Methods and compositions for treating cancer
阅读说明：本技术 用于治疗癌症的方法和组合物 (Methods and compositions for treating cancer ) 是由 J.泰兹 B.塔马里特 P.保利蒂 于 2020-02-05 设计创作，主要内容包括：本发明涉及用于在有此需要的受试者中治疗癌症的组合物和方法。本发明更具体地涉及通过在有此需要的受试者中抑制PLA2GIB来治疗癌症的方法。(The present invention relates to compositions and methods for treating cancer in a subject in need thereof. The invention more particularly relates to methods of treating cancer by inhibiting PLA2GIB in a subject in need thereof.)
1. A compound that inhibits PLA2-GIB for use in treating cancer or neoplasia in a subject in need thereof.
2. The compound for use according to claim 1, wherein the compound is an antibody or fragment or derivative thereof that binds to PLA 2-GIB.
3. The compound for use according to claim 2, wherein the compound is a monoclonal antibody.
4. The compound for use according to claim 3, wherein the monoclonal antibody is human or humanized.
5. The compound for use according to claim 1, wherein the compound is 3- (2-amino-1, 2-dioxoethyl) -2-ethyl-1- (phenylmethyl) -1H-indol-4-yl) oxy) acetic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof, such as a sodium salt thereof.
6. The compound for use according to claim 1, wherein the compound is a vaccine against PLA 2-GIB.
7. The compound for use according to claim 1, wherein said compound is a cyclic peptide selected from the group consisting of FLSYK, FLSYR and (2NapA) LS (2NapA) R.
8. The compound for use according to any one of claims 1 to 7, wherein said compound is administered in combination with a further anti-cancer agent, vaccine or treatment.
9. The compound for use according to claim 8, wherein the compound is administered in combination with chemotherapy or hormone therapy.
10. The compound for use according to claim 8, wherein the compound is administered in combination with radiation therapy, ultrasound therapy or nanoparticle therapy.
11. The compound for use according to claim 8, wherein said compound is administered in combination with a checkpoint inhibitor, immunotherapy or anti-cancer vaccine.
12. The compound for use according to claim 8, wherein the compound is administered in combination with an inhibitor of the co-factor of PLA 2-GIB.
13. The compound for use according to claim 12, wherein the inhibitor is an antagonist of PLA2-GIB cofactor or is a cytostatic or cytotoxic agent or vaccine against PLA2-GIB cofactor or against prokaryotic or eukaryotic cells or viruses expressing PLA2-GIB cofactor.
14. The compound for use according to any one of claims 1 to 13, wherein the compound is administered before, during or after surgery.
15. The compound for use according to any one of claims 1 to 14, wherein the compound is administered repeatedly.
16. The compound for use according to any one of the preceding claims, wherein the cancer is a solid cancer.
17. The compound for use according to any one of claims 1 to 16, wherein the PLA2-GIB cofactor or a prokaryotic or eukaryotic cell or virus expressing the PLA2-GIB cofactor is present in the subject.
18. The compound for use according to any one of claims 1 to 17, wherein PLA2-GIB or PLA2-GIB cofactor is present in the cancer microenvironment or blood.
19. The compound for use according to any one of claims 1 to 18, wherein the subject has a PLA2 GIB-associated CD 4T cell deficiency.
20. The compound for use according to claim 16, wherein the cancer is selected from pancreatic cancer, melanoma, lung cancer, esophageal or throat cancer, retinoblastoma, liver cancer, breast cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, uterine cancer, cervical cancer, thyroid cancer, bladder cancer, prostate cancer, bone cancer, brain cancer or colorectal cancer.
21. The compound for use according to claim 20, wherein the cancer is pancreatic cancer.
22. The compound for use according to claim 21, wherein the cancer is selected from pancreatic adenocarcinoma, neuroendocrine tumors, intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, and severe cystic neoplasms.
23. The compound for use according to claim 1, wherein the cancer induces gastrointestinal and metabolic conditions.
24. A compound for use according to any one of claims 1 to 23 for use in the prevention or reduction of the incidence of cancer.
25. A compound for use according to any one of claims 1 to 23 for use in reducing the rate of cancer progression.
26. A compound for use according to any one of claims 1 to 23 for use in the reduction or prevention or treatment of cancer metastasis.
27. A compound for use according to any one of claims 1 to 23 for use in killing cancer cells.
28. A compound for use according to any one of claims 1 to 23 for use in the treatment of risk factors for cancer, in particular oral-gastro-intestinal inflammation or infection, such as pancreatitis.
29. The compound for use according to any one of claims 1 to 28, wherein the subject is a human.
30. The compound for use according to any one of claims 1 to 29, wherein the level of PLA2-GIB or PLA2-GIB cofactor in the tumor or body fluid is measured to guide a treatment regimen.
Technical Field
The present invention relates to compositions and methods for treating cancer in a subject in need thereof. The invention more particularly relates to methods of treating cancer by inhibiting PLA2GIB in a subject in need thereof.
Background
Group IB pancreatic secreted phospholipase a2(PLA2-GIB) is a low molecular weight (14kD), highly stable (7 disulfide bonds) secreted protein that catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids to release free fatty acids and lysophospholipids (Lambeau & Gelb 2008). The inventors identified PLA2-GIB as being involved in CD 4T inactivation in HIV-infected patients (WO 2015/097140).
By continuing the study, the inventors have discovered the presence of the PLA2-GIB cofactor in cancer patients. The inventors have shown that such cofactors together with PLA2-GIB induce the inactivation of immune cells in cancer patients, allowing tumors to evade immune responses. The inventors have further shown that inhibition of PLA2-GIB can restore an effective immune response and thus effectively contribute to cancer treatment.
Disclosure of Invention
An object of the present invention relates to a method for treating cancer in a subject comprising exposing the subject to a compound that inhibits PLA 2-GIB.
A further object of the present invention relates to a compound inhibiting PLA2-GIB for use in treating cancer in a subject.
A further object of the invention relates to the use of a compound that inhibits PLA2-GIB for the manufacture of a medicament for treating cancer in a subject.
Compounds that inhibit PLA2-GIB may be used alone or in combination with one or more other treatments, such as further anti-cancer agents, surgery, vaccines, radiotherapy, ultrasound therapy or inhibitors of PLA2-GIB co-factors.
The invention can be used for the treatment of any tumor or cancer, such as in particular pancreatic (pancreatic cancer), melanoma (melanoma), lung, oesophageal or throat cancer, retinoblastoma, liver, breast, ovarian, kidney, stomach, duodenal, uterine, cervical, thyroid, bladder, prostate, bone, brain or colorectal cancer. The invention may be used in any mammalian, in particular human, subject.
Drawings
FIG. 1: determination of PLA2-GIB concentrations (pre, active and total) in plasma from PDAC cohort compared to control cohort.
FIG. 2: in vitro effect of PDAC plasma on IL-7 response of CD 4T cells from healthy donors (A). Influence of PLA2-GIB inhibitor (B).
FIG. 3: in vitro effect of PDAC plasma on IL-2 response of CD 4T cells from healthy donors (A). Influence of PLA2-GIB inhibitor (B).
Detailed Description
The present invention relates to compositions and methods for treating cancer and neoplasia (neoplasma) in a subject in need thereof.
Definition of
As used herein, "treatment" or "treating" refers to clinical intervention in an attempt to alter the natural course of disease in the individual receiving treatment, and may be performed for prophylactic or curative purposes. Desirable effects of cancer treatment include, but are not limited to, preventing the occurrence or recurrence of cancer, alleviating symptoms, preventing and treating metastasis, reducing or slowing the progression of cancer, ameliorating or palliating the state of cancer, causing or allowing remission of cancer, or causing or inducing destruction of cancer cells, or providing a synergistic effect for other anti-cancer therapies. Treatment also encompasses any delay in the development of the cancer.
"subject" refers to a mammal. Examples of mammals include humans and non-human animals, such as, but not limited to, farm animals (e.g., cows, sheep, cats, dogs, and horses), non-human primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats). The invention is particularly suitable for treating humans.
As used herein, the term "isolated" refers to molecules (e.g., nucleic acids or amino acids) that are removed, separated, or separated from components of their natural environment and are at least 60% free, preferably 75% free, and most preferably 90% free of other components with which they are naturally associated. An "isolated" polypeptide (or protein) is a polypeptide that has been separated, for example, from components of its natural environment, and preferably purified to greater than 90% or 95% purity as determined, for example, by migration by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). An "isolated" nucleic acid is a nucleic acid molecule that is isolated from a component of its natural environment and/or assembled in a different construct (e.g., a vector, expression cassette, recombinant host, etc.).
The term "sequence identity" as applied to Nucleic acid or protein sequences refers to the quantification (usually as a percentage) of nucleotide or amino acid residue matches between at least two sequences aligned using a standardized algorithm such as the Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al, (1994) Nucleic Acids Res 22:4673-4680), or BLAST2(Altschul et al, (1997) Nucleic Acids Res 25: 3389-3402). BLAST2 can be used in a standardized and reproducible manner to insert gaps in one of the sequences in order to optimize the alignment and achieve a more meaningful comparison between them.
PLA2-GIB
As used herein, "PLA 2-GIB" (or "PLA 2 GIB" or "GIBsPLA 2") represents group IB pancreatic phospholipase a2. PLA2-GIB has been identified and cloned from various mammalian species. Human PLA2-GIB protein is disclosed in, for example, Lammbeau and Gelb (2008). The sequence thereof is available on Genbank No. NP-000919. The amino acid sequence of an exemplary human PLA2-GIB is shown below as SEQ ID NO: 1.
Amino acids 1 to 15 of SEQ ID NO:1 (underlined) are the signal sequence and amino acids 16 to 22 of SEQ ID NO:1 (bold) are the propeptide sequence. The mature form of PLA2-GIB is usually produced by proteolytic cleavage and thus lacks a signal sequence and a propeptide sequence. Thus, a typical example of a mature form of PLA2-GIB comprises (or consists essentially of) amino acid residues 23-148 of SEQ ID NO:1, as represented in SEQ ID NO:2 below.
In the context of the present invention, the term "PLA 2-GIB" preferably denotes human PLA2-GIB in any of its forms (pre-or mature forms), as well as variants thereof, such as any natural variants due to polymorphism or splicing.
In a particular embodiment, the term "PLA 2-GIB" denotes human PLA2-GIB comprising SEQ ID NO:1 or 2 as well as natural variants due to polymorphism or splicing.
Naturally occurring variants of a reference protein include any protein having one or more amino acid substitutions, additions and/or deletions of one or several (typically 1,2 or 3) amino acid residues as compared to the reference protein. Preferably, a variant comprises no more than 10 different amino acid substitutions, additions and/or deletions of one or several (typically 1,2 or 3) amino acid residues as compared to the reference protein. Typical naturally occurring variants retain the biological activity of the reference protein. In this regard, in some embodiments, PLA2-GIB has at least one activity selected from: inducing the formation of membrane Microdomains (MD) in CD 4T cells from healthy subjects, or rendering CD 4T cells of healthy subjects non-responsive to interleukin signaling, such as IL-2 signaling or IL-7 signaling.
In a particular embodiment, the term PLA2-GIB denotes a human protein (in particular a protein comprising or consisting of SEQ ID NO:1 or 2), or a naturally occurring variant thereof.
Treatment of cancer
The present invention relates to methods for treating cancer in a subject comprising administering to the subject a compound that inhibits PLA 2-GIB. The inventors have shown that PLA2-GIB co-factor is present in cancer patients, and that this co-factor together with PLA2-GIB induces the inactivation of immune cells. The inventors have further shown that inhibition of PLA2-GIB can restore an effective immune response in the plasma of the patient and thus effectively contribute to cancer treatment.
In a particular embodiment, the present invention relates to a method for treating cancer or neoplasia in a subject in need thereof comprising administering to the subject a compound that inhibits PLA 2-GIB.
The present invention also relates to a compound that inhibits PLA2-GIB for use in treating cancer or neoplasia in a subject in need thereof.
In particular embodiments, the methods of the invention are used to prevent or reduce the incidence of cancer in a subject in need thereof (e.g., a subject at risk of neoplasia or cancer). In this regard, the present invention may be used to treat risk factors for cancer, thereby avoiding or reducing the risk/incidence of cancer. Such risk factors include, but are not limited to, oral, gastric and/or intestinal inflammation and infections, such as pancreatitis.
The present invention also relates to a compound that inhibits PLA2-GIB for use in preventing or reducing the incidence of cancer in a subject in need thereof.
In another specific embodiment, the methods of the invention are used to reduce the rate of cancer progression in a subject having cancer.
In another particular embodiment, the present invention relates to a compound that inhibits PLA2-GIB for use in reducing the rate of cancer progression in a subject having cancer.
In another particular embodiment, the method of the invention is for reducing or preventing or treating cancer metastasis, or for killing cancer cells, in a subject having cancer.
In another particular embodiment, the present invention relates to a compound that inhibits PLA2-GIB for use in reducing or preventing or treating cancer metastasis in a subject having cancer, or for use in killing cancer cells in a subject having cancer.
The inventors have analyzed several cohorts of patients with solid tumors (e.g., pancreatic cancer). It has been found that there is no statistical difference in PLA2-GIB levels in plasma from the patients compared to plasma from healthy donors. However, surprisingly, they found that plasma from the patients can render immune cells susceptible to inactivation by physiological concentrations of PLA 2-GIB. More specifically, by exposing T cells to plasma from the cancer patient and PLA2-GIB, the immune cells become inactive and fail to initiate an immune response. Thus, the patient's plasma contains one or more cofactors which render immune cells susceptible to inactivation of PLA 2-GIB. The inventors have further shown that by inhibiting PLA2-GIB, the immune cells are restored and no such inactivation occurs.
Thus, the present invention demonstrates the presence of PLA2-GIB cofactor in the plasma of human cancer patients.
The present invention further demonstrates that PLA2-GIB inhibition can be used to treat such cancers.
In this regard, the present invention may be used to treat any cancer.
In a particular embodiment, the cancer is a solid cancer.
In particular embodiments, the method is for treating a subject having cancer and expressing the PLA2-GIB cofactor. In a preferred embodiment, the method is for treating cancer in a subject, wherein the PLA2-GIB cofactor or a prokaryotic or eukaryotic cell or virus expressing the PLA2-GIB cofactor is present in said subject.
In another specific embodiment, the method is for treating a subject having cancer, wherein the PLA2-GIB or PLA2-GIB cofactor is present in the cancer microenvironment or blood.
The invention is also particularly suitable for treating cancer or neoplasia in a subject having a PLA2 GIB-associated CD 4T cell deficiency.
The invention can be used to treat cancer at any stage of development. In this regard, most solid cancers develop via four stages:
stage i this stage is typically a small cancer or tumor that has not yet grown deep into nearby tissues. It also has not spread to lymph nodes or other parts of the body. It is commonly referred to as early stage cancer.
Generally, these 2 stages indicate a larger cancer or tumor that has grown deeper into nearby tissues. It may also have spread to lymph nodes, but not to other parts of the body.
Stage iv. this stage means that the cancer has spread to other organs or parts of the body. It may also be referred to as advanced or metastatic cancer.
Some cancers also have stage 0. Stage 0 cancers remain where they begin and have not spread to nearby tissues. Cancer at this stage is often highly curable, usually by surgical removal of the entire tumor.
The invention may be used to treat tumors or cancers at stage 0, I, II, III or IV.
The invention may be used to prevent or reduce or treat metastasis of cancer at stage 0, I, II or III.
The invention may be used to reduce the rate of progression of cancer at stage 0, I, II or III.
The invention may be particularly useful for the treatment of a solid cancer selected from pancreatic cancer, melanoma, lung cancer, esophageal or throat cancer, retinoblastoma, liver cancer, breast cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, uterine cancer, cervical cancer, thyroid cancer, bladder cancer, prostate cancer, bone cancer, brain cancer or colorectal cancer.
In particular embodiments, the methods of the invention are used to treat pancreatic cancer. Pancreatic cancer is classified according to which part of the pancreas is affected: causing digestive substances to cause exocrine cancer, and causing insulin and other hormones to cause endocrine cancer. Although there are several different types of pancreatic cancer, 95% of cases are due to exocrine cancer (pancreatic ductal adenocarcinoma (PDAC)).
PDACs rank fourth among the major causes of death due to cancer. Researchers predicted that PDAC will be the second leading cause of cancer-related death in the united states by 2030. The incidence has doubled over 30 years, currently increasing by 5% each year. The 5-year relative survival rate is about 5%, and surgery is the most effective option for treating PDAC. The limited availability of diagnostic methods and the possibility of surgery as the only available cure option with only 10% of diagnosed patients increases the scare of the disease. The poor prognosis of the disease can be explained by the lack of effective biomarkers for screening and early detection, as well as aggressive behavior and resistance to currently available chemotherapy.
The present invention shows that PLA2-GIB inhibition can be used to treat pancreatic cancer. The present invention represents a novel strategy to prevent pancreatic cancer progression and metastasis. The present invention can be used for pancreatic cancers of any type/stage, such as pancreatic adenocarcinoma, neuroendocrine tumors, intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, and severe cystic neoplasms. The invention is particularly suitable for treating pancreatic adenocarcinoma at any stage.
The invention is also particularly suitable for the treatment of colorectal cancer, lung cancer and rapidly growing cancers. Colorectal cancer is one of the most common cancers of all sexes. At all stages, the 5-year survival probability is about 55%. (Bossard N, 2007). In fact, more than 180,000 new cases of colorectal cancer were diagnosed in france, japan, usa, germany, italy, spain and uk in 2010. Colorectal cancer is divided into four stages: stage I, which is minimally progressive and is treated primarily by surgery; phases II and III, during both of which the patient receives combined radiochemical therapy (RCT); and phase IV, which is a very advanced and metastatic phase. When a patient is diagnosed with locally advanced (stage II or III) colorectal cancer, the patient is usually treated with RCT prior to surgical resection. The invention is suitable for the treatment of stage I, II, III and IV colorectal cancer. The invention is particularly suitable for treating colorectal cancer at stage II, III or IV.
The invention is also suitable for treating cancers that induce gastrointestinal and metabolic conditions.
For use in the present invention, the PLA2-GIB inhibitor may be administered by any suitable route. Preferably, administration is by injection, such as systemic or parenteral injection or infusion, e.g., intramuscular, intravenous, intraarterial, subcutaneous, intratumoral, etc. Administration is usually repeated or continuous. In particular embodiments, the level of PLA2-GIB or PLA2-GIB cofactor in the tumor or body fluid is measured during the course of treatment to guide the treatment regimen.
PLA2-GIB inhibitors may be used alone or in combination with further cancer treatments.
In a particular embodiment, the present invention relates to a method for treating cancer in a subject comprising administering a compound that inhibits PLA2-GIB to a subject having cancer in combination with chemotherapy or hormone therapy.
In another specific embodiment, the present invention relates to a method for treating cancer in a subject comprising administering a compound that inhibits PLA2-GIB to a subject having cancer in combination with radiochemistry, ultrasound therapy, or nanoparticle therapy.
In another specific embodiment, the present invention relates to a method for treating cancer in a subject comprising administering a compound that inhibits PLA2-GIB to a subject having cancer in combination with a checkpoint inhibitor, immunotherapy, or anti-cancer vaccine.
In another specific embodiment, the present invention relates to a method for treating cancer in a subject comprising administering to a subject having cancer a compound that inhibits PLA2-GIB in combination with an inhibitor of a PLA2-GIB cofactor. The inhibitor of the PLA2-GIB cofactor may be an antagonist of said cofactor or a cytostatic or cytotoxic agent or vaccine against said PLA2-GIB cofactor or against a prokaryotic or eukaryotic cell or virus expressing said PLA2-GIB cofactor. In this regard, where the cofactor is a bacterium, the inhibitor may be an antibiotic directed against the bacterium.
In "combination" therapy, multiple active agents may be used simultaneously or sequentially, together or alternately. Each active agent may be used according to a specific schedule. In other examples, all active agents may be formulated and/or administered together, such as in perfusion.
In further embodiments, the compound is administered before, during or after surgery (tumor resection or removal).
The compounds for use in the present invention may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers.
"pharmaceutical composition" refers to a formulation of a compound of the present invention (active ingredient) with a vehicle generally accepted in the art for delivering biologically active compounds to a subject in need thereof. Such carriers include all pharmaceutically acceptable carriers, diluents, vehicles or supports thereof. Conventional pharmaceutical practice may be employed to provide a suitable formulation or composition to a subject, for example in unit dosage form.
The compounds or compositions according to the invention may be formulated in the form of: ointments, gels, pastes, liquid solutions, suspensions, tablets, gelatin capsules, suppositories, powders, nasal drops or aerosols, preferably formulated in the form of injectable solutions or suspensions. For example, for injectable formulations, the compounds are typically packaged in the form of a liquid suspension, which may be injected by syringe or infusion. In this regard, the compounds are typically dissolved in saline, physiological, isotonic or buffered solutions that are compatible with pharmaceutical use and known to those skilled in the art. Thus, the composition may contain one or more agents or excipients selected from dispersing agents, solubilizing agents, stabilizing agents, preservatives and the like. Agents or excipients which can be used in liquid and/or injectable formulations are, in particular, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia and the like. The carrier may also be selected from, for example, methyl- β -cyclodextrin, polymers of acrylic acid (such as carbomer (carbopol)), mixtures of polyethylene glycol and polypropylene glycol, monoethanolamine, and hydroxymethylcellulose.
The composition typically comprises about 1 μ g to 1000mg of PLA2-GIB inhibitor, such as 0.001-0.01, 0.01-0.1, 0.05-100, 0.05-10, 0.05-5, 0.05-1, 0.1-100, 0.1-1.0, 0.1-5, 1.0-10, 5-10, 10-20, 20-50 and 50-100mg, for example between 0.05 and 100mg, preferably between 0.05 and 5mg, for example 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1,2, 3,4 or 5 mg. The skilled artisan can adjust the dosage depending on the modulator and the disease.
The composition may be formulated in any suitable form or in any suitable container (syringe, ampoule, flask, bottle, sachet, etc.).
In a preferred embodiment, the treatment comprises one or several injections of a liquid formulation containing a PLA2-GIB inhibitor, optionally in combination with one or more anti-cancer agents or treatments. The injection is preferably systemic, such as intravenous, intraarterial, intramuscular, intracancerous, intradermal, and the like.
The practitioner can adjust the dosage and frequency of administration.
Inhibitors of PLA2-GIB
PLA2-GIB inhibitors suitable for use in the present invention may be any compound that inhibits or neutralizes the expression or activity of PLA2-GIB, such as an expression inhibitor, antagonist or spacer (sequestrator). Preferred classes of inhibitors include PLA2-GIB ligands (covalent or non-covalent), anti-PLA 2-GIB antibodies (and fragments and derivatives thereof), nucleic acids encoding anti-PLA 2-GIB antibodies (or fragments and derivatives thereof), inhibitory nucleic acids, peptides or small drugs, soluble receptors, or combinations thereof. Alternatively or additionally, the PLA2-GIB inhibitor may be a PLA2-GIB antigen that induces production of anti-PLA 2GIB antibodies upon administration to a subject.
Inhibition of PLA2-GIB generally means a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more reduction in PLA2-GIB levels or activity, as well as complete blocking or inhibition of PLA2-GIB levels or activity. Depending on the circumstances, inhibition may be transient, persistent, or permanent.
Antibodies to PLA2-GIB
Specific examples of PLA2-GIB inhibitors are anti-PLA 2-GIB antibodies, e.g., antibodies that bind to PLA2-GIB and/or are produced by immunizing a mammal with a PLA2-GIB antigen.
Antibodies can be synthetic, monoclonal or polyclonal, and can be prepared by techniques well known in the art. Such antibodies specifically bind (as opposed to non-specific binding) through the antigen binding site of the antibody. PLA2-GIB polypeptides, fragments, variants, fusion proteins, and the like may be used as immunogens to generate antibodies that immunoreact therewith. More specifically, the polypeptides, fragments, variants, fusion proteins, etc., contain antigenic determinants or epitopes that elicit the formation of antibodies.
The term "antibody" is intended to include polyclonal antibodies, monoclonal antibodies, fragments thereof (such as F (ab')2 and Fab fragments, single chain variable fragments (scFv), single domain antibody fragments (VHH or nanobodies), bivalent antibody fragments (diabodies)), as well as any recombinantly and synthetically produced binding partner, human antibody or humanized antibody.
Preferably, if the antibody is present in an amount greater than or equal to about 107M-1 Ka binds to PLA2-GIB, and the antibody is defined as specifically binding. The affinity of the antibody can be readily determined using conventional techniques, such as those described by Scatchard et al, ann.n.y.acad.sci.,51:660 (1949).
Polyclonal antibodies can be readily generated from a variety of sources (e.g., horses, cattle, donkeys, goats, sheep, dogs, chickens, rabbits, mice, or rats) using procedures well known in the art. Generally, purified PLA2-GIB or an appropriately conjugated peptide based on the PLA2-GIB amino acid sequence is administered to a host animal, typically by parenteral injection. The immunogenicity of PLA2-GIB can be enhanced by the use of an adjuvant (e.g., freund's complete adjuvant or incomplete adjuvant). Following booster immunizations, a small serum sample was collected and tested for reactivity to the PLA2-GIB polypeptide. Examples of various assays that can be used for such determinations include Antibodies such as those described in A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; and procedures such as Countercurrent Immunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assay, and sandwich assay. See U.S. Pat. nos. 4,376,110 and 4,486,530.
Monoclonal antibodies can be readily prepared using well-known procedures. See, for example, U.S. patent nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; mononal antigens, hybrids: A New Dimension in Biological analytes, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980.
For example, a host animal (e.g., a mouse) can be injected intraperitoneally with an isolated and purified wild-type or mutant PLA2-GIB protein or a conjugated PLA2-GIB peptide, optionally in the presence of an adjuvant, at intervals of about 3 weeks, at least once and preferably at least twice. Mouse sera were then assayed by conventional dot blot techniques or antibody capture (ABC) to determine which animals were best suited for fusion. Approximately two to three weeks later, mice were given an intravenous boost of protein or peptide. The mice are then sacrificed according to established protocols and the spleen cells are fused with commercially available myeloma cells (e.g., Ag8.653 (ATCC)). Briefly, myeloma cells were washed several times in culture and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusogenic agent may be any suitable agent used in the art, such as polyethylene glycol (PEG). The fusions are plated in plates containing media that allows selective growth of the fused cells. The fused cells may then be allowed to grow for about eight days. Supernatants from the resulting hybridomas were collected and added to plates first coated with goat anti-mouse Ig. After washing, a label (e.g., a labeled PLA2-GIB polypeptide) is added to each well, followed by incubation. Positive wells can then be detected. Positive clones can be grown in bulk culture (bulk culture) and the supernatant subsequently purified on a protein A column (Pharmacia).
Alternative techniques may be used to generate Monoclonal antibodies of the disclosure, such as those described by Alting-Mees et al, "Monoclonal Antibody Expression antibodies: A Rapid Alternative to hybrids," variants in Molecular Biology 3:1-9(1990), which is incorporated herein by reference. Similarly, the binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of the genes encoding the specific binding antibodies. Such techniques are described in Larrick et al, Biotechnology,7:394 (1989).
Antigen-binding fragments of such antibodies, which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F (ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
Monoclonal antibodies include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibody is administered to a human. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or only its antigen binding site) and a constant region derived from a human antibody. Alternatively, the humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen binding site) derived from a human antibody. Procedures for generating chimeric antibodies and further engineered monoclonal antibodies include those described in: riechmann et al, (Nature 332:323,1988); liu et al, (PNAS 84:3439,1987); larrick et al, (Bio/Technology 7:934,1989); and Winter and Harris (TIPS 14:139, May, 1993). Procedures for transgenic antibody production can be found in GB 2,272,440, U.S. Pat. nos. 5,569,825 and 5,545,806.
Antibodies comprising both human and non-human portions produced by genetic engineering methods, such as chimeric and humanized monoclonal antibodies, can be used, which can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example using the methods described in: robinson et al, international publication No. WO 87/02671; akira et al, European patent application 0184187; taniguchi, m., european patent application 0171496; morrison et al, European patent application 0173494; neuberger et al, PCT International publication No. WO 86/01533; cabilly et al, U.S. Pat. Nos. 4,816,567; cabilly et al, European patent application 0125023; better et al, Science 240: 10411043,1988; liu et al, PNAS 84: 34393443,1987; liu et al, J.Immunol.139: 35213526,1987; sun et al, PNAS 84: 214218,1987; nishimura et al, Canc.Res.47: 9991005,1987; wood et al, Nature 314: 446449,1985; and Shaw et al, j.natl.cancer inst.80: 15531559,1988); morrison, S.L., Science 229: 12021207,1985; oi et al, BioTechniques 4:214,1986; winter U.S. Pat. nos. 5,225,539; jones et al, Nature 321: 552525,1986; verhoeyan et al, Science 239:1534,1988; and Beidler et al, J.Immunol.141: 40534060,1988.
With respect to synthetic and semi-synthetic antibodies, such terms are intended to cover, but are not limited to, antibody fragments, isotype-switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies with multiple specificities, and fully synthetic antibody-like molecules.
Human monoclonal antibodies having human constant and variable regions can be generated by immunizing transgenic animals containing human immunoglobulin genes. See Jakobovits et al, Ann NY Acad Sci 764: 525-. Human monoclonal antibodies to the PLA2-GIB polypeptide can also be prepared by constructing combinatorial immunoglobulin libraries (e.g., Fab phage display libraries or scFv phage display libraries) using immunoglobulin light and heavy chain cDNAs prepared from mRNAs derived from lymphocytes of a subject. See, e.g., McCafferty et al, PCT publication WO 92/01047; marks et al (1991) J.mol.biol.222: 581597; and Griffhs et al, (1993) EMBO J12: 725734. Alternatively, combinatorial libraries of antibody variable regions can be generated by mutating known human antibodies. For example, the variable regions of human antibodies known to bind PLA2-GIB can be mutated, e.g., using randomly altered mutagenic oligonucleotides, to generate a library of mutated variable regions, which can then be screened for binding to PLA 2-GIB. Methods of inducing random mutagenesis in the CDR regions of immunoglobulin heavy and/or light chains, methods of hybridizing randomized heavy and light chains to form pairings, and screening methods can be found, for example, in Barbas et al, PCT publication WO 96/07754; barbas et al, (1992) Proc.nat' l Acad.Sci.USA 89: 44574461.
The immunoglobulin library may be expressed from a population of display packages (preferably derived from filamentous phage) to form an antibody display library. Examples of methods and reagents particularly suitable for generating antibody display libraries can be found, for example, in Ladner et al, U.S. patent nos. 5,223,409; kang et al, PCT publication WO 92/18619; dower et al, PCT publication WO 91/17271; winter et al, PCT publication WO 92/20791; markland et al, PCT publication WO 92/15679; breitling et al, PCT publication WO 93/01288; McCafferty et al, PCT publication WO 92/01047; garrrard et al, PCT publication WO 92/09690; ladner et al, PCT publication WO 90/02809; fuchs et al, (1991) Bio/Technology 9: 13701372; hay et al (1992) Hum antibody hybrids 3: 8185; huse et al, (1989) Science 246: 12751281; griffhs et al, (1993) supra; hawkins et al, (1992) J Mol Biol 226: 889896; clackson et al, (1991) Nature 352: 624628; gram et al, (1992) PNAS 89: 35763580; garrad et al, (1991) Bio/Technology 9: 13731377; hoogenboom et al, (1991) Nuc Acid Res 19: 41334137; and Barbas et al, (1991) PNAS 88: 79787982. Once displayed on the surface of a display package (e.g., filamentous phage), the antibody library is screened to identify and isolate packages expressing antibodies that bind to the PLA2-GIB polypeptide. In a preferred embodiment, the primary screening of the library involves panning with an immobilized PLA2-GIB polypeptide and selecting a display package that expresses an antibody that binds to the immobilized PLA2-GIB polypeptide.
Preferred antibodies for use in the present invention are directed to the PLA2-GIB epitope and/or have been generated by immunization with a PLA2-GIB epitope-containing polypeptide selected from the group consisting of: mature PLA2-GIB protein, PLA2-GIB fragment comprising at least 8 consecutive amino acid residues of SEQ ID NO:1 or 2 (or the corresponding residues of a natural variant of SEQ ID NO:1 or 2), said fragment preferably comprising at least amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71 or a combination thereof (numbering with reference to SEQ ID NO: 2).
The specific anti-PLA 2-GIB antibody used in the present invention binds to mature human PLA2-GIB, even more preferably to an epitope comprised in the domain of PLA2-GIB, which comprises amino acid residues selected from the group consisting of: amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or combinations thereof (numbering according to SEQ ID No: 2). Particular antibodies for use in the present invention bind to a polypeptide comprised between amino acid residues 50-71 (see SEQ ID NO:2) of mature human PLA2-GIB or SEQ ID NO:2 between the corresponding residues of the natural variants. Examples of anti-PLA 2-GIB antibodies suitable for use in the present invention have been disclosed in WO 2015/097140.
A further specific anti-PLA 2-GIB antibody for use in the present invention binds to an epitope comprising at least one amino acid residue selected from the group consisting of W3, R6, K7, K10, C77, Y75, G79 and S80 (numbered with reference to SEQ ID NO:2) of human mature PLA2-GIB, more preferably to an epitope comprising at least 2 or at least 3 amino acid residues selected from the group consisting of W3, R6, K7, K10, C77, Y77, G77 and S77 of human mature PLA2-GIB, even more preferably to an epitope comprising at least 4, at least 5, at least 6 or at least 7 amino acid residues selected from the group consisting of W77, R77, K77, C77, Y77, G77 and S77 of human mature PLA 77-GIB. Particular antibodies for use in the present invention bind to a polypeptide comprised between amino acid residues 1-10 or 75-80 of mature human PLA2-GIB (see SEQ ID NO:2) or SEQ ID NO:2 between the corresponding residues of the natural variant of 2. Such antibodies exhibit potent neutralizing activity and represent valuable therapeutic agents for use in the present invention. Examples of anti-PLA 2-GIB antibodies suitable for use in the present invention have been disclosed in EP18305229, which has not been published so far.
In particular embodiments, the antibody or derivative for use in the present invention is monoclonal antibody 14G9 or an anti-PLA 2-GIB antibody that competitively inhibits the binding of monoclonal antibody 14G9 to human PLA2-GIB, said monoclonal antibody 14G9 comprising a light chain variable region comprising SEQ ID No:3 and a heavy chain variable region comprising SEQ ID No: 4. The antibody may be human or humanized.
In another particular embodiment, the antibody or derivative for use in the present invention is monoclonal antibody #2B or an anti-PLA 2-GIB antibody that competitively inhibits the binding of monoclonal antibody #2B to human sPLA2-GIB, said monoclonal antibody #2B comprising or consisting of a light chain comprising or consisting of SEQ ID No. 5 and a heavy chain comprising or consisting of SEQ ID No. 6.
In another particular embodiment, the antibody or derivative for use in the present invention is monoclonal antibody #2B1 or an anti-PLA 2-GIB antibody that competitively inhibits the binding of monoclonal antibody #2B1 to human sPLA2-GIB, said monoclonal antibody #2B1 comprising or consisting of a light chain comprising or consisting of SEQ ID No. 5 and a heavy chain comprising or consisting of SEQ ID NO. 7.
In another particular embodiment, the antibody or derivative for use in the present invention is monoclonal antibody #2B2 or an anti-PLA 2-GIB antibody that competitively inhibits the binding of monoclonal antibody #2B2 to human sPLA2-GIB, said monoclonal antibody #2B2 comprising or consisting of a light chain comprising or consisting of SEQ ID No. 5 and a heavy chain comprising or consisting of SEQ ID NO. 8.
The term "competitively inhibits" indicates that the antibody may reduce or inhibit or replace binding of the reference antibody to sPLA 2-GIB. Competition assays can be performed using standard techniques such as, for example, competition ELISA or other binding assays. Typically, competitive binding assays involve purified target antigen, unlabeled test antibody, and labeled reference antibody, which typically bind to a solid substrate or cell. Competitive inhibition is measured by determining the amount of bound labeled antibody in the presence of the test antibody. Typically the test antibody is present in excess, e.g., about 5 to 500 times the amount of the reference antibody. Typically, the antibodies are tested in 100X excess for ELISA and 10X excess for enzymatic methods. A test antibody that is present in excess is considered to competitively inhibit a reference antibody when it inhibits or displaces at least 70% of its binding to the antigen. In a specific embodiment, a test antibody present in an excess of 100X is considered to competitively inhibit a reference antibody when it inhibits or displaces at least 70%, more preferably at least 80%, of its binding to antigen in an ELISA. Preferred competing antibodies bind epitopes that share common amino acid residues.
In a particular embodiment, the inhibitor is a monoclonal antibody comprising:
(i) a light chain variable region comprising CDR-L1, CDR-L2, CDR-L3 and FR-L, wherein the CDR-L1, CDR-L2 and/or CDR-L3 consists of or consists essentially of CDR-L1, CDR-L2 and CDR-L3, respectively, of the light chain variable region of SEQ ID NO:3 or 5, and wherein FR-L is of human immunoglobulin sequence; and
(ii) a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 and FR-H, wherein the CDR-H1, CDR-H2 and/or CDR-H3 consists of or consists essentially of CDR-H1, CDR-H2 and CDR-H3, respectively, of the heavy chain variable region of SEQ ID NOs 4, 6, 7 or 8, and wherein FR-H is of human immunoglobulin sequence.
The "variable region" of an antibody refers to the amino-terminal domain of a heavy or light chain ("VH" or "VL") that contains an antigen-binding site. Light or heavy chain variable regions (VL or VH) are typically composed of framework regions ("FRs") interrupted by three hypervariable regions, called "complementarity determining regions" or "CDRs". The extent of framework regions and CDRs has been precisely defined, for example, in Kabat (see "Sequences of Proteins of Immunological Interest," E.Kabat et al, U.S. department of Health and Human Services, (1983)), and in Chothia.
Referring to SEQ ID No. 3, the three CDR regions correspond to the following amino acid residues:
CDR-L1: amino acid residues QDVSTA (residues 27-31 of SEQ ID NO: 3),
CDR-L2: amino acid residues WAS (residues 50-52 of SEQ ID NO: 3),
CDR-L3: amino acid residue QQDYSTPPT (residues 89-97 of SEQ ID NO: 3),
referring to SEQ ID No. 4, the three CDR regions correspond to the following amino acid residues:
CDR-H1: amino acid residues GYTFTNYW (residues 26-33 of SEQ ID NO: 4),
CDR-H2: amino acid residues IDPSDTRT (residues 51-58 of SEQ ID NO: 4),
CDR-H3: amino acid residues ARQTLYYEALDY (residues 97-108 of SEQ ID NO: 4).
In a particular embodiment, the invention relates to a monoclonal antibody selected from the group consisting of:
a monoclonal antibody (14G9) comprising, consisting essentially of, or consisting of a light chain comprising, consisting essentially of SEQ ID No. 3 and a heavy chain comprising, consisting essentially of, or consisting of SEQ ID No. 4;
a monoclonal antibody (#2B) comprising, consisting essentially of, or consisting of SEQ ID No. 5 and a heavy chain comprising, consisting essentially of, or consisting of SEQ ID No. 6;
a monoclonal antibody (#2B1) comprising, consisting essentially of, or consisting of SEQ ID No. 5, and a heavy chain comprising, consisting essentially of, or consisting of SEQ ID No. 7;
a monoclonal antibody (clone #2B2) comprising, consisting essentially of, or consisting of SEQ ID No. 5 and a heavy chain comprising, consisting essentially of, or consisting of SEQ ID No. 8; and
a derivative thereof.
As used herein, the term "antibody derivative" refers to an antibody that retains the antigenic specificity of a reference antibody but in which one or more amino acid residues are (chemically or biologically) modified to improve its properties.
Examples of such chemical modifications include, for example, by alkylation, pegylation, acylation, ester or amide formation, and the like. In particular, a derivative is an antibody as disclosed herein that has been modified to contain one or more additional non-protein moieties, such as a water-soluble polymer. Examples of water soluble polymers include, but are not limited to, PEG, copolymers of ethylene/propylene glycol, carboxymethyl cellulose, dextran, and polyvinyl alcohol.
Derivatives may also be generated to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed. Where the antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically attached by N to Asn297 of the CH2 domain of the Fc region (see, e.g., Wright et al, TIBTECH,1997,15: 26-32). Oligosaccharides may include a variety of carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.
In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%). The amount of fucose was determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue at about position 297 in the Fc region (Eu numbering of Fc region residues); however, due to slight sequence variations in the antibody, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include, but are not limited to, Okazaki et al, j.mol.biol.336: 1239-; 1:230-236. Examples of cell lines capable of producing defucosylated antibodies include Led 3CHO cells lacking protein fucosylation (Ripka et al, arch, biochem, biophysis, 249: 533-.
In certain embodiments, it may be desirable to create cysteine engineered antibodies (e.g., "thiomabs") in which one or more residues of the antibody are substituted with cysteine residues. In particular embodiments, the substituted residue is present at an accessible site of the antibody. By replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody and can be used to conjugate the antibody to other moieties (such as a drug moiety or linker-drug moiety) to create an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No. 7,521,541.
The term derivative also includes immunoconjugates comprising a conjugate to one or more heterologous molecules; or a solid support such as agarose beads or the like, including but not limited to cytotoxic agents, detectable moieties (e.g., fluorescent moieties), diagnostic radioisotopes, or imaging agents. Examples of cytotoxic agents include, but are not limited to, chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins; enzymatically active toxins of bacterial, fungal, plant or animal origin; or fragments thereof), or radioisotopes. Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents well known to the skilled artisan. The linker may be a "cleavable linker" which facilitates the release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari et al, Cancer Res.52:127-131 (1992)).
Antibodies for use in the present invention are typically "isolated", e.g., have been separated from at least one component of their natural environment. In particular, the antibody can be purified to a higher (e.g., at least 95%, at least 96%; at least 97%, at least 98%, or at least 99%) purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatographic (e.g., ion exchange or reverse phase HPLC) techniques. For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromanogr.b 848:79-87 (2007).
Preferred antibodies of the invention are substantially neutralizing antibodies, i.e., they are capable of at least partially inhibiting the activity of PLA 2-GIB.
sPLA 2GIB catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids to release free fatty acids and lysophospholipids. Particular antibodies of the invention inhibit sPLA2-GIB enzymatic activity, such as hydrolysis of the sn-2 fatty acyl bond of a phospholipid. Methods for testing such properties are disclosed in detail in the experimental section. The specific antibodies used in the present invention inhibit the binding of sPLA2-GIB to its substrate. Further specific antibodies for use in the present invention inhibit sPLA2-GIB mediated inhibition of IL-7 induced phosphostat 5 nuclear translocation in CD 4T cells. Methods for testing such properties are disclosed in detail in the experimental section.
The neutralizing activity of an antibody or derivative can be determined in vitro or in vivo using, for example, binding or a bioassay (such as the assays described in the experimental section). Inhibition/neutralization may be complete or partial. In particular, the antibody may inhibit the activity tested by 10% or more, preferably 20% or more, 30% or more, 40% or more, 50% or more.
In preferred embodiments, the antibody is an IgG, e.g., gG1, IgG2, IgG3, or IgG 4.
Conventional methods and media can be used to isolate and preserve the antibody or derivative. They may be lyophilized. They may also be frozen.
Nucleic acids encoding recombinant antibodies, vectors and host cells
In another aspect, the PLA2-GIB inhibitor is or comprises or consists of: a nucleic acid molecule encoding an anti-PLA 2-GIB antibody, or a light or heavy chain thereof, or a variable domain thereof; or a nucleic acid complementary to the coding sequence.
The nucleic acid may be DNA (cDNA or gDNA), RNA or a mixture thereof. It may be in single stranded form or in duplex form or in a mixture of both. It may comprise modified nucleotides comprising, for example, a modified linkage, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to those skilled in the art, including chemical synthesis, recombination and mutagenesis. The nucleic acids according to the invention can be deduced from the sequences of the antibodies according to the invention and the codon usage can be adjusted depending on the host cell in which the nucleic acid should be transcribed. These steps can be carried out according to methods well known to the person skilled in the art and some of these methods are described in the reference manual Sambrook et al, (Sambrook J, Russell D (2001) Molecular cloning: a laboratory Manual, third edition Cold Spring Harbor).
The nucleic acid may encode an amino acid sequence comprising a light chain and/or an amino acid sequence comprising an antibody heavy chain, or may be complementary to such a coding sequence.
Specific examples of such nucleic acid sequences include the sequences provided as SEQ ID NOS 9-12.
The invention further provides a vector comprising a nucleic acid of the invention. Optionally, the vector may comprise several nucleic acids of the invention. In particular, the vector may comprise a nucleic acid of the invention operably linked to a regulatory region (i.e., a region comprising one or more control sequences). Optionally, the vector may comprise several nucleic acids of the invention operably linked to several regulatory regions.
The term "control sequences" means nucleic acid sequences necessary for expression of a coding region. The control sequences may be endogenous or heterologous. Control sequences well known to those skilled in the art and currently used will be preferred. Such control sequences include, but are not limited to, promoters, signal peptide sequences, and transcription terminators.
The term "operably linked" means a configuration in which control sequences are placed in an appropriate position relative to the coding sequence in such a manner that the control sequences direct the expression of the coding sequence.
The invention also relates to the use of a nucleic acid or vector according to the invention for transforming, transfecting or transducing a host cell.
The invention also provides host cells comprising one or several of the nucleic acids according to the invention and/or one or several of the vectors according to the invention.
The term "host cell" also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.
Suitable host cells for cloning or expressing the antibody-encoding vector include prokaryotic or eukaryotic cells, such as bacteria, yeast, insect cells, mammalian cells, and the like.
Inhibitory nucleic acids
In another embodiment, the PLA2-GIB inhibitor is an inhibitory nucleic acid, i.e., any nucleic acid molecule that inhibits expression of a PLA2-GIB gene or protein. Preferred inhibitory nucleic acids include antisense nucleic acids, short interfering RNAs (sirnas), small hairpin RNAs (shrnas), micrornas, aptamers, or ribozymes. In a particular embodiment, the inhibitory nucleic acid is a small interfering RNA that prevents translation of PLA2-GIB mRNA. In another specific embodiment, the inhibitory nucleic acid is an antisense oligonucleotide that prevents translation of PLA2-GIB mRNA. In another specific embodiment, the inhibitory nucleic acid is a small hairpin RNA that prevents translation of PLA2-GIB mRNA.
The siRNA comprises a sense nucleic acid sequence and an antisense nucleic acid sequence of the polynucleotide of interest. The siRNA is constructed such that a single transcript (double stranded RNA) has both sense and complementary antisense sequences from the target gene. The nucleotide sequence of the siRNA can be designed using an siRNA design computer program available, for example, from the Ambion website on the world Wide Web.
In some embodiments, the antisense oligonucleotide or siRNA is less than or equal to 10 nucleotides in length. In some embodiments, the antisense oligonucleotides and sirnas are as long as the naturally occurring transcripts. In some embodiments, the antisense oligonucleotides and sirnas have 18-30 nucleotides. In some embodiments, the antisense oligonucleotides and sirnas are less than 25 nucleotides in length.
Preferred inhibitory nucleic acid molecules comprise a domain having a nucleotide sequence that is fully complementary to a region of the PLA2-GIB gene or RNA. Such domains typically contain 4 to 20 nucleotides, allowing specific hybridization and optimal inhibition of gene transcription or RNA translation. The sequence of the inhibitory nucleic acid can be derived directly from the sequence of the gene encoding PLA2-GIB, as shown in SEQ ID NO: 2. Alternatively or additionally, the inhibitory nucleic acid may hybridize to regulatory elements (e.g., promoters, splice sites, etc.) in the PLA2-GIB gene or RNA and prevent its effective regulation.
Specific examples of inhibitory nucleic acid molecules of the invention include isolated single-stranded nucleic acid molecules consisting of 10 to 50 contiguous nucleotides of a sequence encoding SEQ ID NO. 1. Specific examples of inhibitory nucleic acid molecules of the invention are antisense nucleic acids, which consist of the following nucleotide sequences or the complete complementary strand thereof:
peptides and small drugs
In another alternative embodiment, the PLA2-GIB inhibitor is a peptide or small drug that inhibits the activity of PLA 2-GIB. Peptides or small drugs are typically molecules that selectively bind to PLA2-GIB, or a substrate of PLA2-GIB, or a cofactor of PLA2-GIB, or a degradation product or metabolite of the PLA2-GIB pathway.
Preferably, the peptides comprise 3 to 20 amino acid residues and their sequence may be identical to the domain of PLA2-GIB (decoy peptide) or to the domain of PLA2-GIB substrate, cofactor, degradation product or metabolite. Preferred peptides of the invention contain 4 to 30 consecutive amino acid residues of SEQ ID NO:1 or 2 (or of the corresponding sequence of a natural variant of SEQ ID NO:1 or 2). The most preferred peptides of the invention comprise 5 to 25 consecutive amino acid residues of SEQ ID NO:2 (or of the corresponding sequence of the natural variant of SEQ ID NO:2) and further comprise at least one of the following amino acid residues of SEQ ID NO:2 (or of the corresponding sequence of the natural variant of SEQ ID NO: 2): amino acid 3, amino acid 6, amino acid 7, amino acid 10, amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, amino acid 75, amino acid 77, amino acid 79, amino acid 80, or a combination thereof. Specific examples of peptides of the invention are peptides of less than 25 amino acids comprising any one of the following sequences:
other peptides for use in the present invention include pentapeptides as disclosed in WO2017/060405 (which is incorporated herein by reference). In a specific embodiment, the compound is a cyclic peptide selected from FLSYK (SEQ ID NO:32), FLSYR (SEQ ID NO:33) and (2NapA) LS (2NapA) R (SEQ ID NO: 34).
The peptides of the invention may comprise peptide bonds, non-peptide bonds and/or modified peptide bonds. In a particular embodiment, the peptide comprises at least one peptidomimetic bond selected from methylene (-CH)2-) or phosphate (-PO)2-) groups, secondary amines (-NH-) or oxygen (-O-), alpha-aza peptides, alpha-alkanesA peptidyl, an N-alkyl peptide, a phosphoramidate, a depsipeptide, a hydroxymethylene, a hydroxyethylene, a dihydroxyethylene, a hydroxyethylamine, a retro-inverso peptide (retro-inverso peptide), a methyleneoxy group, a cetomethylene, an ester, a phosphinate (phosphonates), a phosphinics, or a phosphonamide (phosphonamides). The peptide may also comprise protected N-terminal and/or C-terminal functions, e.g. obtained by acylation, and/or amidation and/or esterification.
The peptides of the invention may be produced by techniques known per se in the art, such as chemical, biological and/or genetic synthesis.
Each of these peptides in isolated form represents a particular object of the present invention.
The preferred small drug is a hydrocarbon compound that selectively binds to PLA 2-GIB.
Examples of small drugs include indole compounds such as those disclosed in WO2017/037041 (which is incorporated herein by reference). In a particular embodiment, the compound is 3- (2-amino-1, 2-dioxoethyl) -2-ethyl-1- (phenylmethyl) -1H-indol-4-yl) oxy) acetic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof, such as the sodium salt thereof (Varespladib).
Preferably, the small drugs and peptides are obtainable by a method comprising: (i) contacting a test compound with PLA2-GIB or a fragment thereof, (ii) selecting a test compound that binds to PLA2-GIB or said fragment thereof, and (iii) selecting a compound of (ii) that inhibits the activity of PLA 2-GI. Such a method represents a particular object of the present invention.
Small drugs and peptides can also be obtained by a method comprising: (i) contacting a test compound with a PLA2-GIB substrate, cofactor, or degradation product, or fragment thereof, (ii) selecting a test compound that binds to said PLA2-GIB substrate, cofactor, or degradation product, or fragment thereof, and (iii) selecting a compound of (ii) that inhibits the activity of PLA 2-GIB. Such a method represents a particular object of the present invention.
Vaccination
In an alternative (or cumulative) embodiment, the PLA2-GIB inhibitor is a PLA2-GIB antigen. As a result of vaccination or immunization of the subject with the antigen, the subject produces antibodies (or cells) that inhibit PLA 2-GIB. In particular, injection of PLA2-GIB antigen (e.g., immunogenic PLA2-GIB with substantially no biological activity) can generate antibodies in a subject receiving treatment. These antibodies will prevent PLA2-GIB from being overexpressed, and can be used together as an immunotherapy or vaccine prophylaxis.
Accordingly, the present invention is directed to a method of treating a solid cancer in a subject having a solid cancer comprising administering to the subject a PLA2-GIB antigen.
A further object of the present invention relates to a PLA2-GIB antigen for use in the treatment of a solid cancer in a subject in need thereof.
In particular embodiments, the PLA2-GIB antigen is an inactivated immunogenic molecule that induces an immune response in a subject against PLA 2-GIB. For example, inactivation may be obtained by chemically or physically altering PLA2-GIB or by mutating or truncating the protein, or both; and immunogenicity may be obtained as a result of inactivation and/or by further conjugating the protein to a suitable carrier or hapten (e.g., KLH, HSA, polylysine, virus detoxication toxin, etc.) and/or by polymerization, etc. Thus, the antigen may be chemically or physically modified, for example to increase its immunogenicity.
In a preferred embodiment, the PLA2-GIB antigen comprises PLA2-GIB or an epitope-containing fragment or mimotope thereof.
In a particular embodiment, the PLA2-GIB antigen comprises a full-length PLA2-GIB protein. In a further specific embodiment, the PLA2-GIB antigen comprises a protein comprising SEQ ID No:2 or a sequence having at least 90% identity to SEQ ID No: 2.
In an alternative embodiment, the PLA2-GIB antigen comprises a fragment of the PLA2-GIB protein comprising at least 6 contiguous amino acid residues and containing an immunogenic epitope or a mimotope thereof. In a preferred embodiment, the PLA2-GIB antigen comprises at least 6 to 20 amino acid residues. Preferred peptides of the invention contain 4 to 30 consecutive amino acid residues of SEQ ID NO:2 (or of the corresponding sequence of a natural variant of SEQ ID NO: 2). The most preferred peptides of the invention comprise 5 to 25 consecutive amino acid residues of SEQ ID NO:2 (or of the corresponding sequence of the natural variant of SEQ ID NO:2) and further comprise at least one of the following amino acid residues of SEQ ID NO:2 (or of the corresponding sequence of the natural variant of SEQ ID NO: 2): amino acids 3, 6, 7, 10, 70, 121, 50, 52, 54, 71, 75, 77, 79, 80, or a combination thereof. Specific examples of peptides of the invention are peptides of less than 50 amino acids comprising any of the following sequences:
the PLA2-GIB antigen may be in various forms, such as free form, polymerized, chemically or physically modified, and/or conjugated (i.e., linked) to a carrier molecule. Conjugation to a carrier can increase immunogenicity and (further) inhibit the biological activity of the PLA2-GIB polypeptide. In this respect, the carrier molecule may be any carrier molecule or protein conventionally used in immunology, such as, for example, KLH (keyhole limpet hemocyanin), ovalbumin, Bovine Serum Albumin (BSA), a viral or bacterial detoxified toxin (such as the toxoid tetanos, the toxoid diphtheria B cholera toxin, a mutant thereof (such as diphtheria toxin CRM 197)), an outer membrane vesicle protein, a polylysine molecule or a virus-like particle (VLP). In a preferred embodiment, the carrier is KLH or CRM197 or VLP.
Conjugation of PLA2-GIB to the carrier can be performed by covalent chemistry using linking chemical groups or reactions (such as, for example, glutaraldehyde, biotin, etc.). Preferably, the conjugate or PLA2-GIB protein or fragment or mimotope is subjected to formaldehyde treatment in order to complete inactivation of PLA 2-GIB.
In a particular embodiment, the PLA2-GIB antigen comprises a full-length PLA2-GIB protein, optionally coupled to a carrier protein. In a preferred embodiment, the PLA2-GIB antigen comprises a protein comprising SEQ ID No. 2 or a sequence having at least 90% identity to SEQ ID No. 2 coupled to a carrier protein.
In another particular embodiment, the PLA2-GIB antigen comprises an immunogenic peptide or mimotope of PLA2-GIB, optionally coupled to a carrier protein. In a more preferred embodiment, the PLA2-GIB antigen comprises a polypeptide of at least 10 amino acids in length comprising at least one of the following amino acid residues of SEQ ID NO:2 (or of the corresponding sequence of the natural variant of SEQ ID NO: 2): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof, optionally coupled to a carrier molecule.
The immunogenicity of the PLA2-GIB antigen can be tested by various methods, such as by immunizing a non-human animal that has been transplanted with human immune cells, and then verifying the presence of antibodies; or by sandwich ELISA using human or humanized antibodies. The lack of biological activity can be verified by any activity test described in the present application. In preferred embodiments, the PLA2-GIB antigen has less than 20%, more preferably less than 15%, 10%, 5% or even 1% of the activity of wild-type PLA2-GIB protein in an in vitro method (i) inducing the formation of Membrane Microdomains (MMDs) in CD 4T cells or (ii) rendering CD 4T cells refractory to IL-2 signaling or refractory to IL-7 signaling.
Such molecules and conjugates and vaccines represent powerful reagents for immunizing subjects, thereby causing sustained PLA2-GIB inhibition. After repetition, such methods can be used to cause permanent PLA2-GIB inhibition.
Further aspects and advantages of the present invention are disclosed in the following experimental section, which should be considered illustrative.
Examples
A. Materials and methods
A1.PLA2GIB sandwich ELISA
Sandwich ELISA specific for pre-PLA 2-GIB, PLA2GIB, or both formats was performed using previously generated mabs. For detection of pre-PLA 2GIB, #8G11 mAb was used as capture antibody and #1C11 mAb was used as revealing mAb (rejuvenation mAb). For detection of PLA2GIB, #14G9 mAb was used as the capture antibody, and #1C11 mAb was used as the revealing mAb. For both formats of detection, #7a10 mAb was used as the capture antibody and #1C11 mAb was used as the revealing mAb. Typical assay conditions are as follows:the #8G11 or #14G9 coated microplates were incubated with recombinant human pre-PLA 2GIB or PLA2GIB standards (different concentrations of protein in assay buffer) for 1 hour to generate a calibration curve. EDTA plasma samples were diluted (5 to 50 fold) in dilution buffer into their respective wells and the ELISA plates were incubated for 1 hour at room temperature. After aspiration, wells were washed 3 times with PBS containing Tween 200.05% and biotinylated #1C11 conjugate was added to the wells at room temperature for 30 minutes. After washing with PBS containing 0.05% Tween 20, mAb binding was detected by treatment with streptavidin-HRP conjugate for 15 minutes at room temperature. TMB was added, the reaction stopped, and the absorbance at 450nm was determined on a microplate reader. Using PrismTMThe software (Graph Pad), analyzes the signal by 4-parameter fit analysis.
IL7-induced Nuclear translocation of phosphoSTAT 5
Human CD4 was purified using the RosetteSep isolation kit (Stemcell Technologies, # 15062). CD 4T cells were treated with 106The cells/ml were resuspended in RPMI 1640 medium (Lonza, Verviers, Belgium) (complete medium) supplemented with 5% Fetal Bovine Serum (FBS), 50mM HEPES pH 7.4, glutamine, penicillin, streptomycin, and amphotericin B (fungizone) and equilibrated at 37 ℃ for at least 2 hours in a humidified atmosphere of 5% CO 2.
Confocal microscopy was used to track pSTAT5 nuclear translocation. After equilibration, the immobilized cells were plated on polylysine-coated slides in the presence of plasma (final concentration 1% or 3%) and then activated with 2nM IL-7 for 15 min.
For the PLA2GIB neutralization experiments, 10 μ G (final concentration) of #14G9 mAb was first incubated with plasma (1 or 3% dilution v/v in PBS) for 25 minutes at room temperature, followed by 5 minutes at 37 ℃. The equilibration mixture was then added to CD 4T cells isolated from healthy donors over 30 minutes at 37 ℃. Cells were then activated with 2nM IL-7 for 15 min.
Cells were fixed in 4% PFA for 15 min at 37 deg.C, then fixed for 15 min at Room Temperature (RT), and permeabilized in 90% methanol/water solution at-20 deg.C. pSTAT5 was then revealed by staining with rabbit anti-pSTAT 5(CST, #9359) labeled with donkey anti-rabbit AlexaFluor 555(Life Technologies, # A31572). For human CD 4T lymphocytes, cells were stained with goat anti-mouse AlexaFluor 488(Life Technologies, # A11029) labeled mouse anti-human CD4(eBioscience, # 14-0042-82). For mouse CD 4T lymphocytes, cells were stained with chicken anti-rat AlexaFluor 488(Life Technologies, # A21470) labeled rat anti-mouse CD4(eBioscience, # 14-0042-85). On an inverted laser scanning confocal microscope (LSM700, Zeiss), images above the diffraction limit were acquired with a water immersion apochromatic 40x/1.2NA objective (Zeiss) or an oil immersion plane apochromatic 63x/1.4NA objective (Zeiss). Images were acquired and analyzed using ZEN software (Zeiss).
IL2-induced phosphorylation STAT5 nuclear translocation
The same protocol as in A2 was used except that 2nM IL-2 was used instead of IL-7.
B. Results
B1. Detection of Pre-PLAGIB, PLA2GIB or both forms in plasma from PDAC patients by ELISA
ELISA sandwich was used to examine pre-LA 2GIB and PLA2GIB distribution in plasma from patients with pancreatic ductal adenocarcinoma ("PDAC"). The results are presented in figure 1.
In the control population (n ═ 99 healthy donors), mean (SD) pre-PLA 2GIB levels were 7.8(2.8) ng/mL, and the median was 7.5 ng/mL. The mean (SD) PLA2GIB was 287(243) pg/mL and the median was 170 pg/mL. The mean (SD) value of total PLA2GIB was 9.3(3.2) ng/mL, and the median was 9.1 ng/mL.
In the PDAC population (n ═ 19), mean (SD) pre-PLA 2GIB levels were 10.1(10.4) ng/mL, and median 8.9 ng/mL; mean (SD) PLA2GIB levels of 310(345) pg/mL and a median of 194 pg/mL; the mean (SD) total PLA2GIB level was 11.7(10.5) ng/mL with a median of 10.4 ng/mL.
In this experiment, there was no statistically significant difference in plasma PLA2GIB levels between the control and PDAC cohorts.
PDAC plasma pairIn vitro effect of the activity of human CD 4T cells.
We tested the ability of plasma from PDAC patients to modulate the IL-7 response of CD 4T cells by measuring phosphostat 5 nuclear translocation (pSTAT5 Nt).
As shown in figure 2A, we observed the inhibitory effect of PDAC plasma on CD 4T cell IL-7 response at 1% and 3% dilutions. This result demonstrates that in cancer patients, the tumor microenvironment or plasma provides immunomodulation, e.g., inhibition.
Effect of PLA2-GIB inhibitors on PDAC plasma-induced inactivation of human CD 4T cells.
The ability of neutralizing anti-PLA 2GIB mAb (clone 14G9) to counteract the inhibitory effect of plasma isolated from PDAC patients on IL-7-induced phosphostat 5 nuclear translocation in CD 4T cells was tested by incubating plasma samples from PDAC patients with #14G9 mAb.
As depicted in figure 2B, in human CD 4T cells exposed to healthy donors from plasma samples of PDAC patients, #14G9 mAb restored efficient IL-7-induced nuclear translocation of phosphostat 5. Thus, PLA2-GIB inhibitors can improve cancer subjects. This finding indicates that cancer contains PLA2-GIB cofactor, which renders T cells susceptible to inactivation by PLA 2-GIB.
In vitro effect of pdac plasma on IL 2-induced nuclear translocation of phosphostat 5 in human CD 4T cells.
We tested the ability of plasma from PDAC patients to modulate the IL-2 response of CD 4T cells by measuring phosphostat 5 nuclear translocation (pSTAT5 Nt).
As shown in fig. 3A, inhibitory effects of PDAC plasma on CD 4T cell IL-2 response at 1% and 3% dilutions were observed. This result demonstrates that in cancer patients, the tumor microenvironment or plasma provides immunosuppression.
Effect of PLA2-GIB inhibitors on PDAC plasma-induced inactivation of human CD 4T cells.
The ability of neutralizing anti-PLA 2GIB mAb (clone 14G9) to counteract the inhibitory effect of plasma isolated from PDAC patients on IL 2-induced phosphostat 5 nuclear translocation in CD 4T cells was tested by incubating plasma samples from PDAC patients with #14G9 mAb.
As depicted in figure 3B, in human CD 4T cells exposed to healthy donors from plasma samples of PDAC patients, #14G9 mAb restored efficient IL 2-induced nuclear translocation of phosphostat 5. Thus, PLA2-GIB inhibitors can improve cancer subjects. This finding indicates that cancer contains PLA2-GIB cofactor, which renders T cells susceptible to inactivation by PLA 2-GIB.
B6. In vitro effect of plasma from cancer patients on human CD 4T cells.
The effect of plasma from patients with lung and duodenal cancers on human T cells was tested, as disclosed in B2 above. More specifically, dilutions (1%) of the plasma were prepared and such samples were contacted with CD 4T cells from healthy donors. Such samples were analyzed for the ability to modulate the activity of CD 4T cells by measuring phosphostat 5 nuclear translocation (pSTAT5 Nt) in the presence of IL 7.
The inhibitory effect of the plasma samples may show that in lung cancer patients, the tumor microenvironment or plasma provides immunosuppression, which may be restored according to the present invention.
Sequence listing
<110> Diya Kurett Co
<120> methods and compositions for treating cancer
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<170> PatentIn version 3.5
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Met Lys Leu Leu Val Leu Ala Val Leu Leu Thr Val Ala Ala Ala Asp
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Ser Gly Ile Ser Pro Arg Ala Val Trp Gln Phe Arg Lys Met Ile Lys
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Cys Val Ile Pro Gly Ser Asp Pro Phe Leu Glu Tyr Asn Asn Tyr Gly
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Cys Tyr Cys Gly Leu Gly Gly Ser Gly Thr Pro Val Asp Glu Leu Asp
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Lys Cys Cys Gln Thr His Asp Asn Cys Tyr Asp Gln Ala Lys Lys Leu
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Asp Ser Cys Lys Phe Leu Leu Asp Asn Pro Tyr Thr His Thr Tyr Ser
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Asp Asn Cys Tyr Asp Gln Ala Lys Lys Leu Asp Ser Cys Lys Phe Leu
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Leu Asp Asn Pro Tyr Thr His Thr Tyr Ser Tyr Ser Cys Ser Gly Ser
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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
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Pro Thr Val Ser Ile
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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
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Trp Ile His Trp Val Lys Arg Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Ser Asp Thr Arg Thr His Tyr Asn Gln Lys Phe
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Lys Asp Lys Ala Thr Leu Ala Val Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys
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Ala Arg Gln Thr Leu Tyr Tyr Glu Ala Leu Asp Tyr Trp Gly Gln Gly
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Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr
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Pro Leu
130
<210> 5
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Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
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Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
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Tyr Trp Ala Ser Thr Arg His Thr Gly Ile Pro Ala Arg Phe Ser Gly
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Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ser
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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Thr Pro Pro
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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
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Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
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Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
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Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
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Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
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Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
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Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 6
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Ser Asp Thr Arg Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Thr Leu Tyr Tyr Glu Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
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Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
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Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
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Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
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Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
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Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
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Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
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Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
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Lys
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Ser Asp Thr Arg Thr His Tyr Asn Gln Lys Phe
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Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Gln Thr Leu Tyr Tyr Glu Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
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<211> 449
<212> PRT
<213> Artificial sequence
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<223> 2B2 H
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Asp Pro Ser Asp Thr Arg Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Thr Leu Tyr Tyr Glu Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 9
<211> 726
<212> DNA
<213> Artificial sequence
<220>
<223> 2B L
<400> 9
gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60
cagtgtgaga tcgtgatgac gcagtcgccg gcgacgctga gcgtgagtcc gggggagcgg 120
gcgacgctga gctgcaaggc gtcgcaggac gtgagcacgg ccgtcgcgtg gtatcaacag 180
aagccggggc aagcgccgcg gctgctgatc tactgggcga gcacgcggca cacgggcatc 240
ccggcgcgat tctcggggag ccggagcggg acggagtaca cgctgacgat ctcgtcgctg 300
caaagcgagg acttcgccgt ctactactgc cagcaggact actcgacgcc gccgacgttc 360
ggggggggta ccaaggtcga gatcaaacgt acggtcgcgg cgccttctgt gttcattttc 420
cccccatctg atgaacagct gaaatctggc actgcttctg tggtctgtct gctgaacaac 480
ttctacccta gagaggccaa agtccagtgg aaagtggaca atgctctgca gagtgggaat 540
tcccaggaat ctgtcactga gcaggactct aaggatagca catactccct gtcctctact 600
ctgacactga gcaaggctga ttacgagaaa cacaaagtgt acgcctgtga agtcacacat 660
caggggctgt ctagtcctgt gaccaaatcc ttcaataggg gagagtgctg atagtaaaag 720
ctttga 726
<210> 10
<211> 1431
<212> DNA
<213> Artificial sequence
<220>
<223> 2B H
<400> 10
gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60
cagtgtcaag tccagctcgt ccagagcggg gcggaagtca agaagccggg ggcgagcgtg 120
aaagtctcgt gcaaggcgag cggatatacg ttcacgaact actggattca ctgggtccgg 180
caagcgccgg ggcaggggct ggagtggatc gggaacatcg acccgtcgga cacgcggacg 240
cactacaacc agaagttcaa ggaccgggcg acgctgaccg tcgacaagag cacgagcacg 300
gcgtacatgg agctgtcgag cctgcggagc gaggacacgg ccgtctacta ctgcgcgcgg 360
cagacgctgt actacgaggc gctggactac tgggggcagg ggacgctcgt cacggtctcg 420
agcgctagca caaagggccc tagtgtgttt cctctggctc cctcttccaa atccacttct 480
ggtggcactg ctgctctggg atgcctggtg aaggattact ttcctgaacc tgtgactgtc 540
tcatggaact ctggtgctct gacttctggt gtccacactt tccctgctgt gctgcagtct 600
agtggactgt actctctgtc atctgtggtc actgtgccct cttcatctct gggaacccag 660
acctacattt gtaatgtgaa ccacaaacca tccaacacta aagtggacaa aagagtggaa 720
cccaaatcct gtgacaaaac ccacacctgc ccaccttgtc ctgcccctga actgctggga 780
ggaccttctg tgtttctgtt cccccccaaa ccaaaggata ccctgatgat ctctagaacc 840
cctgaggtga catgtgtggt ggtggatgtg tctcatgagg accctgaggt caaattcaac 900
tggtacgtgg atggagtgga agtccacaat gccaaaacca agcctagaga ggaacagtac 960
aattcaacct acagagtggt cagtgtgctg actgtgctgc atcaggattg gctgaatggc 1020
aaggaataca agtgtaaagt ctcaaacaag gccctgcctg ctccaattga gaaaacaatc 1080
tcaaaggcca agggacagcc tagggaaccc caggtctaca ccctgccacc ttcaagagag 1140
gaaatgacca aaaaccaggt gtccctgaca tgcctggtca aaggcttcta cccttctgac 1200
attgctgtgg agtgggagtc aaatggacag cctgagaaca actacaaaac aaccccccct 1260
gtgctggatt ctgatggctc tttctttctg tactccaaac tgactgtgga caagtctaga 1320
tggcagcagg ggaatgtctt ttcttgctct gtcatgcatg aggctctgca taaccactac 1380
actcagaaat ccctgtctct gtctcccggg aaatgatagt aaaagctttg a 1431
<210> 11
<211> 1426
<212> DNA
<213> Artificial sequence
<220>
<223> 2B1 H
<400> 11
gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60
cagtgtcaag tccagctcgt ccagagcggg gcggaagtca agaagccggg ggcgagcgtg 120
aaagtctcgt gcaaggcgag cggatatacg ttcacgaact actggattca ctgggtccgg 180
caagcgccgg ggcaggggct ggagtggatc gggaacatcg acccgtcgga cacgcggacg 240
cactacaacc agaagttcaa ggaccgggcg acgctgaccg tcgacaagag cacgagcacg 300
gcgtacatgg agctgtcgag cctgcggagc gaggacacgg ccgtctacta ctgcgcgcgg 360
cagacgctgt actacgaggc gctggactac tgggggcagg ggacgctcgt cacggtctcg 420
agcgctagca caaagggccc tagtgtgttt cctctggctc cctcttccaa atccacttct 480
ggtggcactg ctgctctggg atgcctggtg aaggattact ttcctgaacc tgtgactgtc 540
tcatggaact ctggtgctct gacttctggt gtccacactt tccctgctgt gctgcagtct 600
agtggactgt actctctgtc atctgtggtc actgtgccct cttcatctct gggaacccag 660
acctacattt gtaatgtgaa ccacaaacca tccaacacta aagtggacaa aagagtggaa 720
cccaaatcct gtgacaaaac ccacacctgc ccaccttgtc cggcgcctga agcggaagga 780
ggaccttctg tgtttctgtt cccccccaaa ccaaaggata ccctgatgat ctcgcgaacc 840
cctgaggtga catgtgtggt ggtggatgtg tctcatgagg accccgaagt caaatttaat 900
tggtatgtcg acggcgtcga ggtgcataat gccaaaacca agcctagaga ggaacagtac 960
aattcaacct acagagtcgt cagtgtgctg actgtgctgc atcaggattg gctgaatggc 1020
aaggaataca agtgtaaagt ctcaaacaag gccctgcctg ctccaattga gaaaacaatc 1080
tcaaaggcca agggacagcc tagggaaccc caggtctaca ccctgccacc ttcaagagag 1140
gaaatgacca aaaaccaggt gtccctgaca tgcctggtca aaggcttcta cccttctgac 1200
attgctgtgg agtgggagtc aaatggacag cctgagaaca actacaaaac aaccccccct 1260
gtgctggatt ctgatggctc tttctttctg tactccaaac tgactgtgga caagtctaga 1320
tggcagcagg ggaatgtctt ttcttgctct gtcatgcatg aggctctgca taaccactac 1380
actcagaaat ccctgtctct gtctcccggg aaatgataag ctttga 1426
<210> 12
<211> 1503
<212> DNA
<213> Artificial sequence
<220>
<223> 2B2 H
<400> 12
gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60
cagtgtcaag tccagctcgt ccagagcggg gcggaagtca agaagccggg ggcgagcgtg 120
aaagtctcgt gcaaggcgag cggatatacg ttcacgaact actggattca ctgggtccgg 180
caagcgccgg ggcaggggct ggagtggatc gggaacatcg acccgtcgga cacgcggacg 240
cactacaacc agaagttcaa ggaccgggcg acgctgaccg tcgacaagag cacgagcacg 300
gcgtacatgg agctgtcgag cctgcggagc gaggacacgg ccgtctacta ctgcgcgcgg 360
cagacgctgt actacgaggc gctggactac tgggggcagg ggacgctcgt cacggtctcg 420
agcgctagca caaagggccc tagtgtgttt cctctggctc cctcttccaa atccacttct 480
ggtggcactg ctgctctggg atgcctggtg aaggattact ttcctgaacc tgtgactgtc 540
tcatggaact ctggtgctct gacttctggt gtccacactt tccctgctgt gctgcagtct 600
agtggactgt actctctgtc atctgtggtc actgtgccct cttcatctct gggaacccag 660
acctacattt gtaatgtgaa ccacaaacca tccaacacta aagtggacaa aagagtggaa 720
cccaaatcct gtgacaaaac ccacacctgc ccaccttgtc cggcgcctga agcggaagga 780
ggaccttctg tgtttctgtt cccccccaaa ccaaaggata ccctgatgat ctcgcgaacc 840
cctgaggtga catgtgtggt ggtggatgtg tctcatgagg accccgaagt caaatttaat 900
tggtatgtcg acggcgtcga ggtgcataat gccaaaacca agcctagaga ggaacagtac 960
aattcaacct acagagtcgt cagtgtgctg actgtgctgc atcaggattg gctgaatggc 1020
aaggaataca agtgtaaagt ctcaaacaag gccctgcctg cttcaattga gaaaacaatc 1080
tcaaaggcca agggacagcc tagggaaccc caggtctaca ccctgccacc ttcaagagag 1140
gaaatgacca aaaaccaggt gtccctgaca tgcctggtca aaggcttcta cccttctgac 1200
attgctgtgg agtgggagtc aaatggacag cctgagaaca actacaaaac aaccccccct 1260
gtgctggatt ctgatggctc tttctttctg tactccaaac tgactgtgga caagtctaga 1320
tggcagcagg ggaatgtctt ttcttgctct gtcatgcatg aggctctgca taaccactac 1380
actcagaaat ccctgtctct gtctcccggg aaatgataag ctttgatgtc atgcatgagg 1440
ctctgcataa ccactacact cagaaatccc tgtctctgtc tcccgggaaa tgataagctt 1500
tga 1503
<210> 13
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 13
atgaaactcc ttgtgctag 19
<210> 14
<211> 14
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 14
acagcggcat cagc 14
<210> 15
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 15
ttccgcaaaa tgatcaa 17
<210> 16
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 16
cccggggagt gacccc 16
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 17
tacggctgct actgtggctt 20
<210> 18
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 18
gacacatgac aactgctacg acc 23
<210> 19
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 19
acccacacct attcatactc gt 22
<210> 20
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 20
atcacctgta gcagca 16
<210> 21
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 21
agctccatat aacaaggca 19
<210> 22
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> inhibitory nucleic acid
<400> 22
caagaagtat tgtcagag 18
<210> 23
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 23
Asn Asn Tyr Gly Cys Tyr
1 5
<210> 24
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 24
Cys Tyr Cys Gly Leu Gly
1 5
<210> 25
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 25
Tyr Asn Asn Tyr Gly Cys Tyr Cys Gly Leu Gly Gly Ser Gly
1 5 10
<210> 26
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 26
Phe Leu Glu Tyr Asn Asn Tyr Gly Cys Tyr Cys Gly Leu Gly Gly Ser
1 5 10 15
Gly Thr Pro Val
20
<210> 27
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 27
Gln Thr His Asp Asn
1 5
<210> 28
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 28
Cys Gln Thr His Asp Asn Cys
1 5
<210> 29
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 29
Glu Cys Glu Ala Phe Ile Cys Asn Cys
1 5
<210> 30
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 30
Asp Arg Asn Ala Ala Ile
1 5
<210> 31
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 31
Asp Arg Asn Ala Ala Ile Cys Phe Ser Lys Ala Pro Tyr Asn Lys Ala
1 5 10 15
His Lys Asn Leu
20
<210> 32
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 32
Phe Leu Ser Tyr Lys
1 5
<210> 33
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<400> 33
Phe Leu Ser Tyr Arg
1 5
<210> 34
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> inhibitory peptide
<220>
<221> MISC feature
<222> (1)..(1)
<223> X is 2-naphthylalanine
<220>
<221> MISC feature
<222> (4)..(4)
<223> X is 2-naphthylalanine
<400> 34
Xaa Leu Ser Xaa Arg
1 5
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