Q + HV: N107A;
HV: I31F + HV: Q45R + HV: K50Q + HV: V108R;
HV: I31F + HV: Q45R + HV: N107A + HV: V108R;
HV: I31F + HV: K50Q + HV: N107A + HV: V108R;
HV: Q45R + HV: K50Q + HV: N107A + HV: V108R;
HV: I31F + HV: Q45R + HV: K50Q + HV: N107A + HV: V108R.
In a third aspect, the present disclosure relates to the GIP molecular antagonist according to the second aspect, wherein the heavy variable domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and SEQ ID NO: 71.
In a fourth aspect, the present disclosure relates to the GIP molecular antagonist according to the third aspect, comprising a heavy chain with an amino acid sequence as represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.
In a fifth aspect, the present disclosure relates to the GIP molecular antagonist according to any one of the first to fourth aspects, wherein the heavy chain variable domain is part of a single-chain variable fragment (scFv) , an F (ab') 2 fragment, a Fab or Fab' fragment, a diabody, a triabody, a tetrabody, or a monoclonal antibody.
In a sixth aspect, the present disclosure relates to the GIP molecular antagonist according to any one of the first to fourth aspects, further comprising a light chain variable domain, wherein said light chain variable domain comprises:
a first CDR as represented by SEQ ID NO: 4
a second CDR as represented by SEQ ID NO: 5, and
a third CDR as represented by SEQ ID NO: 6.
In a seventh aspect, the present disclosure relates to the GIP molecular antagonist according to the sixth aspect, wherein the light chain variable domain comprises an amino acid sequence having at least 95%identity to SEQ ID NO: 9.
In an eighth aspect, the present disclosure relates to the GIP molecular antagonist according to the seventh aspect, comprising a light chain with an amino acid sequence having at least 95%identity to SEQ ID NO: 10.
In a ninth aspect, the present disclosure relates to the GIP molecular antagonist according to any one of the sixth to eighth aspects, wherein the heavy chain variable domain and the light chain variable domain are parts of a single-chain variable fragment (scFv) , an F (ab') 2 fragment, a Fab or Fab' fragment, a diabody, a triabody, a tetrabody, or a monoclonal antibody.
In a tenth aspect, the present disclosure relates to a DNA sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NOs: 7-72.
In an eleventh aspect, the present disclosure relates to the DNA sequence according to the tenth aspect, which have at least 85%identity to a DNA sequence selected from the group consisting of SEQ ID NOs: 73-137.
In a twelfth aspect, the present disclosure relates to a pharmaceutical composition comprising the GIP molecular antagonist according to any one of the first to ninth aspects.
In a thirteen aspect, the present disclosure relates to a method of treating diseases related to glucose metabolism disorders such as obesity, diabetes and metabolic syndrome, comprising: administering to a person a composition comprising a pharmaceutically effective amount of a molecular antagonist of gastric inhibitory polypeptide (GIP) according to any one of the first to ninth aspects.
The present disclosure will further be illustrated in the following non-limiting working examples, it being understood that these examples are intended to be illustrative only and that the disclosure is not intended to be limited to the materials, conditions, process parameters and the like recited herein. All proportions are by weight unless otherwise indicated.
Examples
Example 1: Sequence Design
The parental sequence used in the present disclosure is Protein X, with the full length amino acid sequence of its heavy chain (SEQ ID NO: 8) and the amino acid sequence of its heavy chain variable region (SEQ ID NO: 7) disclosed in Figure 1. a, and the full length amino acid sequence of its kappa light chain (SEQ ID NO: 10) the amino acid sequence of its light chain variable region (SEQ ID NO: 9) disclosed in Figure 1. b. The CDRs of Protein X is SEQ ID NO: 1 for HC CDR1, SEQ ID NO: 2 for HC CDR2, SEQ ID NO: 3 for HC CDR3; SEQ ID NO: 4 for LC CDR1, SEQ ID NO: 5 for LC CDR2, and SEQ ID NO: 6 for LC CDR3.
The Antibody Modeler tool within the Molecular Operating Environment (MOE) was used to generate a Fab model of Protein X. The Fv framework pair chosen as a template from the Protein Data Bank (rcsb. org) was 4XAK. L for the light chain and 4XAK. H for the heavy chain. The CDR templates chosen were: L1: 4M1G.L, L2: 3LS5.L, L3: 3SO3.B, H1: 12E8.P, H2: 1QOK.A Domain 1, H3: 1KEM.H. The Fab model was built using default parameters, with ten main chain and three side chain models generated per main chain. Intermediate models were subjected to the Medium minimization protocol, while the final structure chosen with GB/VI scoring, was subjected to the Fine minimization protocol. The AMBER99 (R-Field) force field was used. Fab structure model of Protein X constructed is shown in Figure 2.
Based on humanization, germline analysis, hot spot analysis and surface patch analysis, the inventor of the present disclosure designed 31 variants as candidates. Protein and DNA coding sequences of the heavy chains (HC) and heavy chain variable regions (VH) of the 31 variants are shown in the following table 1.
Table 1
All variants share the same kappa light chain, the protein and DNA coding sequences of which are shown in the following table 2.
Table 2
Ab Structure IDs | LC Prot. | LC Nuc. | VL Prot. | VL Nuc. |
SEQ ID Nos: | SEQ ID Nos: | SEQ ID Nos: | SEQ ID Nos: | |
ProteinX and Protein X 001-031 | 10 | 138 | 9 | 137 |
Example 2: Cloning, transfection and antibody production
Production of the optimized antibodies using CHO stable transfection system: An expression plasmid pDL5 with the coding sequence of the antibody heavy chain (GenScript) and an expression plasmid pDL2 with the coding sequence of the antibody light chain (GenScript) were co-transfected with a helper plasmid pDL4 into the CHO-K1 host cells using Lipofectamine 3000 (Invitrogen L3000-015) following the manufacturer recommended procedure. The transfected cells were incubated in media without selection pressure for 1 day, then in media with 20mg/L puromycin for 6 days. The recovered cells were cultured in production media to allow the accumulation of the secreted antibody in the spent media.
Materials used in Examle 3 were obtained from small scale purification: The spent media, after clarification by centrifugation, were used for antibody purification with Protein-A column (Nab Protein A Plus Spin Column, Thermo Scientific, Cat#89952) following manufacture’s protocol. Purified antibody was then subjected to endotoxin removal by a high-capacity endotoxin removal column (Thermo Scientific, Cat#88274) . Endotoxin level after the removal step was tested using E-Toxate Kit (Sigma-Aldrich Cat#ET0100, ET0200 and ET0300) and verified to be less than 0.1 EU/mL. Antibody concentration was measured by OD280.
Materials used in Examples 4, 5 and 6 were obtained from fed-batch production: The stable pools of transfected cells were thawed and expanded for generation of materials in fed-batch. Stable pools from each molecule were expanded into shake flask and passaged in medium M1 (CD FortiCHO + 4mM Glutamine + 5μg/mL of Blasticidin + 10 μg/mL of Puromycin) for approximately two weeks before inoculated for fed-batch cultures. Upon fed-batch inoculation, cells were directly diluted into medium M2 (ActiPro + 4mM Glutamine +1X GS supplement) at a density about 10x10
6 cells/mL in shake flask with initial working volume of 200mL on day 0. The fed-batch cultures were in incubated in InFors HT incubator (37.0℃, 85%humidity, 5%CO2, 110 RPM, 50mm orbital throw) . Feed media were added into the fed-batch cultures on day 1, 3, 5, 7 and 9. Glucose was added into the cultures to maintain its level at >3 g/L and GlutaMAX was added into the cultures to maintain glutamine level at > 2 mM. Culture temperature was shifted to 32℃ on day 2 when cell density reached about 20x10
6 cell/mL. Fed-batch cultures were harvested on day 10. Supernatants from harvested cultures were measured for titers by Cedex Bio analyzer. Supernatants of fed-batch cultures were purified by one-step Protein A chromatography.
Example 3: Variant Characterization and Selection
A series of biophysical techniques designed to interrogate conformational and colloidal stability were conducted for these 31 variants as well as the parental Protein X to select variants with more beneficial properties. The following table 3 shows characteristics and desired outcomes for all biophysical methods used for variant characterization. Additional details are contained in the following subsections from each method.
Table 3: Analytical methods for IgG analysis
3.1 Size Exclusion Chromatography (SE-HPLC)
SE-HPLC separates proteins based on differences in their hydrodynamic volumes. Molecules with larger hydrodynamic volumes, such as dimers, elute earlier than molecules with smaller volumes, such as monomers. Undiluted samples were loaded onto a Waters XBridge Protein BEH SEC 200A column (3.5 μm, 7.8 x 300 mm) , separated isocratically with a 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8 running buffer and the eluent monitored by UV absorbance at 220 nm. Purity was determined by calculating the percentage of each separated component as compared to the total integrated area. The following table 4 contains the percentage of high molecular weight (HMW, dimer + oligomer) and main peak for each of the variants. All variants tested showed less %HMW.
Table 4. Protein X Size Exclusion Data
Ab Structure | %HMW | %Main |
ProteinX_SP_K | 11.55 | 88.45 |
ProteinX_SP_K. 001 | 10.52 | 89.48 |
ProteinX_SP_K. 002 | 2.67 | 97.33 |
ProteinX_SP_K. 003 | 8.79 | 91.21 |
ProteinX_SP_K. 004 | 4.09 | 95.91 |
ProteinX_SP_K. 005 | 10.46 | 89.54 |
ProteinX_SP_K. 006 | 3.68 | 96.32 |
ProteinX_SP_K. 008 | 5.09 | 94.91 |
ProteinX_SP_K. 009 | 4.06 | 95.94 |
ProteinX_SP_K. 010 | 2.05 | 97.95 |
ProteinX_SP_K. 011 | 4.27 | 95.73 |
ProteinX_SP_K. 012 | 2.51 | 97.49 |
ProteinX_SP_K. 013 | 3.17 | 96.83 |
ProteinX_SP_K. 014 | 7.93 | 92.07 |
ProteinX_SP_K. 015 | 4.08 | 95.92 |
ProteinX_SP_K. 016 | 3.35 | 96.65 |
ProteinX_SP_K. 017 | 5.82 | 94.18 |
ProteinX_SP_K. 018 | 3.90 | 96.10 |
ProteinX_SP_K. 019 | 1.67 | 98.33 |
ProteinX_SP_K. 023 | 3.14 | 96.86 |
ProteinX_SP_K. 025 | 0.98 | 99.02 |
ProteinX_SP_K. 026 | 1.80 | 98.20 |
ProteinX_SP_K. 027 | 2.10 | 97.90 |
ProteinX_SP_K. 028 | 1.55 | 98.45 |
ProteinX_SP_K. 029 | 2.75 | 97.25 |
ProteinX_SP_K. 030 | 1.29 | 98.71 |
ProteinX_SP_K. 031 | 0.94 | 99.06 |
3.2 Differential Scanning Fluorimetry (DSF)
DSF is a high throughput technique that is used to estimate a protein’s relative conformational stability and by ranking the results, can be used as a tool to select candidates with more favorable stability properties. The DSF technique consists of measuring the intrinsic fluorescence intensity of a molecule at gradually increasing temperatures to determine the transition temperature based on exposure of the hydrophobic regions of a protein. The data is reported as Tm1 and Tm2 with the first transition correlating with the CH2 domain and the 2nd transition correlating with the unfolding of the Fab and CH3 domain regions. Higher unfolding temperatures are desirable and have been linked with an increase in a product’s conformational stability. Lack of a Tm2 is indicative of the Fab unfolding at the same or similar temperature to the CH2 domain, reported as Tm1. Additional information is also obtained from a proprietary parameter termed the weighted shoulder score which accounts for multiple pieces of information from the unfolding curve. Again, higher values are indicative of greater conformational stability.
The DSF analysis for the variants was conducted in PBS buffer using a Prometheus NT.48 instrument, samples were heated from 20℃ to 100℃ at a rate of 1.5 degrees per minute. The weighted shoulder score and transition temperatures are summarized in the following Table 5. Improvement was observed in many of the variants as compared with the parent molecule.
Table 5: Protein X DSF Data
3.3 Self-interaction Nano Particle Spectroscopy (SINS)
Protein-protein self-interaction has been correlated to low solubility, high viscosity and increased manufacturability difficulties. Therefore, it is important to understand and screen out molecules early in development that self-interact. The SINS assay utilizes gold colloid surfaces to measure protein-protein interaction. Gold colloids have unique optical absorption properties dependent on their aggregation state, absorbing light at longer wavelengths as nanoparticle aggregation occurs. Protein self-interaction is assessed by capturing test antibodies on the surface of a gold colloid and measuring the shift of the absorbance maxima. If the immobilized antibodies self-interact, the absorbance maximum of the spectrum red shifts to longer wavelengths, or blue shifts to shorter wavelengths if less interaction occurs. SINS requires minimal sample mass and has a high throughput advantage over traditional B22 measurements for protein self-interaction.
The wavelength maximum of the SINS data is summarized in the following table. Some improvement in protein-protein interactions as measured by SINS was observed for Protein X variants when tested in PBS. Data produced in-house have demonstrated that antibodies with high viscosity typically have red shifts in the 20-30 nm range compared with the PBS control sample, and therefore variants that showed a reduction in SINS values may experience less manufacturing challenges when exposed to a high salt/pH buffering system.
Table 6: Protein X SINS Data
3.4 Low pH Aggregation
The low pH aggregation assay can be used to help select candidates that are suitable for low pH viral inactivation. In this assay, samples are titrated to pH 3.3 using 2 M acetic acid and held for 30 minutes before being neutralized to pH 5 with 2 M tris base. The samples are characterized using SE-HPLC as described in section 4.1. Samples that were diluted with PBS using the same volume of acetic acid and tris base were used as a control. Molecules that demonstrated a significant increase in high molecular weight would be considered as unstable during low pH exposure and needing further method development if low pH viral inactivation were used. The following table contains the low pH data for Protein X variants. All Protein X variants showed little to no aggregation after low pH stress.
Table 7: Protein X Low pH Stress Data
3.5 Conclusion
Optimized molecules were identified based on demonstrating improvement in multiple biophysical attributes as compared with the parent Protein X. Two representative optimized molecules, ProteinX_SP_K. 003 and ProteinX_SP_K. 017 were selected and subjected to further biological activty evaluation.
Example 4: hGIP Binding affinity
In order to analyze the binding affinity of the variants of the present disclosure, the following procedure is conducted for a binding ELISA assay.
4.1 Main reagents and materials:
1) Gastric inhibitory polypeptide, GIP (Phoenix pharmaceuticals, Inc. Cat No. : 027-02)
2) 96-well plate (Corning, Cat No. : 3590)
3) Goat anti-Human IgG-Fc HRP conjugated (Bethyl Laboratories, Cat No. : A80-104P)
4) TMB substrate (SurModics, Cat No. : TMBW-1000-01)
4.2 Procedure
Coat Gastric inhibitory polypeptide GIP (Phoenix pharmaceuticals, Inc. Cat No. : 027-02) in 96-well plate (4ng GIP in 50 μl 1xPBS per well, as 80ng/ml) , sealed with plate sealer film, incubate at 4℃ overnight. Wash the wells of the plate with 300ul/well PBST for 4 times. Prepare 3%BSA-PBST. Pipette 200 μl of 3%BSA-PBST into each well. Seal the plate with plate sealer film, incubate at 37℃ for 2 hours. Wash the wells of the plate with 300 μl/well PBST for 4 times.
Prepare primary antibody dilutions using 1%BSA-PBST. Prepare the following 1: 4 serial dilutions: 320.0 μg/ml, 80.0 μg/ml, 20.0 μg/ml, 5.0 μg/ml, 1.25 μg/ml, 0.31 μg/ml, 0.078 μg/ml, 0.020 μg/ml, 0.0049 μg/ml, 0.0012 μg/ml, 0.00031 μg/ml, 0 μg/ml, 50 μl/well, run in duplicate. Sealed with plate sealer film. Incubate for two hours at 37℃. Wash the wells of the plate with 300 μl/well PBST for 4 times.
Read the plate on EnVision multimode plate reader using the parameters λ1: 450nm and λ2: 650nm. Save the raw data file and analyze the result.
The result is shown in Figure 3. Both variants shown similar binding affinity comnparing to parental control.
Example 5: Functional cell-based assay using reporter cell line
The effectiveness of selected derivatives of humanized anti-GIP mAbs (2 variants of the present disclosure) is evaluated in a cell-based reporter assay. The ability of candidates to neutralize GIP and prevent ligand-receptor interaction, receptor activation, and receptor-dependent signaling was tested using the GIPR-expressing reporter cell line.
Briefly, purified antibodies at various concentrations were mixed with increasing amounts of GIP in DMEM containing 10%FBS. After incubation, the mixtures were added to LGIPR reporter cells. These reporter cells were derived by the transduction of LVIP cells with lentiviral pseudoparticles containing cDNAs encoding the GIPR (GIP Receptor) . Upon GIP stimulation, LGIPR cells will increase LacZ gene expression. The cells were incubated before the mixtures were removed, and the cells were then washed and reporter activity was assayed by measuring accumulated beta-galactosidase. More details can be found with reference to Am J Physiol Endocrinol Metab 309: E1008–E1018, 2015, Endocrinology 135: 2662–2680, 1994. and Endocrinology 133: 2861–2870, 1993.
The result is shown in Figure 4. Data were plotted as Schild regreesion to describe the ligand-receptor signaling (GIP, y-axis) in the presence of avarious concentration of the antagonist (GIP antibody, x-axis) . Data indicate that both variants shown comparable cell specific activities comparing to parental control, as the ligand induced signaling events respond similarly to the change of antibody concentration for each molecule.
Example 6: Oral glucose tolerance test
The glucose tolerance test measures the clearance of an oral glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to human conditions such as obesity, diabetes or metabolic syndrome.
Materials and Methods
110 six-week old male C57BL/6J mice were purchased from Shanghai SLAC (Lingchang) Laboratory Animal Co. LTD. (Fengxian, Shanghai, P. R. China) for acclimation, and 90 of them were used for dosing when they were nine-week old.
Animals are fed with Rats and rats Growth and Reproduction Diet provided by XIETONG ORGANISM (Lot#2018) , with principal raw materials of Peru fish meal, chicken, corn, bean pulp, plant oil, bran, vitamin, mineral substance, essential amino acid etc. Chemical composition of the diet is shown in the following table 8.
Table 8
Mice were selected based on body weight. On the first day of the study, each mouse weighed between 20 and 25 grams. The mice were then subsequently divided at random into the following five dose groups, with 18 mice per dose group.
Table 9
On the day of the test, mice were moved to procedure room from 8: 00 am and placed on the bench and these mice were kept on the same bench where the glucose tolerance experiment was conducted so that they can be accustomed to the area to reduce stress during the procedure.
Blood glucose level was determined immediately prior the dosing. Dose the mice with vehicle or test article at ~9 am by IP injection.
Five hours later, blood glucose level were determined (t=0 min) , then immediately the animals were challenged with glucose (2g/kg) by oral administration. Blood glucose was measured at 15 and 30min post glucose dose.
The result is shown in Figure 5. The tested variant showed the same effect on insulin and glucose as the parental molecule: attenuated GIP induced insulin elevation while not impacting glucose level.
While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intented to embrace all such alternatives, modifications, variations, improvements, and substantial equivalents.
Claims (13)
- A molecular antagonist of gastric inhibitory polypeptide (GIP) , comprising a heavy chain variable domain having an amino acid sequence with at least 95%identity to SEQ ID NO: 7, wherein said heavy variable domain comprises:a first CDR as represented by SEQ ID NO: 1,a second CDR as represented by SEQ ID NO: 2,a third CDR as represented by SEQ ID NO: 3, andone or more mutations selected from the group consisting of HV: I31F, HV: Q45R, HV: K50Q, HV: N107A and HV: V108R, and the numbering of the amino acid sequence is calculated based on the numbering system as shown in Figure 1.
- The GIP molecular antagonist according to claim 1, wherein the one or more mutations is selected from the following groups:HV: I31F;HV: Q45R;HV: K50Q;HV: N107A;HV: V108R;HV: I31F + HV: Q45R;HV: I31F + HV: K50Q;HV: I31F + HV: N107A;HV: I31F + HV: V108R;HV: Q45R + HV: K50Q;HV: Q45R + HV: N107A;HV: Q45R + HV: V108R;HV: K50Q + HV: N107A;HV: K50Q + HV: V108R;HV: N107A + HV: V108R;HV: I31F + HV: Q45R + HV: K50Q;HV: I31F + HV: Q45R + HV: N107A;HV: I31F + HV: Q45R + HV: V108R;HV: I31F + HV: K50Q + HV: N107A;HV: I31F + HV: K50Q + HV: V108R;HV: I31F + HV: N107A + HV: V108R;HV: Q45R + HV: K50Q + HV: N107A;HV: Q45R + HV: K50Q + HV: V108R;HV: Q45R + HV: N107A + HV: V108R;HV: K50Q + HV: N107A + HV: V108R;HV: I31F + HV: Q45R + HV: K50Q + HV: N107A;HV: I31F + HV: Q45R + HV: K50Q + HV: V108R;HV: I31F + HV: Q45R + HV: N107A + HV: V108R;HV: I31F + HV: K50Q + HV: N107A + HV: V108R;HV: Q45R + HV: K50Q + HV: N107A + HV: V108R;HV: I31F + HV: Q45R + HV: K50Q + HV: N107A + HV: V108R.
- The GIP molecular antagonist according to claim 2, wherein the heavy variable domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and SEQ ID NO: 71.
- The GIP molecular antagonist according to claim 3, comprising a heavy chain with an amino acid sequence as represented by SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 72.
- The GIP molecular antagonist according to any one of claims 1 to 4, wherein the heavy chain variable domain is part of a single-chain variable fragment (scFv) , an F (ab') 2 fragment, a Fab or Fab' fragment, a diabody, a triabody, a tetrabody, or a monoclonal antibody.
- The GIP molecular antagonist according to any one of claism 1 to 4, further comprising a light chain variable domain, wherein said light chain variable domain comprises:a first CDR as represented by SEQ ID NO: 4a second CDR as represented by SEQ ID NO: 5, anda third CDR as represented by SEQ ID NO: 6.
- The GIP molecular antagonist according to claim 6, wherein the light chain variable domain comprises an amino acid sequence having at least 95%identity to SEQ ID NO: 9.
- The GIP molecular antagonist according to claim 7, comprising a light chain with an amino acid sequence having at least 95%identity to SEQ ID NO: 10.
- The GIP molecular antagonist according to any one of claims 6 to 8, wherein the heavy chain variable domain and the light chain variable domain are parts of a single-chain variable fragment (scFv) , an F (ab') 2 fragment, a Fab or Fab' fragment, a diabody, a triabody, a tetrabody, or a monoclonal antibody.
- A DNA sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NOs: 7-72.
- The DNA sequence according to claim 10, which have at least 85%identity to a DNA sequence selected from the group consisting of SEQ ID NOs: 73-137.
- A pharmaceutical composition comprising the GIP molecular antagonist according to any one of claims 1 to 9.
- A method of treating diseases related to glucose metabolism disorders such as obesity, diabetes and metabolic syndrome, comprising:administering to a person a composition comprising a pharmaceutically effective amount of a molecular antagonist of gastric inhibitory polypeptide (GIP) according to any one of claims 1 to 9.
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JP2013138638A (en) * | 2011-12-28 | 2013-07-18 | Meneki Seibutsu Kenkyusho:Kk | Anti-bioactive gip antibody and use of the same |
CN106068125A (en) * | 2013-12-17 | 2016-11-02 | Mhs克尔创新有限责任公司 | The compositions for the treatment of fatty tissue accumulation and method |
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CN101119749A (en) * | 2004-10-25 | 2008-02-06 | 赛托斯生物技术公司 | Gastric inhibitory polypeptide (gip) antigen arrays and uses thereof |
JP2013138638A (en) * | 2011-12-28 | 2013-07-18 | Meneki Seibutsu Kenkyusho:Kk | Anti-bioactive gip antibody and use of the same |
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